PHENOTYPIC AND MOLECULAR CHARACTERIZATION OF THE LAND RACES OF CULTIVATED IN NORTHERN

MOHAMMAD ISLAM

DEPARTMENT OF BOTANY HAZARA UNIVERSITY MANSEHRA 2015

HAZARA UNIVERSITY MANSEHRA

Department of Botany

PHENOTYPIC AND MOLECULAR CHARACTERIZATION OF THE LAND RACES OF PEARS CULTIVATED IN NORTHERN PAKISTAN

BY

Mohammad Islam

This research study has been conducted and reported as partial fulfillment for the requirements of PhD degree in Botany awarded by Hazara University Mansehra, Pakistan

The Tuesday 15, October 2014

CONTENTS

2. 6. RNA Analyses ...... 29 2. 6.1 Amplification of RNA ...... 29 2. 6. 2Electrophoresis and Visualization of DNA ...... 32 2.6.3. 1X TAE Buffer (pH. 8.4) ...... 32 2. 6. 4. Ethedium Bromide Solution ...... 32 2. 6. 4 Preparation of Gel Casting Tray ...... 32 2. 6. 5 Preparation of Gel Solution ...... 32 2. 6. 6 Gel Casting………...... 33 2. 6. 7 Gel Electrophoresis ...... 33 2. 6. 8 Visualization of Gel/DNA and Photography ...... 33 2. 6. 9 Extraction of DNA from the Gel ...... 33 2. 6. 10 Confirmation of DNA purification ...... 35 2.7. Data Analyses ...... 35 2.8. The rRNA Sequencing ...... 37 2.9. Consensus Sequence Development ...... 38 Chapter 3 ...... 39 RESULTS ...... 39 3.1 NUMERICAL TRAIT ANALYSIS...... 39 3.1.1PETIOLE LENGTH…… ...... 39 3. 1. 2LEAF AREA………...... 39 3. 1.3 PEDICEL LENGTH ...... 40 3.1.4 LENGTH……...... 40 3.1.5 FRUIT WIDTH……...... 40 3.1.6 FRUIT WEIGHT…… ...... 41 3.2 TAXONOMIC DESCRIPTION OF THE TAXA ...... 50 A. ORIENTAL PEARS ...... 51

3. 2. 1 Buchanan-Hamilton ex D. Don, ...... 51 3.2.2. Pyrus bretschneideri Rehder…………………………………………………………55 3.2.4 Decaisne………………………………………………………...... 69 B. OCCIDENTAL PEARS ...... 61 3.2.6 L…...... 63 3.2.7 Pyrus sinkiangensis …………………………………………………………………...66 3.2.8 Pyrus hopeienses ……...... 69 3.2.9 Pyrus armeniacaefolia…...... 71 3.2.10 Pyrus serrulata……...... 73 3.2.11 Pyrus ovoidea Rehder ...... 75 3.2.13 ………...... 79 3. 2. 14 Pyrus xerophila ……………………………………………………………………..81 3.3.1 DNA Extraction……...... 82 3.3.2 Amplification ofRAPD primers ...... 84 3.3.3 Primers specificity… ...... 90 3.3.4 Similarities among the land races...... 92 3.3.5Phylogenetic Relationship of Pyrus Land races ...... 95 3. 4. Nucleotide Sequencing ...... 100 3. 4.1 Kacha Tora Tangai ………………………………………………………………...100 3.4.2 Zira Mamoothay…… ...... 103 3. 4. 3. Khan Tango (Kt)…...... 106 3. 4. 4. Ghata Zira Tangai...... 109 3. 4. 5. Nashpati…………...... 111 3. 4. 6 Parawoo Tango…… ...... 114 3. 4. 7 Pekhawry Tango…...... 117 3.4. 8 Nak Tango………… ...... 120 3. 4. 9 Spina Mamothy…… ...... 123 3. 4. 10 Asmasy Tango…...... 126 3. 4. 11 Mamosay Batal-8...... 129

3. 4. 12 Mamosay Bat_12… ...... 132 3. 4. 13 Mamoosay Bat_14 ...... 135 3. 4. 14 Mamoosay B15…...... 138 3. 4. 15 Gultar Tango……...... 141 3. 4. 16 Hary Tango Batal...... 144 3. 4. 17 Kado Batang (Kb)...... 147 3. 4. 18 Malyzay Tango…...... 150 3. 4. 19 Mamosranga…… ...... 153 3. 4. 20 Guraky Tango…...... 156 3. 4. 21 Ghata Tora Tangai (Gtt) ...... 159 3. 4. 22 Shaker Tango…… ...... 162 3. 4. 23 ―Pak-24‖………… ...... 165 3. 4. 24 Shaker Tango……...... 168 Chapter 4 ...... 170 DISCUSSION ...... 170 4.1. Numerical variations ...... 170 4.1.1 Petiole length……...... 170 4.1.2 area…………...... 171 4.1.3 Pedicel length……...... 171 4.1.4 Fruit length ...... 172 4.1.5 Fruit width………...... 172 4.1.6 Fruit weight………...... 172 4.2 Markers assisted assay ...... 173 4.2.1. DNA Isolation……...... 173 4.2.2 The RAPD Assay…...... 175 4.2.3 Molecular characterization through 18S rRNA……………………..……………188

REFERENCES ...... 195 APPENDICES ...... 212

TABLE OF FIGURES Fig. 1.1. Map of the study area ...... 3 Fig. 2.5. A skeetch of the conditions of PCR set for RAPD…………………………………27 Fig. 2.6. Flow Chart of the PCR conditions...... 30 Fig. 3. 1. Phylogenetic relationship among 13 Pyrus land races ...... 49 Fig. 3. 2. Pyrus pashia:...... 54 Fig. 3. 3. Pyrus bretschneideri:...... 56 Fig. 3.4. : ...... 58 Fig. 3. 5. Pyrus calleryana: ...... 60 Fig. 3.6. Pyrus pseudopashia:...... 62 Fig. 3.7. Pyrus communis: ...... 65 Fig. 3.8. Pyrus sinkiangensis: …………………………………………………………………..68 Fig. 3.9. P. hopeiensis: ………………………………………………………………………….70 Fig.3.10. P. armeniacaefolia:...... 72 Fig.3.11. Pyrus serrulata: ...... 74 Fig.3.12. Pyrus ovoidea: ...... 76 Fig. 3.13. P. turcomanica: ...... 78 Fig. 3.14: Pyrus ussuriensis:...... 80 Fig. 3.15. Gel documentation images of genomic DNA ...... 82 Fig. 3.16. PCR product of 500 bp fragment...... 83 Fig. 3.17. PCR product of 18s rRAN ...... 83 Fig. 3.16 Showing the presence of the cleaned genes...... 84 Fig. 3. 17. Homology tree...... 94 Fig. 3. 18. Phylogenenetic tree ...... 97 Fig. 3.19. Phylogenetic analysis of Kacha Tora Tangai ...... 102 Fig. 3.20. Phylogenetic analysis of Zira Mamoothay ...... 105 Fig. 3.21 Phylogenetic analysis of Khan Tango ...... 108 Fig. 3.22. Phylogenetic analysis of Ghata Zira Tangai ...... 110 Fig.3.23. Phylogenetic analysis of Nashpati ...... 113

Fig.3.24. Phylogenetic analysis of Parawoo Tango ...... 116 Fig.3.25. Phylogenetic analysis of Pekhawry Tango ...... 119 Fig.3.26 Phylogenetic analysis of Nak Tango ...... 122 Fig.3.27. Phylogenetic analysis of Spina Mamothy...... 125 Fig. 3.28: Phylogenetic analysis of Asmasy Tango ...... 128 Fig 3.29. Phylogenetic analysis of Mamosy Batal_8 ...... 131 Fig.3.30. Molecular phylogenetic analysis of Mamosay B12 ...... 134 Fig. 3.31. Phylogenetic analysis of Mamosy Batal-14...... 137 Fig.3.32. Phylogenetic analysis of Mamosy B15...... 140 Fig.3.33. Phylogenetic analysis of Gultar_Tango ...... 143 Fig.3.34. Phylogenetic analysis of Hary Tango-Batal ...... 146 Fig.3.35. Phylogenetic analysis of Kado Batang ...... 149 Fig.3.36. Phylogenetic analysis of Malyzay Tango ...... 152 Fig.3.37. Phylogenetic analysis of Mamosranga ...... 155 Fig. 3.38. Phylogenetic analysis of Guraky Tango...... 158 Fig. 3.39. Molecular phylogenetic analysis of Ghata Tora Tangai ...... 161 Fig. 3.41.Phylogenetic analysis of Pak-24 ...... 167

LIST OF TABLES

Table 2.1. Reagents used for isolation of DNA ...... 25 Table 2.2. List of the primary information ...... 25 Table 2.4. Basic information of the decamers sequences ...... 27 Table 2.5. The 25µl volume of each PCR reaction mixture ...... 31 Table 2.6. Forward and reverse primers ...... 31 Table 2.7. Reagents for 1X TAE buffer ...... 32 Table 2.8. Information about the similarity of the coefficients ...... 36 Table 3.1. Mean values ...... 47 Table 3.2. Analysis of variance...... 48 Table 3.3. List of Pyrus land races ...... 85 Table 3.4. Allelic distribution ...... 87 Table 3.5. List of primers specificity ...... 88 Table 3.6. Performance of the markers ...... 89 Table 3.7. Markers associated with the amplification patterns of DNA ...... 91 Table 3.8. Distance matrix of 36 sequences ...... 98 Table 3.9. Homology matrix of 36 sequences ...... 99

DEDICATION

I dedicate the success of this endeavor to my FATHER (late) and MOTHER who spent their lives to educate and prepare me as to become an honorable human being for the service of mankind. I present this thesis to those who strirved to instil a perfect character in me.

ACKNOWLEDGEMENT

All praises to Almighty ALLAH (Jalla-Jalalaho), who induced the man with intelligence, knowledge, sight to observe and mind to think. Peace and blessing of ALLAH be upon the Holy Prophet Hazrat Muhammad (S.A.W.W) who exhorted the humanity as a whole to seek for knowledge from cradle to grave.

It is a pleasure and great honor for me to express my deepest and profound gratitude to my research supervisor Prof. Dr. Habib Ahmad TI, Chairman, Department of Genetics, Hazara University, Mansehra Pakistan, for selection of topic. My heartiest appreciation and sense of gratitude goes to him for his sustained interest, guidance, orientation, motivation, advice, suggestions, criticism, inspiration and unforgettable cooperation throughout my research work. I found him a wonderful supervisor who really goes out of his way to show his care for his students.

I express the depth of my cordial gratitude to my respected co-supervisor, Dr. Imtiaz Ahamad Khan, Foreign Faculty Professor, Department of Botany, Karakuram International University, Gilgit Baltistan for his guidance and supervision.

I express the depth of my cordial gratitude to Dr. Shahid Nadeem and Khushi Mohammad, Assistant Professors Department of Genetics, Hazara University, Mansehra for guidance and facilitation in molecular work. I also express the cordial gratitude to Dr. Inamullah, Assistant Professor, Department of Genetics, Hazara University, Mansehra for facilitation in chemicals, use of his lab equipment‘s and guidance in data analysis. I am very thankful to Dr. Jan Alum, Dr. Azhar Hussain Shah and Dr. Haidar Ali Assistant Professors, Department of Botany, Hazara University, Mansehra and Center of Biodiversity and Botany, University of Swat, KP, respectively for guidance and facilitation in taxonomical work. I am also thankful to Mr. Sajidul Ghafoor, Assistant Professor, Department of Genetics, Hazara University in protocols optimization and computational work. I am also thankful to Dr. Niyaz Ali, Assistant Professor,

i Department of Botany, Hazara University, Mansehra for guidance in references development in Endnote software.

I express the depth of my cordial gratitude to Dr. Abdul Nasar Khalid, Associate Professor, Department of Botany, Punjab University Lahore for providing me an excellent working environment in his Mycobiotechnology research lab, guidance and comfortable accommodation in the hostel. I am cordially thankful to Dr. Mohammad Hanif, Assistant Professor, Department of Botany, GC University Lahore for guidance in molecular data analysis and hospitality. I am also thankful to Dr. Abdur Rehamn Khan Niazi, Assistant Professor, Department of Botany, University of the Punjab for their company, facilitation and hospitality.

I am thankful to my teachers and Department colleagues and supporting staff for cooperation and facilitation during my Ph.D work.

In particular I would like to acknowledge my friends Mr. Abdur Razaq, Lecturer, Center of Plant Biodiversity, University of Peshawar, Mr. Amjad Khan, Mr. Ajmal Khan, Research Associate, Department of Botany, University of Malkand and Mr. Inamullah, Research Associate, Department of Genetics, Hazara University Mansehra for their company in field visits during research materials collection.

I am also thankful to Mr. Amjad Khan, Molecular Lab Attendant, Department of Genetic, Hazara University, Mansehra for facilitation and cooperation in Lab and Saqib Iqbal, Head Mali, Punjab University, Lahore for good company in hostel during stay in Lahore.

My humble obligations are due to my Father (late) and Mother and elder Brother Mr. Mohammad Amin who brought me to the level to be able to present this work.

MOHAMMAD ISLAM

ii ABSTRACT

Biosystematic treatment of 110 taxonomically unknown specimens collected from the land races of Pyrus trees available in nature and traditional farms of moist temperate region of Northern Pakistan is presented here. The landraces were surveyed in 100,565 Km2 area and best representative trees of different types were selected for morphological, DNA and ribosomal gene analyses. For morphological trait analyses, numerical parameters viz. petiole length, leaf area, pedicel length, fruit length, fruit width and fruit weight were considered. The morphological analyses sorted out all the collected specimens into 14 species viz., Pyrus pashia, P. calleryana, P. bretschneideri, P. pyrifolia, P. pseudopashiae, P. communis, P. sinkiangensis, P. hopeienses, P. serrulata, P. ovoidea, P. turcomanica, P. ussuriensis, P. xerophila and P. armeniacaefolia. Only two species i.e. P. pashia and P. communis were previously known from Pakistan. A substantial amount of genetic diversity was observed in all the land races with respect to all the parameters, except the leaf area. Mean values shows that the landraces Kushbago Batang (Kbb), Atti Bating (Ab) and Shardi Tanchi (Srt) had longer petioles with mean of 56 mm, 49 mm and 48 mm, respectively while the landraces Batangi and Glass Batang (Gb) had minimum values of 30.60 mm and 30.67 mm for petiole length, respectively. For pedicel length, landraces Batangi, Ghata Zira Tangai (Gzt), Klak Nak (Kn) Shardi Tanchi (Srt) had minimum value of 15-20 mm where as in Kushbago Batang (Kbb) a maximum pedicel length of 65.5 mm was recorded. For fruit length, Glass Batang (Gb) had the highest means (95.2 mm) followed by Kado Batang (Kb) and batang with a mean of 73.1 mm and 72.2 mm, respectively. The land races Batangi, Gzt and Srt had the minimum fruit length. For fruit weidth, the land race Cb had highest (59.3 mm) value and the land races Batangi, Srt and Kzt had the lowest value of 23 mm, 23 mm and 25 mm, repectively while land races Kado batang (Kb) and Glass batang (Gb) proved similar in fruit width, 50.50 mm and 50.37 mm, respectively. The fruit weight was maximum in Cb and Gb which ranged from 148.0-163.7 g, followed by Kb, Kbb and Nhs while the minimum values were showed by land race Batangi, Srt, Kb and Kzt, ranging

iii from 8.7-11.8 g. The numerical parameters like petiole length, pedicel length, fruit length, fruit width and fruit weight provides strong basis for the identification of Pyrus species and should be kept under consideration in taxonomic studies. For molecular characterizations a handy protocol for DNA isolation was optimized and tested on herbarium specimens using bark, wood and , yielding 100, 68 and 53µg/µl quantity of DNA, respectively. The DNA yield was used both for marker assisted elaboration of the specimens and nucleotide sequencing of 18S RNA. PCR amplification of 36 landraces with 60 RAPD primers showed that only 28 primers successfully generated 304 reproducible bands, with the band sizes ranging from 150-2600 bp. The average bands per primer were 10.85 with 100% polymorphism. Fourteen among the primers showed land race specificity by producing 35 different size bands ranging from 150-2100 bp. Out of the 14 land race specific primers, 8 primers showed specificity to single land races with 1-2 loci. The primers D-16, K-09, J-05 and F-13 were specific to three different groups of landraces in the range of 4, 5 and 6, respectively. The homology tree based upon the reproducible bands categoriged all the 36 Pyrus landraces into 6 major groups with 62%-100% homology. The clustering pattern showed that most of the land races shared 80%-100% phylogeny and lineage similarity with each other. Results based upon 24 land races evaluated through 8S rRNA showed that Ktt was closely related to P. pyrifolia cv. Shinil, whereas Gtt occupied an intermediate position between P. pyrifolia cvs. Nijisseiki and Okusankichi. The accession Gzt showed its close relationship with P. pyrifolia cv. Mansoo, Zm showed its close relationship with P. communis cv. Clap‘s Favourite. Khan Tango was closely related to P. pyrifolia cv. Nijiseeki and P. pyrifolia cv. Okusankichi. The Pakistani Nashpati had close affinities with P. pyrifolia cv. Minibae. Parawoo Tango occupied an independent position in Pyrus sub clade I and Pekhawry Tango showed its close affinity with P. pyrifolia cv. Mansoo whereas the Pakistani land race Nak Tango showed its close resemblance with P. communis cv. Favorite.

iv Asmasy Tango showed close relation with P. communis cv. Beurre. Mamosay-8 showed close relation with P. pyrifolia cv. Shinsui. Mamosay-12 occupied an intermediate position between P. pyrifolia cv. Miwang and P. communis cv. Conference, ―Mamosay Batal-14‖ showed the land race lies in between P. pyrifolia cvs. Shinsui and Niitaka. Mamosay-B15 showed close relation with P. communis cv. Clapps-Favourite. The Pakistani land race Gultar Tango was similar to P. communis cv. Clapp's Favorite. Hary Tango-Batal was closely related to P. communis cv. Clapp's Favorite. Kado Batang showed close resemblance with P. communis cv. Clapp's Favorite. The Pakistani land race of Malyzay Tango showed close relationship with P. communis cv. and Mamosranga showed close relationship with P. pyrifolia cv. Gamcheonbae. Guraky Tango showed close relationship with P. pyrifolia cv. Nijisseiki. Shaker Batang occupied an intermediate position in between P. pyrifolia cvs. Nijisseiki and Okusankichi. Similarly, the land race Pak-24 was in between the P. pyrifolia cv. Nijisseiki and Okusankichi. Shaker Tango proved to be closely related with P. communis Pachkan's Triumph.The biosystematics analysis of all the 110 specimens collected from northern Pakistan, added 12 new species to Pyrus from Pakistan. The study established phylogenetic relationship of Pakistani land races of pears with the recorded available in different parts of the world. We concluded that the land races Ktt, At, St and Pak-24 were hybrid in nature and their origin can be traced from their potential proginators as provided in their respective phylogentic trees.

v Chapter 1 INTRODUCTION 1 The study Area Pears belong to the genus Pyrus (subfamily Pomoideae of family ) with basic chromosome number x= 17 (Challice & Westwood, 1973). Most of the belonging to this genus are shrubs or trees. The trees are 4-20 m tall, pyramidal in outlook, umbrella like or narrow and straight in shape. On the basis of morphological and chemical characters, the genus is divided into four groups, the East Asian Pea Pears, the larger fruited East Asian Pears, the North African Pears and the European and West Asian Pears (Challice & Westwood, 2008). Morphologicaly and geographicaly the genus Pyrus is divided in to two groups i.e the Oriental and Occidental pears (Zhukovsky & Zeelinski, 1965). Oriental Pears are characterized by sepals, woody pedicels and subglobose . Such pears have been reported from Eastern Asia through , from Hindu Kush mountains to Japan. Occidential pears on the other hand have persistent sepals, fleshy pedicels and pyriform fruit. The group has been reported from Europe, North Africa, Central Asia, Turkey, , and Kashmir.

The northern part of Pakistan is blessed with a wide diversity of scientifically unexplored germplasm of and its allies, traditionally grown in kitchen gardens and field boundaries of the subsistence farms. The scientific exploration and evaluation of these resources is not only of academic interest but could also contribute to the livelihood of marginal communities. Keeping in view, both the scientific and commercial importance of Pyrus resources, a scientific endeavor was undertaken for taxonomic exploration and characterization of the available biodiversity through DNA and protein analyses. The Districts of Northern Pakistan included in this study were Swat, Shangla, Battagram, Mansehra and Abbottabad of Khyber Pakhtunkhwa (KP) province and Muzafar Abad, Bagh and Palandri Districts of Azad Jammu and Kashmir (AJK). Line sketch of the study area is given in Figure 1.1.

1 Historically, the study area has been visited by a large number of invaders, visitors, traders and pilgrims etc. of different origins. Archeological remains, ruins, inscriptions and petroglyphs are scattered throughout the area. A brief historical sketch of the inhibitants of the area is presented in Table 1.1. Besides cultural and other biological resources, these people had introduced a variety of crops into the area, some of which like the pears are still available in the traditional agricultural farmlands of the area.

Table 1.1. Historic profile of the northern Pakistan.

S. No Period Duration 1 Pre-historic 40,000 - 4000 before present (B.P) 40,000 – 10,000 40,000 – 10,000 upper, lower, middle Palaeolithic 6000 - 4000 Neolithic 2 Protohistoric 3280 -1750 BC

nd 3 Aryan Middle of the 2 millennium B.C 4 Buddhism Gandhara Indo-Greek: 190 B.C - 90 B.C Civilization Scythians: 90 B.C-25 A.D Parthians: 25 A.D- 49 A.D Kushans: 49 A.D-458 A.D White Huns (Epthalite): 458 A.D 5 Turk Shahi 666 - 822 A.D 6 Hindu Shahi 822 - 977 A.D 7 Islam 977 A.D

2

Fig. 1.1. Map of the study area showing collection sites of the Pyrus samples

3 Pears are one of the most important fruit plants worldwide, cultivated in Europe and Asia for two to three thousand years. The genus Pyrus contains at least 22 to 26 widely recognized primary species, all indigenous to Europe, Asia and the mountainous area of North America (Bailey, 1917). Among Pyrus species, only a few species have been domesticated for commercial production (Bailey, 1917; Bell & Itai, 2011). On the basis of geographical distribution and morphological characters, most cultivated pears are native to East Asia (Teng & Tanabe, 2002). Pears are commercially cultivated in temperate regions of more than 50 countries in the world (Bell, 1990; Bell et al., 1996). According to Cuizhi & Spongberg (2003), there are 25 species of pears in Asia. A total of 14 species have been reported from China which includes 8 endemic species. Based on morphological traits, 14 land races and 14 species belonging to the genus Pyrus have been reported from Northern Pakistan (Islam & Ahmad, 2012; 2014). Pyrus pashia Buchanan-Hamilton ex D. Don, Prodr, Pyrus bretschneideri Rehder, Pyrus pyrifolia (N. L. Burman) Nakai, Pyrus betulifolia Bunge, Pyrus phaeocarpa Rehder, Pyrus calleryana Decaisne, Pyrus communis L., Pyrus serrulata Rehder, Pyrus sinkiangensis T. T. Yu, Pyrus armeniacifolia T. T. Yu, Pyrus pseudopashia T. T. Yu and Pyrus xerophila T. T. Yu are very common are among the species reported from China (Burman, 1926). From North Africa, Asia and Europe about 25 species are reported.

1.2 The genus Pyrus

Pyrus L. Sp. Pl. 1: 479. 1753; Hook, Fl. Brit. Ind. 2: 387. 1878; Rehder, Proc. Am. Acad. Arts. Sci. 50: 223. 1915; Terpo and Franco, Fl. Europaea 2: 65. 1968; Schonbeck- Temesy, Fl. Iran. 66: 27. 1969. Malvee, Fl. of U.S.S.R. 9: 259. 1985; Cuizhi, Fl. China 9: 173. 2003.

The term Pyrus is derived from a Latin root referring to pears. Pears are mostly shrubs or trees; trees may be 4-20 m tall, pyramidal, umbrella like or cylindrical in shape. The members of the genus may either be deciduous or semi-evergreen, sometime thorny at the lower parts in young stage. Young leaves are mostly tomentose or pubescent which

4 become glabrous at latter stages. Leaves are simple, petiolate, stipulate, base ovate- elliptic or ovate orbicular, rounded or elliptic. Leaf margin may be serrate, serrulate, crenulated or dentate to entire. Dorsal surfaces of leaves are generally dark green while ventral surfaces are light green. Inflorescence is corymbose raceme, appearing either later or before leaves and born on short lateral spurs. Hypanthum copular, bell shape or somewhat flattened. There are 5 sepals, green, mostly triangular, some time hooded, spread at mature , forming crown at fruit. Apex is acute to acuminate. Margin is glandular or dentate, mostly tomentose inside and slightly so outside. It can some time be glabrous, free or united at base, persistent or deciduous in fruit and villous on both sides. are white or pinkish in bud condition. They are sub-sessile, narrow at the base, rounded, broad oblong or lobed at apex. are 15-35 and filaments are usually white. Anthers are radish to pinkish in color. Styles are 2-5, free, united together with the hypanthum. is inferior, 2-5 loculed having 2 ovules per locule. Fruits is a pome which is succulent, usually pyriform, subglobose, globose, turbinate, depressed globose or oval, flesh may be with sclerenchymatous cells, rich in stone cells, 2-5 celled, with cartilaginous endocarp. are black or nearly so, testa cartilaginous, coltyledons plano-convex. Flowering time ranges from March, 20 to April 30, depending on altitudinal variation while fruits maturation time ranges from July to September depending on species, variety and altitudinal variation.

Basic chromosome number (x) is 17; however there may be some triploid and tetraploids. Most species of Pyrus require cross due to self-incompatability.

1.3 Origin and Distribution

It is generally accepted opinion that pears originated in the Caucasus and spread to Europe and Asia. They were first cultivated more than 4000 years ago. Different species belonging to the genus Pyrus are present in most of the countries of the world. The Corvallis (Oregon, USA) gene bank maintains 2031 clonal Pyrus accessions, 327 lots of 36 taxa from 53 countries. The clonal accessions include 844 European cultivars, 144 Asian cultivars, 87 hybrid cultivars and 159 root stock selections of Pyrus species

5 (Postman, 2008). European pear cultivars were well established in Greece and cultivars with distinct names were propagated as early as 300 B.C. Oriental pears, which arose independently, were also grown in China for more than 2000 years (Kikuchi, 1946). Pears have been cultivated in Europe since 1000 B.C., when Homer wrote about the garden of Alcinous (Hedrick et al., 1921).

In Britain its cultivation was started during Roman occupation and by the 13th century many varieties of pears had been imported from France and the fruit was used mainly for cooking rather than eating raw. At the end of the 14th century, the ―Warden‖ pear had been bred and became famous for its use in pies.

By 1640, at least 64 varieties of pears were under cultivation in England. In the 18th century new and improved strains were introduced. However, the majority of pears continued to be used for cooking. Dessert pears were grown mainly in private gardens but were unsuitable for commercial cultivation. One exception was the‖William's Pear‖, which became very popular by 1770 and is still produced on a limited scale. It is reported that the Royal Horticultural Society encouraged pear growing and in 1826 there were 622 varieties in their gardens at Cheswick. The first significant English Pear to be produced by controlled breeding was ―Fertility‖ in 1875, although this variety is no longer produced commercially. In early 19th century the renowned horticulturist Thomas Andrew Knight began to develop pear varieties. It has been reported that by the middle of the 20th century, the scale and production of ―Comic‖ cultivar decreased while that of ―Conference‖ increased both in scale and production and now a days represents more than 90% commercial production in UK (Anonymous, 2012).

Regarding the origin of Japanese pear cultivars, it is generally accepted that, this group of cultivars has been domesticated from wild P. pyrifolia occurring in Japan (Kikuchi, 1948). The genus Pyrus is believed to have arisen during the Tertiary period in the mountainous regions of western China. Dispersal and speciation is believed to have

6 followed the mountain chains both east and west (Yamamoto & Chevreau, 2009). Pyrus sinkiangenses is a natural hybrid between Pyrus communis and P. bretschneideri while Pyrus pashia, Pyrus betulifolia, Pyrus phaeocarpa, Pyrus calleryana, Pyrus xerophila are often used as stock for pear cultivars and Pyrus bretschneideri, Pyrus pyrifolia, Pyrus communis, Pyrus serrulata, Pyrus sinkiangensis, Pyrus armeniacifolia, Pyrus pseudopashia produces excellent fruits (Cuizhi & Spongberg, 2003).

1.4 Cytogenetics

Mitotic and meiotic analysis in Pyrus species has revealed that all the species are diploid, with 2n = 34, though several cultivars, variants are triploid in nature. Pyrus armeniacaefolia is triploid, P. ussuriensis has diploid and triploid cultivars; P. bretschneideri has diploid, triploid, and tetraploid cultivars. Diploid and triploid cultivars of P. pyrifolia also exist. Diploid, triploid and tetraploid cultivars exist in P. sinkiangensis; P. phaeocarpa, P. betulaefolia, P. hopeiensis, P. serrulata, P. pashia, P. xerophilus and P. calleryana. The Chines Pyrus shows variation in chromosome number in the evolutionary process. It will enrich the polyploid composition of Pyrus in the world and may be used in the production, genetics and for the breeding purpose in pear (Shenghua & Chengquan, 1993). Speciation within the genus Pyrus, as in other genera of the Pomoideae, has occurred without a change in chromosome number (Zeelinski & Thompson, 1967). Majority of cultivated apple and pear are diploid (2n = 2x = 34). The whole tribe of Maloideae is characterised by a basic chromosomes number (x = 17), compared to the three other tribes of family Rosacéae, where basic chromosome number is 7, 8, 9 for Rosoideae, Prunoideae and Spiraeoideae, respectively. This number of 17 chromosomes has attracted discussion about the genetic origin of Maloideae. Several hypotheses were put forward on auto or allopolyploid origin of Maloideae. Apple and pear chromosomes are difficult to distinguish because of their small size ranging from 0.5 to 1.5µm (Lespinasse and Salesses, 1973).

7 1.5. Trade and Commerce

During 2005-2006, the world pear exports increased by 6% and reached to 1.6 million tons. Despite a slight decline in pear exports from Argentina. China‘s, export registered a 20-percent increase. In the Northern Hemisphere, exports increased by 12 percent, supported by growth in most reporting countries. Export volumes from the US for 2005- 2006 decreased by 6 percent with lower production level (Anonymous, 2006). In 2010 the share of pear production with respect to Argentina, Australia, Austria, Belgium, Brazil, Canada, Chile, China, France, Germany, Greece, Hungry, , Iran, Italy, Japan, Mexaco, Netherland, New Zeeland, Poland, Portugal, Romania, Russia, South Africa, Spain, Turkey, Ukrain, United Kingdom, United States and Uruguay was 704200, 95111, 48400, 260000, 16367, 7830, 180000, 15231858, 173746, 38895, 93600, 24176, 382000, 160000, 736646, 284900, 24986, 274000, 25100, 57514, 176900, 60375, 41000, 366216, 473400, 380003, 141700, 32800, 732642 and 18072 metric tons, respectively while the net production was 22,644,756 metric tons. Similarly, in 2011 and 2012 the net production of pears from European countries was 2.629 x1000 and 2.060 x 1000 metric tons respectively, (Anonymous, 2013).

In China the pear production during 2005-06 was 11.2 million metric tons (Anonymous, 2006). China produced 10.6 million MT of pears in 2004-05. In China pear production increased due to planting area and improved tree management (Anonymous, 2005). China produces more pear than other countries of the world, having an annual production of 8 million tons with under cultivation being 0.94 million hectares. The prominent cultivars are mainly produced from Pyrus bretschneideri Rehd., P. ussuriensis Maxim. and P. serotina Rehd and many being imported from Japan (Jun & Hongsheng, 2001). According to World pear export production, the share of Argentina, China, Belgium, Netherland, USA, South Afric, Chili and other countries is 18 %, 17%, 13 %, 12%, 10 %, 8 %, 7 %, 16 %, respectively to the world export (Anonymous, 2005). In Pakistan, the total

8 area under cultivation was 2115 ha, 97 ha, 44 ha in Khyber Pakhtunkhwa (KP), Baluchistan and Punjab, respectively while the net production was 28343 tones, in which 27596, 431 and 316 tons produced by Khyber Pakhtunkhwa, Baluchistan and Punjab, respectively (Anonymous, 2010) and only Shawar Valley, Swat (KP), like other fruit crops, pears contributed about 10412 boxes to market and the net income of the valley during 2004 was approximately Rs. 0.72 million (Hussain et al., 2006; Islam & Razziq, 2010).

1.6. Molecular Biology

Molecular genetic analyses can resolve many aspects of species that are critical in conservation (Frankham et al., 2004). However, the so-called wild types are only found near human habitation and supposed to be escapes (Shimura, 1988). In addition, genetics resources have not been fully identified due the low morphological diversity, lack of prominent differentiating characteristics among species and widespread crossability. It is unfortunate that a large number of pear cultivars available in the conventional farms which have neither been described taxonomically nor tagged genetically, due to the low morphological diversity, lack of prominent differentiating characteristics among species and widespread crossability. Classical morphological characters are very useful in but sometimes these characters are very poor or influenced by environmental conditions; hence thay can not serve as tools for genotypes identification (Bailey, 1917). Morphological characterization is the first step in the description and characterization and classification of plants germplasm (Smith & Smith, 1989; Singh & Tripathi, 1985). Morphological characteristics and isozyme analysis have been the major approaches to assess the genetic variation with in Pyrus spp. or cultivated pears (Chevreau et al., 1985; Jang et al., 1991). Nevertheless, their exact variation still remains unclear because isozyme markers and morphological characteristics are limited in number. Identification of Pyrus is mainly carried out through morphological and geographical distribution but identification to species level is difficult because of (i) limited wild population (ii)

9 reduced morphological characteristics and diversity (iii) wide spread crossability and interspecific hybridization and introgression among different species. Estimation of genetic diversity among Pyrus spp., however, is often difficult because it is considered that there are at least 9 interspecific hybrids (Bell et al., 1996). More over estimation of genetic diversity and relationship between germplasm collections are very important for facilitating efficient germplasm collection, evaluation and utilization. Several tools are now available for identifying desirable variation in the germplasm including total seed protein, isozymes and various types of DNA markers. Therefore presently many DNA markers have been used for Taxonomical relationships and evolution in Pyrus (Yamamoto & Chevreau, 2009).

During last few years, many attempts using different research methods, especially DNA markers have been tried to solve the disputes about the origin of pear cultivars native to East Asia. Several different types of DNA markers have been successfully applied for Pyrus cultivars identification and the analysis of genetic relationship (Kim et al., 1997; Kimura et al., 2003). The identification and localization of red colour trait on morphological marker in the linkage maps 4 of Max Red Bartlef for the first time has been mapped out of linkage group 9 in a species of Maloideae (Dondini et al., 2008).

1.6.1 DNA Technolgy

Before starting the detailed biosystematics endeavor of pears, it was imperative to optimize protocol for its DNA isolation of plant parts available at different times of the year, which can be used for genetic characterization of pears, and have not been identified due to low morphological diversity and overlapping traits (Jang et al., 1991; Chevreau et al., 1997; Frankham et al., 2004). The applications of molecular biology have revolutionized due to the use of DNA for better understanding of genome structure, evolution and identification of species, which needs high quality genomic DNA (Kim et al., 1997; Kimura et al., 2003). The isolation of high quality genomic DNA is essential for many molecular biological applications such as PCR (Chakraborti et al., 2006) but DNA

10 isolation from mature trees of high altitude is a difficult task due to the presence of large number of phenolic compounds and polysaccharides etc. (Gupta et al., 2011).

Various protocols have been reported for isolation of high quality DNA (Saghai-Maroof et al., 1984; Doyle, 1991) each having its own limitations and scope. The longer processing time, need of expensive equipments, costly kits and chemicals and low quality yield generally hampers their application in fruit trees (Adams & Graver, 1991; John, 1992; Kim et al., 1997; Shepherd et al., 2002; Aganga & Tshwenyane, 2003; Varma et al., 2007). The low quality yield of DNA can be due to variety of chemical constituents in trees (Scott & Playford, 1996; Shepherd & McLay, 2011) which needs modification in the protocol accordingly (Barzegari et al., 2010; Li et al., 2010; Smyth et al., 2010). Saghai-Maroof et al., (1984) develop a protocol for isolation of nuclei through a lengthy, expensive and low yielding cesium chloride-ethedium bromide ultracentrifugation procedure. However it yielded only degraded DNA from soybean leaves. Doyle & Doyle (1987) applied exactly the same procedure of Saghai Maroof et al., (1984) but failed with soybean leaves. So they modified the said procedure by doubling the concentration of extraction buffer and the modified protocol worked better. This procedure was published in different times (Taylor and Powell, 1982; Doyle & Doyle, 1987; Doyle & Dickson, 1987; Doyle, 1991). It shows that the adjustment of extraction protocol for obtaining high quality DNA some time be necessary. Similarly a modified CTAB was adopted for extraction of high quality DNA which is suitable for RAPD. In such procedure high salt concentration was used for removal of polysaccharide and PVP for polyphenolic compounds (Porebski et al., 1997). DNA degradation is mediated by secondary plant products such as phenolic terpenoids that bind to DNA after cell lysis (John, 1992). The isolation of High quality DNA from plants containing a high content of polyphenolics such as pear, apples, grape and conifers present problems (John, 1992; Pich & Schubert, 1993). Kim et al., (1997) tried to isolate DNA from materials containing highly polyphenolic compounds by modifying several existing methods. Genomic DNA was isolated from dry root of Berberis lyceum by modifying CTAB procedure using 1%

11 PVP to remove polysaccharides and modified purification protocol using low melting temperature agarose. The extracted DNA worked beautifully for RAPD (Kimura et al., 2003). 1.6.2 Randomly Amplified Polymorphic DNA

Random amplification of polymorphic DNA (RAPD) is a PCR reaction in which segments of DNA are amplified randomly by using many arbitrary, short primers (8-10 nucleotides) to amplify DNA fragments to enable differentiation of genetically distinct individuals. In RAPD, primers bind somewhere to the sequence without any prior knowledge of sequence of genomic DNA is not required (Oliveira et al., 1999; Teng & Tanabe, 2002; Weder, 2002; Srivastava & Mishra, 2009). Presently, RAPD has been used to evaluate, trace and determine the phylogenetic relationship of plants and animals species. Many molecular markers are applied for genetic diversity, DNA fingerprinting and plant identification etc. Among these, random amplified polymorphic DNA, generate DNA fingerprints with a single oligo-nucleotide primer. RAPD are dominant markers and inherited in a simple Mendlian fashion. The RAPD technique is less expensive, faster and require only a small amount of DNA. It is reproducible, require less skill for operation and does not need radioisotopes or sequence information. Further RAPD have proven useful for genotype identification, gene mapping and diversity analysis (Demeke & Adams, 1994). RAPDs have been used for taxonomic identification of Allium, cauliflower, cabbage and Brassica at cultivar and species level (Wilkie et al., 1993). Similarly, RAPD have been used for authentication and identification of different species of medicinal plants (Monte-Corvo et al., 2000; Arif et al., 2010; Khan et al., 2010). Monte-Corvo et al., 2000 used RAPD, AFLP for assessing genetic similarity of Pyrus cultivars and varieties of Pyrus (Botta et al., 1997). Similarly different cultivars of P. pyrifolia and P. communis were identified by RAPD and Sequenced Characterized Amplified Region (SCAR) primers (Lee et al., 2004). Molecular and phenotypic characterization of P. communis, P. pyrifolia, P. cordata, P. bourgaeana and P. pyraster were evaluated through RAPD markers has shown that some primers are genotype specific which can be used for cultivar identification (Oliveira et al., 1999). Similarly RAPD

12 markers were also used for classification and identification of P. pyrifolia and P. communis cultivars (Lee et al., 2004). RAPD markers have been found linked to major genes that controlling skin colour in Japanese pear which is an important character with respect to market value and external pressures. Similarly, the RAPD marker (OPH-19425) was found specific to green fruit color genotypes with 92% probability. It can be useful in breeding programs (Inoue et al., 2006). The genetic diversity of 56 accessions was studied through 12 microsatellite markers, among which 8 reference cultivars belonging to the genus Pyrus. The results had showed 106 putative alleles that ranged from 7 to 19 and average value was 11.8 alleles per locus (Ahmed et al., 2010). Pyrus ussuriensis var. aromatica was evaluated for conservation. Five SSR markers were applied on 86 Pyrus individuals including 58 accessions from Iwate for genetic characterization. Due to high allelic frequency, Iwate accessions were genetically more different than the other Japanese pear varieties. A combined analysis of SSR and cpDNA showed high genetic diversity in Iwateyamanashi and coexistence of Iwateyamanashi and hybrid progeny of P. pyrifolia (Katayama et al., 2007). Seven SSRs markers derived from apple were successfully transferred to 25 Tunisian pear genotypes and 6 varieties of Pyrus communis for low chilling requirements and adaptation to dry conditions. All the microsatellites amplify more than one locus in some of the genotypes (Brini et al., 2008). Similarly the genetic diversity and relationship of Pyrus cultivars for 168 putative alleles studied through SSR markers generated from six primer-pairs, showed a high level of genetic polymorphism with a mean of 28 putative alleles per locus. The heterozygosity was noted as 0.63 while the Dice‘s similarity coefficient between cultivars ranged from 0.02 to 0.98 and Occidental pears generally showed low affinities to Asian pears. Similarly, it has been shown that Chinese white pears is a variety or an ecotype of Chinese sand pears (P. pyrifolia var. sinensis) and the progenitor of Japanese pears has come from China (Bao et al., 2007). A seven microsatellite loci (SSR) developed in apple were used for the identification of 63 European pears. A total of 46 fragments were amplified with an average of 6.6 alleles per SSR. Each microsatellite marker amplified more than one locus. The He and Ho

13 heterozygosities over the six single-locus SSRs averaged 0.68 and 0.44, respectively and the number of effective alleles per loci was 3.43 (Wünsch & Hormaza, 2007). Apple and pear have complex genetic constitution with respect to reproductive cycle and total self-sterility is a complicating factor in the genetic knowledge of Pyrus species. Most agronomic features show a continued variation allowing a polygenic heredity conclusion (Janick & Moore, 1996). Observed segregation on several lineages shows a bigenic disomic heredity as well as fixed heterozygosity. These species should have been considered secondary polyploid with disomic behavior (Challice, 1981; Chevreau et al., 1985; Chevreau & Laurens, 1987). Perennial fruit trees such pear require a long-term effort for breeding because of their long generation time and high level of heterozygosity (Bouvier et al., 2002). Fragments of copia-like retrotransposons were obtained from pear, peach and apple and fifty-one non- redundant sequences derived from Japanese pear were classified into 15 groups by 80% nucleotide identity. Phylogenetic survey revealed a high degree of heterogeneity among the groups. Southern bloting demonstrated that several types of retrotransposon-like sequences existed in the genomes of Pyrus species and polymorphisms were detected among Pyrus species as well as within the species. Retrotransposons contributes to the understanding of the genome structures and the principles of mutation in pear as well as other fruit tree species (Shi et al., 2001).

1.6.3 Small Subunit rRNA Sequences

Ribosomal ribonucleic acid (rRNA) play an important role in protein synthesis. It is a major component of ribosome which consist of 60% rRNA and 40% protein. Ribosomes contain two major rRNAs i.e large subunit (LSU) and small subunit (SSU), present in large and small ribosomal subunits respectively. In eukaryotes, total size of ribosome is 80S, large subunit (LSU) consists of 60S (5S, 5.8S nucleotide (nt), 256, 28S: 5070 nucleotide (nt) sequences and small subunit (SSU) consist of 40S (18S: 1869 nt sequences (Wuyts et al., 2004). Eukaryotes possess many copies of RNA genes, organized in tandem repeats and has special structure and transcription behavior. The rRNA gene clusters are

14 commonly known as ribosomal DNA but the actual DNA is not present in them. Ribosomal RNAs are ancient in origin and present in all forms of life. Therefore, in phylogenetic relationship and taxonomic identification, its nucleotides sequences are excessively used (Smit et al., 2007) and the genes that encode the ribosomal RNA (rRNA) are sequenced for taxonomy, estimation of relatedness of groups and for the estimation of species divergence rates. Therefore, thousands of rRNA sequences are available in different databases such as Ribosomal Database project-II (RDP) and SILVA (Cole et al., 2003; Pruesse et al., 2007). The traditional classification of plants into classes, order, family, genus and species is based on morphological, cytological, biochemical and ecological characteristics. The invention and advancement of molecular techniques in molecular biology such as molecular hybridization, gene cloning, nucleotides and proteins sequencing and restriction endonucleas digestion have provided new tools for investigation and phylogentics relationship of plants, animals and microbes.

The strategies adopted for comparative DNA based identification have opened up new windows for classification of prokaryotic and eukaryotic organisms (Summerbell et al., 2005). In this method, a target region of the genomic DNA is amplified through polymerase Chain Reaction (PCR), isolating the amplicon and sequencing the selected amplicons. After developing a consensus sequence, the species are identified by generating denedrogram based on presence of similarity and absence of dissimilarity or through advanced phylogenetic analysis. In interpreting sequence comparison data, a % identity score, single numeric score determined for each aligned pair of sequences and the number of similar matched nucleotides in relation to the length of the alignment are important. A point of termination scores are arbitrary in species identification and the score can vary depending on many factors, quality of the sequence, number and authenticity of existing database library records from the same species and locus, fragment length of the sequence and the software program that are employed for analysis.

15 Identification of different species depends on the success of comparative sequencing strategy and the choice of target gene. The best target gene should have evolved by a common descent (orthologous), high level of interspecific variation combined with low levels variation on intraspecific bases. Except these factors, the target gene must be easily amplified with a standard universal primer set. The size of the amplicon should be within the range of DNA sequencers used for sequencing and capable of easy alignment with a sequence database for comparison. The genome of all living organisms consist of DNA sequences that code for ribosomal RNAs which is an essential for cellular proteins synthesis. In plants, ribosomal DNA (rDNA) is present in chloroplast, nuclear and mitochondrial genomes. The presence of rRNA throughout nature combined with the development of techniques for the rapid determination of primary nucleotides sequences of rRNA make rRNA a good tool for determining evolutionary relationships (Hamby & Zimmer, 1992). In flowering plants, determination of relationships among different species is a difficult task because of less prominent morphological characteristics and a relatively small number of useful molecular markers for phylogenetic relationship in angiosperms. Therefore for solving the above problems, the 18S rRNA sequences techniques were applied for evaluation and identification of genus and species levels relationships in different families (Nickrent & Franchina, 1990). Ro et al., (1997) evaluated 31 and 4 species belonging to family Ranunculaceae and Berberidaceae respectively to demonstrate phylogentic relationships through 26S rDNA sequences. The result strongly support the concept that Thalictrum chromosome group is not monophyletic but comprises three independent lineages i.e Hydrastis, Xanthorhiza and Thalictrum, Aquilegia and Enemion. The results of 26S rDNA topology is compared with results from two previously published DNA sequences of rbcL,atpB, 18S rDNA genes and RFLP data of cpDNA. The 3 topologies are highly similar and match with karyological characters. With regard to evolution, animals, plants and fungi form monophyletic groups which seem to have originated at approximately the same time. In contrast to eukaryotes, the dissimilarities in small ribosomal subunit RNA sequences among protoctist is large and exceed in prokaryotes. On the basis of rRNA

16 data it can be said that Protoctista branch off very easily in eukaryotic evolution while other groups diverge later. Protoctista is considered to be a collection of independent evolutionary lineages. Generally, it has assumed that eukaryotes have recently diverged. However, on the basis of rRNA sequences they seem to be as ancient as prokaryotes (van de Peer et al., 1993). Evolutionary distances measured from comparison of sequences of small subunit (16S rRNA) of Giardia lamblia and other eukaryotes, show similar measurements of evolutionary diversity between archaebacteria and eubacteria and ensure the phylogenetic significance of multiple eukaryotic kingdoms. The Giardia lamblia 16S like RNA possess many of the properties that might have been occured in the common ancestor of eukaryotic and prokaryotic organisms (Sogin et al., 1989). The gene sequences of nuclear encoded small-subunit 18S rRNA were determined for the Mantoniella squanata, Chara foetida etc and Mougeotia scalaris, Marchantia polymorpha, Fossombronia pusilla, and Funaria hygrometrica and Selaginella galleottii for better understanding of sequential evolution from green algae to land plants. After sequential alignments and conclusion of maximum parsimony and maximum likelihood analysis, Charophyceae was continuously placed on the branch going to the land plants (Kranz et al., 1995). Similarly a coding region sequences of SSU, 16S-like rRNA were determined for 8 species belonging to Chlorophyceae. The phylogenetic tree constructed, showed evolutionary relationships between several plant and green algae (Huss & Sogin, 1990). The Ginseng drugs were identified through PCR-RFLP and mutant allele specific amplification (MASA) on the basis of differences in 18S rRNA gene sequence among three Panax species. The 18S ribosomal RNA gene was amplified and digested with restriction enzymes Ban-II and Dde-I . Each piece give unique electrophrotic profiles for each species (PCR-RFLP analysis). The genomic DNA of each species were amplified with specific primer and the expected size of the fragments of corresponding species were determined with the help of PCR conditions. With the help of these two molecular techniques, Ginseng drugs were identified. To insure identification, partial sequence of plastid gene (matK) was determined in addition to the 18S ribosomal RNA gene (Fushimi et al., 1997).

17 DNA sequences from arbuscular endomycorrhizal fungi are reported to have been obtained by direct sequence overlapping of amplified fragments of the nuclear genes coding for SSU RNA. The said sequences were used to develop PCR-based primers which amplifies a portion of the vesicular- arbuscular endomycorrhizal fungus SSU rRNA directly from a mixture of plant and fungal tissues (Simon et al., 1992). In biodiversity and Taxonomic research, DNA barcoding provide an effective tool for species level identifications and the sequences of target genes are collected and provide a horizontal genomic view with vast implications i.e comparing the goals and methods of DNA barcoding with population genetics and molecular phylogeny (Hajibabaei et al., 2007). Identification of species on morphological parameters/characteristics is very difficult task because of poor morphological traits, environmental effects and anthropogenic activities. Therefore DNA barcoding, DNA taxonomy or molecular taxonomy in combination with morphological data generates an integrated picture. DNA barcoding generate a universal molecular identification key on the bases of taxonomic knowledge that is assembled in logical sequences in reference library. The DNA barcoding extensively strengthen the field of molecular identification (Teletchea, 2010). The molecular trees have the capacity to enclose both minimum and maximum periods of time based on the observation that rate of evolution of genes are different and the DNA specifying ribosomal RNA (rRNA) changes relatively slow as compared to mitochondrial DNA (mtDNA). Therefore, rRNA is useful for investigating relationships between taxa that diverged millions of years ago. The procedure adopted in DNA barcoding (a unique sequence of nucleotides that is characterized by species) is simple. Sequences of the barcoding region are obtained from different individuals and the resulting sequence data are then used to construct a phylogenetic tree using a distance- based neighbour-joining method. In such a tree, similar, putatively related individuals are clustered together and each cluster is assumed to represent a separate species (Hebert et al., 2003).

18 1.7 Scope of the Study

Updated Knowledge of genetic diversity is a prerequisite for crop improvement, which can be elaborated through morphological markers, that are usually limited in number, overlapping in expression and influenced by environmental changes. Unfortunately no serious effort has been made with reference to the importance of pears and their allied species in Pakistan. Pears which are traditionally cultivated in temperate parts of the country have been introduced and highly adopted in traditional farming systems of northern parts of Pakistan. During the present research, recently developed molecular techniques were used to study genome structure and to document the amount of existing genetic variability in pear genotypes commonly grown in Northern parts of Pakistan. Techniques for the isolation of total genomic DNA from selected genotypes were developed / optimized. Genetic variation, in terms of Genetic Distance present in various taxons of pears were studied at the DNA level through Polymerase Chain Reaction using Randomly Amplified Polymorphic DNA primers and 18S rRNA. DNA profiles was statistically analyzed to find out phylogenetic relationship among the land races using cluster analysis and dendrogram. Results of the present research will be helpful for developing better strategies for characterization of pear genotypes, their improvement and conservation. Successful completion of the project will result in tagging the pear genotypes in Pakistan. In addition, it will also contribute twards collection and description of germplasm of pear and allied species.

1.8 Objectives

1. Exploration of genetic diversity of Pyrus species and land races. 2. Identification of different species of Pyrus on the basis of morphological data. 3. Identification of different species of pears using molecular techniques. 4. Establishment of relationship among different species and land races of Pyrus.

19 Chapter 2 MATERIALS AND METHODS

2.1 The Plant Materials Frequent field trips were arranged to the field area (Fig. 1.1) for having a general insight of the area and for selecting representative trees. For further observations sampling was done from selected trees for numerical, taxonomic and molecular elaboration of the landraces during 2010-2013. For numerical parameters, plant specimens were collected from five tree of the same landrace available in different localities. The plant specimens were collected, tagged and pressed in plant presser. The plant materials were properly dried and mounted on herbarium sheets for identification and further reference. Accession numbers were allotted to each specimen in accordance with local names. A land race having local name consisting of a single word was kept as such. While those having name consisting of two and three words were abbreviated, with first letters of all the names being written in capital and the others in small letters. For example, Nashpati and Batangai were used as such while Khan Tango and Ghata Zara Tangai were abbreviated as Kt and Gzt, respectively. All the related information were recorded in the data book and are presented in Appendix-6.7.

2.2 Field Record

Data were collected from the previously selected five trees. The fruit and leaf parameters were measured with the help of vernier-caliper and meter rod. It was made sure that the selected trees were of the same land race growing in different localities. The fruit specimens were preserved in specimen jars in 3.00% formaline for future reference. All the research material was submitted to the Herbarium at Hazara University, (HUP) garden campus, Mansehra.

20 2.3 Taxonomic Description Field trips were made each year from March to August for collection of specimens for taxonomic description and identification. The plant materials were collected both at flowering and fruiting stages. Global positioning system (GPS) data was recorded (Appendics 6.7), plant specimens were tagged, numbered and field information were recorded on the specimen tags. Pictures for important taxonomic traits were taken. In almost all cases the taxon were studied in their natural habitats and field data were recorded in a note book. Plant specimens were dried in old news papers pressed in plant presser. The wet news papers were replaced till the plant specimens were dry. Before mounting, the herbarium specimens were treated with Copper Sulpate (CuSo4) and

Mercuric Chloride (HgCl2) in the ratio of 2:1 per litre of methanol solution. The plant specimens were placed in the solution for 5 minutes and then dried on the newspapers. The specimens were mounted on the herbarium sheet with the help of glue. All the related information such as voucher specimen number, GPS data, locality, habitat, local name, date of collection and name of the collector were properly recorded. Taxon were identified with the help of original description, different floras, accessory notes and photographs. The type specimens and duplicates were deposited to the Herbarium Hazara University (HUP), garden campus, Mansehra, KP, Pakistan.

2. 4 DNA Analalysis

2. 4. 1 The Plant Material

Plant materials such as fresh shoots, fallen dry leaves bark, wood and herbarium specimens were collected from selected trees at different locations of Northern Pakistan and Azad Kashmir. Specimens were dried at room temperature and processed further for DNA isolation.

21 2. 4. 2 DNA Isolation

The plant materials were ground into fine powder at room temperature through pestle and mortar. The ground samples were packed and labeled properly. For DNA Isolation, 0.12g of the powdered material was put into 15ml centrifuge tube. Seven (7.00) ml of 3% heated (60ºC) CTAB solution was added to the tuble. The composition of cetyl trimethyl ammonium bromide (CTAB) solution was; 3%CTAB, 100mM Tris-HCl, 2.5M NaCl and 20mM EDTA (both the Tris and EDTA pH was 8.0). The powdered samples were mixed well in the CTAB solution, with the help of micropipette tip. One (1.00) ml of lysis buffer (10% SDS, 0.1M Tris HCl, 20mM EDTA pH. 8.0, 0.1M Tris-HCl pH. 8.0), 200µl β. merceptoethanol and 0.1g PVP were added to each sample. The samples were water heated at 60ºC for 1.00 hr. During incubation tubes were inverted gently after every 15.00 min. 500µl of Chloroform Isoamyl Alcohol (24:1), was added to each sample and centrifuged at 10,000 rpm for 15min. The supernatant was shifted to another eppendorp tube and 500µl of Chloroform Isoamyl Alcohol were again added to each sample. It was again centrifuged at 10,000 rpm for 15 min. The supernatant was shifted to eppendorp tube and 500µl of Isopropenol and 30µl Sodium acetate was again added to each samples. The samples were gently inverted and kept at -20ºC for 1.00 hr and centrifuged again at 12,000 rpm for 15 min to get the DNA pellet. The pellet was kept in 70% ethanol for 10 minutes and slowly inverted few times. The ethanol was discarded and the pellet was dried at 35ºC for 30 minutes. Each pellet was added; with 30µl Tris

EDTA (TE) buffer and water heated at 60ºC for 5 minutes. Only 5.00µl of the extracted genomic DNA was run and checked for quality on 1.00% agarose gel. The gel was visualized on gel documentation system (Fig. 3.15 at page 82).

22 2. 4.3 PCR Amplification

The PCR amplification of 18S rRNA genes was carried out for different Pyrus land races. A typical PCR mix contained 200µM of dNTPs, 25mM of MgCl2, 10x Taq Buffer, 20 pmol of forward and reverse primers each, 50ng of template DNA and 0.5 µl (2.5U) of Taq

DNA Polymerase. The final volume was made upto 25µl dH2O.The conditions for amplification of the said gene in thermal cycler (Applied Biosystems-2720) were adjusted at 94ºC for 4 minutes as pre PCR denaturation step, 35 repeated cycles of denaturation for 40 seconds, annealing temperature was 54ºC for one minute, extension at 72ºC for 1.5 minutes. A final extension step was carried out at 72ºC for 5 minutes. The PCR products of all samples were resolved on 1.5 % agarose gel in gel electrophoresis (Fig. 2.1 & 2.2).

2. 4. 4 Elution of Gel

The PCR amplified fragments were eluted from the gel. The gel along with the amplified fragment was excised with scalpel and kept in eppendorp tube. Gel Elution Kit (K0513, Fermentas) was used for purification of the excised fragments. A 700µl of binding solution was added to each sample and incubated at 56ºC for 5-10 min and gently shaken after every 3 minutes to mix well. Five (5) µl of Salica beads were added to each sample, incubated at 560C for 5 min and centrifuged at 7000 rpm for 1.00 min. The supernatant was discarded and 500µl wash buffer was added to the pellet and centrifuged again at 10000 rpm for 1.00 min. The pellet was air dried after discarding the supernatant. The pellet was dissolved in 50µl of TAE buffer and centrifuged at 10000 rpm for 2 min. The upper liquid layer containing eluted DNA was carefully shifted to another eppendorf tube. The 5µl eluted DNA was loaded into 1.5% agarose gel for the confirmation of DNA and the gel was visualized in gel documentation system as described before in section 2.4.2.

23 2. 5 RAPD Analyses 2. 5. 1 Plant Materials

Plant materials such as small branches and herbarium specimens were collected in polythene bags from different areas of Northern Pakistan and Azad Kashmir. The samples were tagged and placed at -20ºC. The plant materials of thirty Six (36) Pyrus land races (Table 2.2) were selected for further analysis such as DNA extraction, PCR and RAPD etc.

2. 5. 2 DNA Extraction

For DNA isolation, a 3%CTAB method (Islam et al., 2013) was adopted for extraction of genomic DNA.The reagents used for DNA extraction given in Table 2.1.

24 Table 2.1. Reagents used for isolation of DNA from Pyrus materials

S. No Reagents Amount 1 3%CTAB (Cetyl trimethyl Ammonium Bromide) 3g/100ml 2 50mM Tris HCl (pH 8.0) 0.78g/100ml 3 1.4 M NaCl 8.17g/100ml 4 20 mM EDTA (pH 8.0) 0.58g/100ml 5;2 Lysis Buffer (10% SDS,0.1M Tris HCl, 20Mm EDTA) 1.0ml 6 β-merceptoethanol 200µl 7 PVP 1.0g 8 Chloroform Isoamyl Alcohol (24:1) 500µl 9 Isopropenol 500µl 10 Sodium acetate 30µl

Table 2.2 List of Pyrus landraces collected from Northern Pakistan

S. Genotypes Location GPS Position Altitude No (ft) 1 Malezae Tango (Mt) Swat, Shawar 350 08. 368‘ N/720 32. 958‘ E 5160 2 Khan Tango (Kt) (Kt) Swat, Shawar 340 49.044‘ N/720 18. 661‘ E 4830 3 Nashpati (Nashpati) Swat, Chail 350 08.194‘ N/720 38.963‘ E 5149 4 Asmasy (Asmasy) Swat, Shawar 350 08. 364‘ N/720 33. 957‘ E 5150 5 Nak Tango (Nt) Swat, Shawar 350 08. 364‘ N/720 32. 956‘ E 5140 6 Parawoo Tango (Pt) Swat, Chail 350 08. 364‘ N/720 33. 086‘ E 4825 7 Kacha tora tangai (Ktt) Swat, Chail 350 08. 421‘ N/720 33.008‘ E 4720 8 Khapa Tango (Kt) Swat, Shawar 340 49.033‘ N/720 18.650‘ E 4812 9 Mamosranga Swat, Shawar 350 08. 365‘ N/720 32. 957‘ E 5145 10 Guraky Tango (Gkt) Swat, Shawar 340 50.045‘ N/720 19.561‘ E 4916 11 Pekhawary Tango (Pkt) Swat, Shawar 340 50.045‘ N/720 19.561‘ E 4916 12 Tora Tangai (Tt) Ghazi abad, AJK 330 59.821‘ N/730 37.375‘ E 4014 13 Kacha Tora Tangai (Ktt) Batal Mansehra 340 35.265‘ N/730 09.606‘ E 5180 14 Ghata Zira Tangai (Gzt) Ghazi abad, AJK 330 59.822‘ N/730 37.376‘ E 4030 15 Kala Batang (Kb) Balakot-Sangar 340 34.701‘ N/730 22.423‘ E 5166 16 Nak Batang Balakot (Nb- Balakot-Sangar 340 34.674‘ N/730 22.579‘ E 4980

25 Balakot) 17 Kashmiry Batangi(Kshb) Balakot-Sangar 340 34.700‘ N/730 22.613 E 5320 18 Kashmiry Flexble(Kshf) Balakot-Sangar 340 34.700‘ N/730 22.613 E 5320 19 Kado Batang (Kb) Balakot Sangar 340 34.708‘ N/730 22.386‘ E 5157 20 Ghata Zira Tangai (Gzt) Balakot-Sangar 340 34.700‘ N/730 20.611 E 5100 21 Zira Mamothy (Zm) Shawar, Swat 340 40.014‘ N/720 19.621‘ E 4850 (Zm) 22 Spina Mamothy (Sm) Shawar, Swat 340 40.014‘ N/720 19.621‘ E 4850 23 Khapa Tango (Kt) Shawar, Swat, 340 49.033‘ N/720 18.650‘ E 4812 24 Momsay Batal Batal, Mansehra 340 35.299‘ N/730 09.530‘ E 5099 (Mamosay-B) 25 Shaker Batal (SB) Batal, Mansehra 340 35.291‘ N/730 09.511‘ E 5079 26 Batangai (Batangai) Batal, Mansehra 340 35.279‘ N/730 09.588 E 5170 27 Batangai (Batangai) Batal, Mansehra 340 35.279‘ N/730 09.588‘ E 5160 28 Pashakaly batang (Psb) Mansehra, 340 35.267‘ N/730 09.608‘ E 5190 29 Shaker Batang (Sb) Batal, Mansehra 340 35.268‘ N/730 09.609‘ E 5195 30 Mamosy Bat-14 Batal, Mansehra 340 35.277‘ N/730 09.587‘ E 5160 (Mamosy B14) 31 Mamosy Bat-8 (Mamosy Batal, Mansehra 340 35.289‘ N/730 09.290‘ E 5040 B8) 32 Haray Tango (Ht) Batal, Mansehra 340 35.267‘ N/730 09.608‘ E 5190 33 Shaker Bat-10 (Shaker Batal, Mansehra 340 35.291‘ N/730 09.411‘ E 5079 B10) 34 Mamosy Bat12(Mamosy Batal, Mansehra 340 35.277‘ N/730 09.587‘ E 5200 B12) 35 Mamosy Bat-15 Batal, Mansehra 340 35.262‘ N/730 09.600‘ E 5180 (Mamosy B15) 36 Mamosy Bat- Batal, Mansehra 340 35.272‘ N/730 09.587‘ E 5191 13(Mamosy13

2.5.3 RAPD amplification

A total of sixty RAPD primers were tested for reproducibility of amplicons. Among these 60 primers, 28 produced clear, prominent and reproducible bands. The 28 RAPD primers (details in Table 2. 4) were further used for molecular characterization of 36 Pyrus land

26 races. The reaction was carriedout through 2720 Thermal Cycler (Applied Biosystems, U.S.A.) in a total volume of 20µl (Table 2.3). Table 2. 3. The PCR mix for RAPD amplification

S. NO Reagents Volum (R1 X)

1 dNTPs (2mM) 2.0µL

2 Taq Buffer (10x) 2.0µL

3 Mg Cl2 (25mM) 1.5µL

4 RAPD Primer (10 pmol) 1.0 µL 5 Taq Polymerase (5u/µL) 0.5 µL

6 Template DNA (50ng/µL) 1.0 µL

7 H2O 12.0 µL Final Volume 20.0 µL

The optimized conditions for DNA amplification in each PCR reaction is given below

Fig. 2.5. A skeetch of the conditions of PCR set for RAPD amplification

27 Table 2. 4. Basic information of the decamers sequences used for amplification

Sr. No Primer Nucleotides GC Tm MW Names 1 G-07 GAACCTGCGG 60.0% 32.0 3053.0

2 F-17 AACCCGGGAA 60.0% 32.0 3046.0 3 G-06 GTGCCTAACC 60.0% 32.0 2987.9

4 G-08 TCACGTCCAC 60.0% 32.0 2947.9 5 F-13 GGCTGCAGAA 60.0% 32.0 3077.0 6 L-08 AGCAGGTGGA 60.0% 32.0 3117.0 7 J-05 CTCCATGGGG 70.0% 34.0 3044.0 8 I -08 TTTGCCCGGT 60.0% 32.0 3009.9 9 L- 18 ACCACCCACC 70.0% 34.0 2901.9 10 J-20 AAGCGGCCTC 70.0% 34.0 3013.0 11 E-10 ACCAGGTGA 60.0% 32.0 3037.0 12 J-05 ACGCACAACC 70.0% 34.0 3044.0 13 A-11 CAATCGCCGT 60.0% 32.0 2987.9 14 F-02 TGTCATCCCC 60.0% 32.0 3028.0 15 A-02 GCCGAGCTG 70.0% 34.0 3044.0 16 C-09 CTCACCGTCC 70.0% 34.0 2923.9

17 D-16 GGGCGTAAG 60.0% 32.0 3117.0 18 D-15 CATCCGTGCT 60.0% 32.0 2978.9

19 E-15 CGCACAACC 60.0% 32.0 2965.9 20 C-08 GGACCGGTG 60.0% 32.0 3090.0 21 F-07 CCGATATCCC 60.0% 32.0 2947.9 22 B-09 GGGGGACTC 70.0% 34.0 3084.0 23 F-13 GGCTGCAGAA 60.0% 32.0 3077.0 24 L-02 TGGGCGTCAA 60.0% 32.0 3068.0 25 F-19 CCTCTAGACC 60.0% 32.0 2947.9 26 M-06 CTGGGCAACT 60.0% 32.0 3028.0 27 I-16 TCTCCGCCCT 70.0% 34.0 2914.9 28 K-09 CCCTACCGAC 70.0% 34.0 2932.9

28 2. 5. 4 Agarose gel Electrophoresis

For gel electrophoresis, 1X TAE Buffer (pH. 8.4) stock solution as prepared for gel preperation and 2.42g of Tris Base, 0.186g EDTA were added for 250 ml stock solution. The pH of the stock solution was adjusted to 8.4 with the help of 20mM Acetic acid and the volume in the volumetric flask was taken to 250 ml through ddH2O. A 10µl of 10 mg/ml ethidium bromide solution to 200 ml of dd H2O and the bottle was covered with aluminum foil to protect from light.

Gel casting tray was prepered by wrapping the plastic tap or by adjusting their respective side segments and comb. Agarose (0.5g) was added into 50 ml of 1x TAE buffer in a 500 ml conical flask and heated in microwave oven for 2 minutes for dissolving the agarose. The solution was cooled to 50oC and 10-15ul ethedium bromide was added and mixed well. The gel solution was poured into the electrophoresis gel tray and the comb was placed in position.

After solidification, the gel was shifted into the gel tank, 1X TAE buffer was added so that the buffer submerged the gel. The comb was removed carefully in vertical position. A 2ul 6x gel loading dye was added into each PCR vial, mixed well and 20ul PCR product was loaded into each well. A 5µl of 1000bp (Fermentas SM0331) DNA ladder was used as reference for sizing the fragments obtained. The cover was placed on the gel tank and 75Ao current was passed through the gel until the dye reached to the middle of the gel.

After successful running phase, the gel was shifted to the gel documentation system. Pictures were taken with the help of computer and processed in Microsoft Office Power Point for better visualization of DNA fragment.

2. 6. RNA Analyses 2. 6.1 Amplification of RNA

A 500 base pairs (bp) fragment of 18S rRNA of the genus Pyrus amplified through 2720 Thermal Cycler of Applied Biosystems, U.S.A. The optimized conditions of the reaction

29 are provided in Fig. 2.7. Initial denaturation step was 94oC for 4 minutes, denaturation step was at 94oC for 40 seconds, annealing temperature was 540C for one minute, extension at 72oc for 1.5 min. The process was continued for 35 cycles and the final extension was carried out at 72oC for five minutes.

Fig. 2.6. Flow Chart of the PCR conditions optimized for amplification of the double stranded of 18s rRNA

Details of the reaction mix are given in table 2.5 while details of F and R primers are presented in table 2.6.

30

Table 2.5. The 25µl volume of each PCR reaction mixture comprises of the following reagents

S. NO Reagents Volum (R 1X) 1 dNTPs 2µl 2 Taq Buffer (10x) 2.5µl

3 Mg Cl2 (25mM) 2 µl

24 F1P(Forward 1 Primer) (10pmol/µl) 2 µl 5 R1P (Reverse 1 Primer) (10pmol/µl) 2 µl 6 Taq Polymerase (5u/µl) 0.5 µl 7 Template DNA (50ng/µl) 2.0 µl

8 H2O 12 µl Final Volume 25 µl

The following forward and reverse primers were used in PCR amplification

Table 2.6. Forward and reverse primers

No Primers Sequence bp TmoC 1 F1P 5‘ GTC AAA CTG CGA ATG GCT CAT TAA ATC 3‘ 27 63 2 R1P 3‘GCA ACA ACT TAA ATA TAC GCT ATT GGA G 5‘ 28 61 Source: Elim Biopharmacuticals, Inc 25495 Whitesell st. Haywar, CA 94545

31 2. 6. 2 Electrophoresis and Visualization of DNA

The following stock solution were prepared for gel preparation. 2.6.3. 1X TAE Buffer (pH. 8.4)

The following reagents were added into 500 ml of conical flask for preparation of 250 ml stock solution Table. 2.7. Reagents for 1X TAE buffer

S. No Reagents Amount (g)

1 Tris-Base 2.42

2 Ethyline Diamine Tetra Acetic Acid (EDTA) 0.186

3 Acetic Acid (pH adjustment) …….

Final Volume (ml) 500

The pH of the stock solution was adjusted to 8.4 with the help of 20 mM Acetic acid and the final volume was made to 250 ml through ddH2O.

2. 6. 4. Ethedium Bromide Solution

A 10µl of 10mg/ml ethedium bromide solution was added to 200 ml of dd H2O and the stock solution bottle covered with aluminum foil to protect from light. 2. 6. 4 Preparation of Gel Casting Tray

Gel casting tray was developed by wrapping plastic tap or by adjusting its respective side segments and comb. 2. 6. 5 Preparation of Gel Solution

The following reagents were added into 500 ml conical flask and heated in microwave oven for 2 minutes to dissolve the agarose well till the solution became clear.

32 S. No Reagents Amount

1 Agarose 0.5 g

2 1X T A E Buffer 50 ml

2. 6. 6 Gel Casting

The gel solution was cooled to 50oC and 10-15ul Ethedium bromide was added and mixed well. The gel solution was poured into the electrophoresis gel tray and the comb was placed in position.

2. 6. 7 Gel Electrophoresis

When the gel solidified and cooled it was shifted into the gel running assembly. 1x TAE buffer was added that the buffer was added till the gel submerged in the buffer the comb was removed carefully in vertical position. 2ul of dye was added into each PCR vial, mixed well with the help of micropipette. 25ul of PCR product was loaded into each well of the gel along with DNA marker. The cover was placed on the gel tray apparatus and 75 Ao current was passed through the gel until the dye reached to the middle of the gel.

2. 6. 8 Visualization of Gel/DNA and Photography

After successful completion of gel running, the gel was shifted to UV-Tank. UV-rays wer focused and fixed for better visualization of DNA fragments. Pictures were taken with the help of computer and saved for further record and analysis. The DNA fragments of desired size were cut and placed into eppendorp tube and preserved at -20 0C.

2. 6. 9 Extraction of DNA from the Gel

The PCR amplified fragments ware isolated again from the PCR gel as per manufacture‘s (GeneAll Korea ) instructions presented below ;

33 1. The desired gene fragment was isolated from the PCR gel with the help of blade and placed into eppendorp tube and marked with the corresponding number or name. 2. 700µl of binding solution (Sodium Iodide, NaI) was added to the eppendorp tube and the sample was incubated at 56oC for 5-10 minutes. 3. The samples were gently shacken after every three minutes to mix well. 4. 5µl of Silica beads were added to the samples and mixed by inverting the tubes gently. 5. The samples were again incubated at 56oc for 5 minutes. 6. The samples were centrifuged for 1 minute and the supernatant was discarded. 7. 500µl of silica beads wash buffer (diluted and cooled at 00C) was added to the pellet and mixed properly. 8. The sample was centrifuged at 10000 rpm for 1 min and supernatant was again discarded. 9. Step #.7 and 8 wer repeated. 10. The tubes were air dried/incubated at 56oC for about 5 min to remove the water.

11. 50 µl of dH2O or TE buffer was added and mixed well to dissolve the DNA fillet. 12. The sample was centrifuged at 10000 rpm for 2 min. 13. The upper layer was carefully shifted to another eppendorp tube. 14. 8-10µl of final extracted DNA samples were loaded into gel for confirmation, whether the DNA was successfully isolated or not. The following precations were observed during the process; 1. Temperature of 1st incubation was not allowed to go above 60oC. 2. The washing buffer used had a temperature of around 0oC. 3. The liquid layer was carefully separated from silica beads

34

2. 6. 10 Confirmation of DNA purification

After DNA elution (2.6.10), DNA was again run in a gel and visualized in UV tank. The DNA bands of amplified fragments appeared prominant and hence confirmed that DNA was successfully isolated (Fig. 3.18).

2.7. Data Analyses

The numerical data for morphological traits were analyzed through MSTAT-C software; where as, amplified PCR products were separated by electrophoresis alongwith the DNA marker (Fermentas cat# SM0403) on 1.5% agarose gels. The gel was run for 02 h at 100 V and the DNA banding were visualized with UV light using Gel Doc. The DNA amplification and separation for each sample/primer combination was reconfirmed for reproducibility of band scoring.

The sizes of amplified RAPD fragments were estimated with reference to the known DNA marker (Fermentas cat No SM0403). Only RAPD fragments were compared and used for statistical analysis. The presence or absence of fragments was recorded as either 0 (absent), 1 (present). The binary data set was recorded on spreadsheet for further processing using, DNAMAN statistical software (version 5.2.2.0; Lynnon BioSoft). Coefficients for all possible pairs of isolates based on their fingerprinting groups were estimated using the Dice 1945, Nei and Li (1979) coefficient. A dendrogram was constructed from the similarity coefficient data by the (Unweighted Pair-Group Method using the Arithmetic averages) clustering algorithm.

The genetic distances between genotypes were assessted on the bases of scoring the presence (1) or absence (0) of RAPD amplicons and were determined with the help of published formulae

35 (Jaccard, 1901; Russel, 1940; Dice, 1945; Sørensen, 1948; Ochiai, 1957; Sokal, 1958; Rogers and Tanimoto, 1960; Anderberg, 1973) and presented in Table 2.8

Table 2.8. Information about the similarity about the coefficients for documenting

Pyrus.

Microsoft Excel 2007 was used for scoring of RAPD loci. Only clear, prominent and reproducible bands were considered for data analysis. In the gel the band/bands were matched with 100bp DNA ladder with the help of a straight line and the size of respective band was incorporated in the RAPD marker column of Excel sheet. The amplified fragments were denoted as ―1‖ and non-amplified fragments as ―0‖. The collected data was inserted in the Excel sheet in the respective genotypes.

The DNAMAN computer software version 2.10 (Applied Biostatistics Inc.) was used to analyze and develop coefficients. A dendogram was developed from the similarity coefficient data by the Neighbor Joining Method.

36 The binary data collected for the presence of allele (1) and absence of allele (0) was recorded in a Microsoft excel table. The columns were labeled with the names of object or sample while the rows were labelled with the names of marker. The excel table was converted into a form with suitable numbers available without blank cells, such as a form with 0 and 1 in rectangle. Details of the analytical procedures are provided in Annextur 7.1. The similarity coefficient was based on DNA bands profile. These statistics, like the coefficient (Jaccard, 1908; Nei and Li, 1979) compare the number of bands shared between genotypes.

2.8. The rRNA Sequencing

For nucleotides sequencing of the the amplified 500bp DNA fragment of 18S template rRNA, samples of 30 land races of Pyrus were sent to Macrogen Inc. Korea. After successful sequencing of samples, sequences data were retreived and analyzed through Applied Biosystem Sequence Scanner v 1.0, BioEdit Sequence alignment editor, MEGA 5.1 (Tamura et al., 2011) and geneious R6 1.2. The forward (PF1) and reverse (PR1) sequences of each land race obtained through Macrogen were opened in Applied Biosystem Sequence Scanner V 1.0 for creating sequence peaks. The peaks were corrected for overlapping peaks following by trimming of the ambiguous/dirty regions from both ends of the sequences. Only clear nucleotides of both (PF1) and (PR1) sequences were selected, copied and pasted into Microsoft word document. Both Fp and Rp sequences were imported in BioEdit software and ClustalW multiple Alignment. The consensus sequences were created using the two softwares. For plus sequences confirmation, one of the Fp or Rp sequence was BLAST in NCBI and the sequences of the downloaded species were observed.If the sequences showed Plus/Minus, then reverse sequenencing was carried out for candidate sequence. Itherwise consensus sequences were developed rightaway.

37 2.9. Consensus Sequence Development >Accessory Applications (Toolbar) >ClustalW Multiple Allignment > Alignment (Toolbar) > create consensus sequence> copy> paste into MS office file below the conserned FP & RP under the heading of consensus. The consensus sequences were then blasted against the available retrievable sequences of NCBI. The quary and Maximum identity of each reference species were run and the the arranged sequences were trimed through the BioEdit software.

The last digits of the align file were noted for finding out the total characters. The chopped file were then opened and copy the first 20 nucleotides of any sequence copied: On the aligned file, and the exact location from where the extra region was trimmed, were found out. The position was recorded and the number of the last bp of the sequence on the chopped file was noted. The number of extra region was trimmed in align file was added and the last number of chopped file and the sum was subtracted from the total No of the aligned file. The remaining number was considered as a total characters which were subjected for analysis.

The phylogenetic tree of land races were developed as: >Open MEGA 5.1 > Data> click > Open a file/session > upload Choped file> open> New Box >analyze> select nucleotide sequence >Ok > protein coding> No> MEGA 5.1 opened > phylogeny> click > construct/test Max. Likelihood Tree >New window was opened > analysis> phylogeny reconstruction > statistical Method > bootstrap Method >no of bootstrap replication >1000 >substitution type >nucleotide > model/method> maximum composite Likelie hood >complete> press the save button > new window box > save as >tree session file (mts).

38 Chapter 3 RESULTS

Results obtanined for the genetic and taxonomic elaboration of the land races from Northern Pakistan are presented under sections titled as numerical, taxonomic, DNA and RNA below:

3.1 NUMERICAL TRAIT ANALYSIS The numerical traits taken under consideration ware petiole length, leaf area, pedicel length, fruit length, width and weight. Summary of the results obtained thereby is given below.

3.1.1 PETIOLE LENGTH

Analysis of variance showed that variation in petiole length is statistically significant (Table 3.2). Hence, all the land races studied were different with respect to the petiole length. Table of mean (Table 3.1) showed that petiole length ranged from 30.6–56.07 mm and the highest value of mean for petiole length (56.07 mm) was for Kushbago Batang (Kbb) followed by Atti Batang (49.00 mm) and Shardi Tanchi (48.47mm). The lowest value of mean was recorded for Black Batangi (30.60 mm). Franci Batang (42.03 mm) and Ghata Zira Tangai (44.83 mm) had closely related values. Kado Batang (Kb), Taunchi Batang and Nak Batang were also similar with respect to petiole length. The land races, China Batang (Cb), Kacha Zira Tangai (Kzt) and Klak Nak (35-37mm) had little variation among themselves with respect to petiole length.

3. 1. 2 LEAF AREA

Analysis of variance (Table 3.2) for leaf area showed non-significant (0.1345) differences even at 5% level of significance, which showed that the landraces were similar with respect to leaf area. Table of mean (Table 3.1) showed that the highest value of mean (2672A) was for the land race Ghata Zira Tangai (Gzt), while all others land races had closely related values and fall in the same group ―B‖.

39 3. 1.3 PEDICEL LENGTH

Analysis of Variance (Table 3.2) showed that the land races have significant variation at 5% level of probability (0.0000) with respect to pedicel length. Table of mean (Table3.1) showed that highest value of mean for pedicel length was for land race, Kushbago Batang (65.53 mm) followed by Taunchi batang and Glass Batang (50.87 mm ).The lowest value for pedicel length rangeed from 15-17 mm, for land races Klak Nak (Kn), shardi taunchi (Srt) and Batangi. Others land races showed intermediate values with respect to pedecle length.

3.1.4 FRUIT LENGTH

Analysis of variance (Table 3.2) showed significant variation at 5% level of significance. Therefor, land races can be regarded to be different from each other with respect to fruit length. The least significant difference (LSD) value at (5%) was recorded as 10.058. Table of mean (Table3.1) showed that the lowest value of mean was for land races, Batangi (21.47 mm), Shardi taunchi (23.30 mm) and Ghata Zira Tangai (23.80), respectively with respect to fruit length, while highest value was shown by land race, Glass Batang (95.23 mm) followed by Kado Batang (73.17 mm) and China batang (72.23 mm). Land races, Klak Nak (Kn) Shakar batang (Sb), Mamosai and Atti batang (At) showed intermediate values, respectively for fruit length.

3.1.5 FRUIT WIDTH

Analysis of variance (ANOVA), showed significant results (0.0000) (Table 3.2) with respect to fruit width at 5% level of significance, it means that the land races showed statistically significant variation from each other with respect to fruit width. Table of mean (Table 3.1) showed highest value (59.37 mm) for the gennotype, China Batang followed by land race, Klak Nak (54.97 mm). Lowest value was shown by Batangi (23.60 mm), Shardi taunchi (23.40 mm) and Ghata Zira Tangai (Gzt, 25.93 mm) respectively. Kushbago Batang (Kbb), Kado batang (Kb) batang and Glass Batang (Gb) showed intermediate values with respect to fruit width.

40 3.1.6 FRUIT WEIGHT

Analysis of variance (ANOVA), (Table 3.2) showed significant (0.0000) variation at 5% level of probability with respect to fruit weight. Table of mean showed (Table 3.1) that the land races, Glass Batang (163.4g) and China Batang (148.0 g) have highest values followed by Kado Batang (113.2 g), Kushbago (109.2 g) and Klak Nak (Kn) (108.5 g). Land races, Batangi and Shardi Tanchi (Srt) possess lowest values (8.7, 9.3 g), respectively. Similarly land races, Franci Batang (Fb) and Atti Batang (At) have 81.27 g and 52.67g possess intermediate values, respectively with respect to fruit weight.

On the basis of quantitative parameters, using Minitab software cluster analyses was carried out; the dendrogram (Fig. 3.1) divided all the land races into two main groups, A and B. Group-A was further divided into two subgroups A1 and A2. Subgroup A1 consisted of land races 2 (Batangi), 10 (Nb-Balakot), 4 (Cb) and 11 (Nhs) while sub group A2 consisted of land race 5 (Fb), 6 (Gb), 9 (Kbb), 12 (Srt), and 14 (Kb). Group-B comprised of 3 (Gzt), 13 (Tb), 8 (Kb) and 7 (Kzt) land races.

41 Table 3.1. Mean values (mm) of numerical parameters of the Pyrus land races

Sr.No Land races Coded Analyzed Parameters Name Petiole Pedicel Fruit Fruit Fruit Leaf Area Length Length Length width weight

1 Atti Batang Ab 49.00AB 23.57CD 37.53EF 46.17E 52.67 608.2B 2 Batangi Batangi 30.60D 17.23D 21.47G 23.47H 8.700F 818.1B 3 Ghata Zira tangai Gzt 44.83BC 20.83 40.60E 38.43FG 39.43DE 847.2A 4 China Batang Cb 35.90CD 21.57CD 72.23B 59.37 A 148.0A 623.5B 5 Franci Batang Fb 42.87BC 28.63C 65.50C 47.07DE 81.27C 715.6B 6 Glass Batang Gb 30.67D 50.70B 95.23A 50.23CDE 163.7A 417.6B 7 Kacha Zira Tangai Kzt 36.60CD 28.63C 26.30G 25.93H 11.80F 444.5B 8 Kado Batang Kb 40.53BCD 30.73C 73.17B 50.50CD 113.2B 421.5B 9 Kushbago Batang Kbb 56.07A 65.53A 60.00D 54.03BC 109.2 B 698.9B 10 Nak Batang Nb 40.10BCD 25.53CD 38.47EF 40.27F 36.40DE 521.4B 11 Klak Nak Kn 37.90CD 15.57D 60.33D 54.97B 108.5 B 653.0B 12 Shardi Taunchi Srt 48.47AB 16.90D 23.80G 23.60H 9.320F 565.3B 13 Taunchi Batangi Tb 40.37BCD 50.87B 34.07F 35.17G 26.17EF 530.9B 14 Kala Batang Kb 43.033BC 22.20 CD 23.30G 26.00 H 10.60F 460.7B

47

Table 3.2. Analysis of variance of some important traits of the Pyrus land races.

S. No Parameters Degree of Sum of Squares Mean Square F Value Probability

Freedom

1 Petiole length 13 1919.403 147.646 4.0685 0.0011

2 Leaf Area 13 10582799.267 814061.482 4.0685 0.1345

3 Pedicel aength 13 8852.655 680.973 17.2392 0.0000

4 Fruit length 13 20621.807 1586.293 187.6217 0.0000

5 Fruit width 13 6133.518 471.809 79.7416 0.0000

6 Fruit weight 13 114743.600 8826.431 54.7393 0.0000

48

Fig. 3. 1. Phylogenetic relationship among 13 Pyrus land races established through the

analysis of numerical traits.

49

3.2 TAXONOMIC DESCRIPTION OF THE TAXA

Taxonomic analysis of Pyrus species, collected from different areas of northern Pakistan sorted out into two main groups i.e Oriental pears and Occidental pears. Taxonomic details of the taxa from the northern Pakistan have been recorded for the first time and are presented below:

Key to the Species 1+ Pome without persistent sepals, rarely few persistent pome present and style 2-5 in number…………………………………………………………………..…………..…………2 -Pome with persistent sepals and style 3-5 in number……………………………………..6 2+ Leaf margin spiny serrate…………………………………………………………………...3 - Leaf margin non-spiny serrate……………………………………………………………....4 3+Pome yellow; leaf basally cuneate ………………………… ……………4. P. bretschneideri - Pome brown; Leaf basally sub-cordate or rounded ………...... 3. P. pyrifloia 4+Leaf margin sharply serrate……………………………………….……...... 5. P. phaeocarpa - Leaf margin obtusely serrate………………………………………...……….…………….. 5 5+Leaves are corymb glabrous; stamens 20 and styles 2or 3………………....2. P. calleryana -Leaves and corymb initially pubescent; stamens 25-30 and styles 3-5……..…1. P. pashia 6+ Leaf margin spiny serrate……………………………………..……………………….….…7 - Leaf margin serrulate or obtusely serrate and without spins…….….……..………...… 8 7+ Leaf blade long spiny-serrate; styles 5; pome yellow…………………..…7. P. ussuriensis - Leaf blade short spiny-serrate; styles 4; pome brown …………………..…9. P. hopeiensis 8+ Leaf margin serrate…………………………………………………………………...... …… 9 -Leaf margin seteso or obtusely serrate………………………………………..……..…..…10 9+Pome yellowish green, ovoid or obovid, 5-loculed; fruiting pedicel 4-5 cm and thickened distally……………………………………………………….…10. P. sinkiangensis -Pome brown subglobose or obovid, 3-4 loculed; fruiting pedicel 3-4 cm and not

50

thickened distally………………………………………………………...…….8. P. serrulata 10+ Leaf margin seteso serrate………………………………………………...…..11. P. ovoidea - Leaf margin seteso or obtusely serrate………………………………………….….…… 11 11+Fruit thick-skinned and with numerous grit cells…………...………...12. P. turcomanica - Fruit not as above………………………………………………………………….………. 12 12+Pome yellowish green………………..……………………………………...………….…..13 - Pome brown…………………………………………. ………..…………..……....…..……14 13+Pome 1.5-2.5 cm in diam; 5-7 flowers; stamens 25 styles 3-4..………..6. P. pseudopashia - Pome 1-1.5 cm in diam; 3-6 flowers; stamens 25; styles 5(4)……………13. P. xerophila 14+ Pome obovoid or subglobose; leaf blade elliptic to ovoite; petiole thin, 1.5-5 cm long ………………………………………………………………...………..……14. P. communsis -Pome depressed globose; leaf blade broadly ovate to suborbicular; petiole thick, 2-3 cm long……………………………………………………………………15. P. armeniacaefolia

A. ORIENTAL PEARS 3. 2. 1 Pyrus pashia Buchanan-Hamilton ex D. Don, Prodr. Fl. Nepal. 236. 1825; Brandis, Forest Fl. Brit. Ind. 204. 1874; Rehder, Proc. Ame. Acad. of Arts & Sci. 50: 238. 1915; Terpo, Ann. Accd. Horti. Viticult. 22, 6, 2: 30-32. 1960; Temesy, Fl. Iranica. 66: 28. 1969; Cuizhi, Fl. China. 9: 178. 2003.

Synonyms: P. heterophylla Hort. Ex Decaisne, Jard. Fruit I. 328, sub t. 7. 1872. P. nepalensis herb. Hamilton et Hort. f Hook., Fl. Brit. Ind. 2: 374 (1878), Pro syn. P. variolosa Wall., Cat. No. 680 (1828), num. nud.; G. DON, Gen. Syst. 2: 622. 1832. P. verruculosa Bertol., Mam. Accad. Sci. Inst. Bologna, ser. 2, 4: 312. 1864. Local Names: Tangay (Pushto), Batangi (Hindko), Tangi, Tang, Shinder, Kent, Ban-Keint, Katari, Kathu, Kainth, Kaenth, Sheghel, Ku (Punjab), Chalthi (Assam). Trees 5-12 m tall, pyramidal; with branches often armed. Branchlets purplish brown or dark brown when old, prominently brownish lenticellate, young branches woolly later glabrous; terete, lanate when young, glabrous when old. Buds ovoid, apex obtuse; scales

51 puberulous along margin. Stipules caducous, linear-lanceolate, 4-8 mm, membranous, adaxially pubescent, margin entire, apex acuminate; petiole 1.5- 3 cm, initially piolose, soon glabrescent; Leaves 6.5- 12.0 x 4.5x5.5 cm or 4.5-9x2.5-4 cm, oblong ovate to cordate, leaf blade ovate or narrowly elliptic, 4-7x2-5 cm, thickly hairy when young, some time redish when young, glabrescent, base rounded, rarely broadly cuneate, margin obtusely serrate, apex acuminate or acute, glabrous on both surfaces, densely white woolly, when young, on young branches leaves are 3-5 lobed, villous at margin, turn red and fall off, Petioles 1.3-2.5 cm; Raceme umbel-like or corymbose, thickly tomentose, 6-10 flowered; peduncles 1.5 cm long, initally thick tomentose, then become glabrescent; bracts caducous,12.0 x0.5 mm, woolly when young, linear, 8-10 mm, membranous, both surfaces tomentose, margin entire, apex acuminate. Pedicel 2-3 cm long, villous, initially tomentose, glabrescent. Flowers 2-5 cm in diam. hypanthium cupular, 2 mm high, villous on both surfaces, deciduous, abaxially tomentose. Sepals triangular, 3-6 mm, both surfaces tomentose with white hairs, margin entire, hairy, apex acute, acuminate and redish in bud, sepals caducous in fruit. Petals white, pinkish in newly opened condition, suborbiculus, obovate, 8-15 x 4-9 mm or 8x 8 mm, orbicular or rounded, base narrow and shortly clawed, apex rounded and sometime lobed. Stamens 27-31, filaments 4-5 mm long and white, in two whorls in young flower, outer, 15-18, erect and inner, 12-13, short and bending to the center, at maturity both adopt same level, slightly shorter than petals, glabrous. Ovary 3-5-loculed, with 2 ovules per locule; styles 4 (or 5), 8-10 mm long, free at base, base woolly, nearly as long as stamens, glabrous. Fruiting pedicel 2-3 cm, subglabrous; Fruits, pome, calyx caducous, subglobose, solitary or in 2- 3, 2-4 cm across or 1-1.5 cm in diam., rounded, base umblicate, rusty or yellowish brown, to dark brown, when ripe, covered with raised covered with numerous white, brown spot-like lenticels, spots or dots. Fruit is ebible, mature become dark with white dots, several stone like bodies, black flatent seeds, immature frikely, hard to eat. Fl. Apr, later as compared to P. communis L. fr. Aug-Sep. sometime persistent to tree in whole winter season, 2n= 34.

52

Pyrus pashia is wild type and commonly used as stock for grafting. In Yunnan, it is used as stock for grafting pear cultivars. Young and healthy plants possess spines on the branches while mature and old plants lack this property. It is native to the Himalaya extends its range to China and is intermediate in morphology between the occidental and oriental groups.

Occurance: Occurs throughout the Himalayan ranges with Pinus wallichiana, Quercus incana, Quercus baloot forest at altitude of 600 m -2500 m, very common on natural water channels, moist and rocky slopes, mostly in graveyards, land border or road sides.

Distribution: Russia, Afghanistan, Pakistan, China, India, Iran and Bhutan. Laos, Myanmar, Nepal and West Pakistan: Bhutan, Thimphu, Punkha, Bumthang, Mongar and Tashigang districts, Sikkim: Dargeeling district. Cultivated or growing near habitation, 2130-3000 m altitude.

Afghanistan: E: Nuristan: Inter Barikot and Kamdesh, 300 m, in wilvia Quercus baloot, Gill 18111, EDELB. 17891 Ad fluvium in parte inferiore vialia kunar, NEUB. 4585: Gorlask, ad ripas fluvii Pech, NEUB, 51/512:

Pakistan: Swat: Shawar, Beha, Biakand, Sakhara, Madyan, Miandam and Malamjaba, Islmpur Vallies and Saidu Sharif. Shangla: Khawazakhiela to Shangla, 1300-1900 m, Puran, Alpuri, Chakysar and surrounding areas of Besham, Battagram. Mansehra: Batal, Chatarplane, Shinkiyari, Oggi, Ather Sheha, Balakot, Sangar, Lower Kaghan Valley. Abbottabad, on the way to Galyath and Sherwan. Kashmir (AJK): Dheer Kot, Chikkar, Sudden Gahli, Bagh, Rawalakot, Chakoti and Neelum Valley.

Geographical Position: Swat Valley, Madyan (Chail), Altit. 4650 ft., 350 08. 486‘ N and 720 33. 151‘ E , Kuz Shawar, Altit. 4797ft., 340 59.064‘ N and 720 18. 626‘ E , Balakot (Sangar), Altit. 4881ft., 340 34.992‘ N and 730 22.313‘ E , Azad Kashmir (Ghazi Abad), Altit. 4014 ft., 330 59. 821‘ N and 730 37.375‘ E , Chakoti (Oppi Bala), Altit. 4000 ft., 340 06.327‘ N and 730 54.007‘ E.

53

A B C

D E F

Fig. 3. 2. Pyrus pashia: Out look of the tree in the flowering and fruiting stages (A, B), Inflorescence showing flower colour, floral parts and arrangement of flowers (C), hypanthum showing sepals and carpels (D), arrangement of fruits in the tree (E) and close up of the fruits (F).

54

3.2.2. Pyrus bretschneideri Rehder Proc. Am. Acad. Arts and Sci. 50: 231. 1915; Fl. China. 9: 177. 2003.

Local Name: Mamosay (Pashto)

A medium size tree, 5-8 m. tall and umbrella shaped. branches purplish brown when old, quickly glabrous, mature branches dark brown, sparsely lenticellate; buds dark purple, ovate, 4-5 mm. long, apex obtuse, perilous broad ovate clearly mucronulatis chestnust outer margine vallus except glabrous. Stipules caducous, linear or linear lanceolate, apex acuminate; Leaves subchordate, ovate or elliptic ovate, acuminate, base broad cuneate, rarely almost rounded acutely serrate, acutely dentate like initially setoso-acuminatis, atleast clearly acuminates mostely slightly accumbentibus, five- eleven cm. long and 3.5-6 cm. wide, initially on both sides loosely tomentose, quickly glabrous upper surface yellow green, below little while back paler, leviter reticulate, margin spinulose-serrate, apex acuminate; petiole slender 2.5-7 cm long, initially scatterly in hanging position, soon glabrous. Inflorescence raceme-umbel, 7-10 flowers, hypanthium cupular, out side glabrous, pedicel 1.5- 3 cm. long, bracteole 2 subulatis about 2 mm. long; Sepals wide at base, triangular, acuminate and about 4 mm long, glandular-serrate, outside glabrous, inside yellow tomentose, depression present, some time lanceolate; Petals white, oval, apex rounded and irregular erect base slightly clawed and narrow, 12-14 mm. long and 10-12mm wide; stamens about 20, anthers initially pinkish red, bending to the center, soon erect and posses the same level, filaments white, style-5 or 4, 2- long and 3- short in most cases and ½ of the petals, glabrus, Ovary 5- or 4-loculed, with two ovules per locule. Pome subglobose or globose- ovoid, 2.5-3 cm. long and 2.5 cm diam., apex scar impressed, yellow, with fine dots. Calyx deciduous, fruiting pedicel 1.5-3 cm. long glabrous, seeds obovoid, slightly compressed, 6-7 mm long and chestnut. Fruit red in colour and less tasty.

This species seem nearest to P. ovoidea Rehder which is easily distinguished by the persistent calyx and by oblong ovate leaves rounded or even subcordate at the base. In

55 its deciduous calyx it agree with P. phaeocarpa Rheder, but this species in its smaller 3-4 celled brown fruit and the coarser serration of the more oblong ovate leaves.

Distribution: China and Pakistan.

Geographical Position: Swat Valley, Kuz Shawar (Bagh) Altit. 4850 ft., 340 40. 014‘N and 720 19.661‘ E.

A B

C

Fig. 3. 3. Pyrus bretschneideri: Out look of the tree (A), front view of inflorescence (B), side view of inflorescence (C)

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3.2.3 Pyrus pyrifolia (N.L. Burman) Nakai, Bot. Mag. (Tokyo) 40: 564. 1926; Cuizhi, Fl. of China 9: 177. 2003.

Synonyms: Ficus pyrifolia (N. L. Burman) Nakai, Bot. Mag. 40: 564. 1926. Ri vulgo Nas Kaempfer, Amoenit. Exot. V. p. 800 (1712). Pyrus communis (non Linnaeus) Thunberg in Nov. Acta Reg. Soc. Upsal. III. P. 199 (1780); Fl. Jap. P. 207 ( 1784), Siebold and Zuccarini in Abh. Acad. Muench. IV. 3. P. 131. 69. (1846). Local Name: Khan Tango (Kt) (Pashto).

Trees to 7-20 m tall and healthy. Branchlets purplish brown or dark brown when old, terete, tawny villous or tawny tomentose when young, soon glabrescent, glabrous when old, sparsely lenticellate; buds narrowly ovoid, apex obtuse; scales tomentose at margin and apex. Stipules caducous, linear-lanceolate, 1-1.5 cm, membranous, margin villous and entire, apex acuminate; petiole 3-4.5 cm, initially tomentose, glabresecent; leaf blade ovate-elliptic or ovate,7-12x 4-6.5 cm, glabrous or brown lanate when young, base rounded or subcordate, rarely broadly cuneate, margin spinulose-serrate, apex acute. Inflorescence, raceme umbel like, 6-9-flowered; peduncle sparsely pubescent when young; bracts caducous, linear, 1-1.4 cm, membranous, villous at margin, apex acuminate. Pedicel 3.5-5 cm, sparsely pubescent when young. Flower 2.5- 3.5 cm in diam. Hypanthium cupular, some what platend, abaxially glabrous. Sepals triangular-ovate, lobed at the middle, hoded, abaxially glabrous, adaxially brown tomentose, margin glandular denticulate, apex acuminate. Petals white, ovate, 1.5-1.7 cm, base shortly clawed, apex rounded. Stamens 20 or 23-25, ca. 1/2 as long as petals. Ovary 5-or 4- loculed, with 2 ovules per locule; styles 5, rarely 4, nearly as long as stamens, glabrous. Pome brownish, with pale dots, subglobose, 2-2.5 cm in diam., (4- or ) 5-loculed; sepals caducous; fruiting pedicel 3.5-5.5 cm, subglabrous. Fl. Apr, fr.Aug. 2n= 34, 51. Fruit have excellent quality due to high sugar, water contents and cold by nature.

Many varieties of this species are cultivated in the regions of the chang Jiang and Zhu Jian rivers and warm rainy regions of about 100-1400 m altitude. Anhui, Fujian etc. In Pakistan only reported from Swat.

57

Collection Sites:

Pakistan: Swat, Kuz Shawar (Bagh), Altit. 4830 ft., 340 49. 044‘ N and 720 18. 661‘ E . Distribution: China: Anhui, Fujian, Guangxi, Hobei; Japan; Pakistna, Swat.

A B

C D

Fig. 3.4. Pyrus pyrifolia: Out look of the tree (A), inflorescence (B), side view of fruits

(C), top view of fruits (D).

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3.2.4 Pyrus calleryana Decaisne, Jard. Fruit. 1: 329. 1871-72; Maximowicz, Bull. Acad. Sci. St. Petersbourg, XIX: 172. 1873; Mel. Biol. IX. 169. 1873. Rehder, Proc. Amer. Acad. Arts and Sci. 50: 237. 1915; Cuizhi, Fl. of China 9: 178. 2003.

Trees five to eight m tall. Young branches reddish brown and the old are grayish brown , terete, initially tomentose, soon glabrescent, glabrous when old; buds triangular-ovoid, sparsely tomentose, apex shortly acuminate. stipules caducous, linear-lanceolate,4-7 cm, herbaceous, glabrous; margine entire, apex acuminate; petiole 2-4 cm, glabrous; Leaves: small, creneate, leaf blade broadly ovate or ovate, rarely narrowly elliptic, 4-8x3.5-6 cm, glabrous, base rounded or broadly cuneate, margin obtusely serrate, apex acuminate, rarely acute. Raceme umbel-like, 6-12-flowered, small in size, glabrous or nearly glabrous, peduncle glabrous; bracts caducous, linear-lanceolate, 0.8-1.3 cm, membranous, adaxially tomentose, margin initially glandular serrate, apex acuminate. Pedicel 1.5-3 cm or 3-4 cm long, slender glabrous. Flowers 2-2.5 cm in diam. Hypanthium copular and hairless. Sepals lanceolate, abaxially hairless, adaxially tomentose, margin entire, apex acuminate, mostly long acuminate. Petals white, ovate, ca. 1.3x 1 cm, base shortly clawed, apex rounded. Stamens 20, slightly shorter than petals. Ovary 2 (or 3)- loculed, with 2 ovules per locule; style 2 (or rarely 3), nearly as long as stamens, glabrous basally. Pome blackish brown with pale dots, globose, 2 ( or 3)-loculed; sepals caducous; fruiting pedicel 1.5-3 cm, glabrous. Fl. Apr, fr. Aug-Sep. 2n=34.

Present on slopes, plains, mixed valley forests, thickets, 100-1800 m. Anhui, Fujian, Guangdong etc. The wood of this species is hard and close-grained, and is sometime used for making furniture and stools. It is often used as stock to graft Pyrus pyrifolilia. Pyrus calleryana Decaisne is a widely distributed species and seems not uncommon on mountanins at an altitude of 1000 to 1500 m. It is easily recognizable by its comparatively small crenate leaves, inflorescence glabrous or nearly glabrous by small flowers with 2 or 3 styles.

59

In 1908 the species was introduced by Wilson to the Arnold Arboretum. It seem to be most closely related to P. serotina Rehder which differs in its serrulate, not setosely serrate, generally wider leaves and smaller flower having 3-4 styles, shorter sepals and smaller fruit.

The following Japanese pear is referred by Koidzumi as a variety of P. calleryana. Pyrus calleryana var. dimorphophylla Pyrus taiwansis Iketani and H. Ohashi (J. Jap. Bot. 68: 40. 1993), described from Tawan, might be an allied species or an ecotype of P. calleryana.

Geographical Position:

Pakistan, Azad Kashmir, Neelum Valley- Punch Ghar, Altit. 3209 ft., 730 36. 939‘N and 340 26.675‘ E.

A B

Fig. 3. 5. Pyrus calleryana: Branch showing side view (A) and front view of fruits

respectively.

60

B. OCCIDENTAL PEARS 3. 2. 5 Pyrus pseudopashia T.T. Yu, Acta Phytotax. Sin 8: 232. 1963; Guizhi, Fl. of China

9: 176. 2003.

Tree 5-10 m tall, some time shrubby, branchlets purplish brown when old or redish brown, terete, sparsely yellow lanate when young, soon glabrescent, glabrous when old, sparsely pale lenticellate; buds ovoid, apex acuminate; scales pubescent at margin. Stipules caducous, ; petiole 1.5-3.5 cm, yellow lanate or subglabrous; leaf blade ovate or narrowely ovate, rarely lanceolate-ovate, eleptic- lanceolate, 6-8 x 3.5-4.5 cm, lateral veins 7-12 conspicuous pairs, abaxially initially yellow lanate, glabrescent, adaxially glabrous, base rounded or broadly cuneate or elliptic at the base, margin obtusely serrate or serrate, apex acute, acuminate or rounded-obtusely. Raceme umbel-like, 5-7 flowere; peduncle thickly tomentose when young, soon glabrescent; a single or double elongated bracts present soon caducous Pedicel 2-3 cm, initially thikly tomentose then sparsely tomentose. Flowers 9-12 in diam. Hypanthium campanulate, tomentose when young, soon glabresent. Sepals triangular-ovat, ca. 2-3 mm, tomentose on both sides, adaxially densely, margin sparsely glandular deniculate, apex acute, acuminate or obtuse. Petels white, broadly ovate, 5-8 mm, base narrow and shortly clawed, apex rounded with two lobes. Stamens 20- 25, ca. 1/2 as long as petals. Ovary 3- or 4-loculed, with 2 ovules per locules per locule; Style 4 or 5 nearly as long as stamens, glabrous. Pome brown, with pale dots, subglobose or rounded 1.5-2.5 cm in diam., 3- 4, 4-loculed, 2-3 in group; sepals persistent; fruting pedicel 3-4.5 cm, thickened distally, glabrous. Fl.Apr, fr. Aug- Sep. In mixed forests, thickets; 500-3000 m. Guizhou, Yunnan. This species is similar to Pyrus pashia var pashia, which differs by its smaller leaves, tomentose pedicels, caducous sepals, and small fruit. This species is rarely found as compared to P. pashia and used as a stock for grafting other varieties.

Geographical Position: Balakot (Sangar), Altit. 4401 ft. 340 34. 986‘ N and 730 22. 201‘ E , 4818 ft. 340 59.02‘ Nand 720 18. 68‘ E. Azad Jammu and Kashmir (Ghazi Abad), Altit. 4014 ft. 330 59. 821‘ N and 730 37. 375‘ E

61

A B

C D E

Fig. 3.6. Pyrus pseudopashia: Front (A) and dorsal (A) views of inflorescence, out look of the tree (C), branch showing fruits (D) and close uo of the fruits (E).

62

3.2.6 Pyrus communis L. Sp. Pl. 1: 479. 1753; Fl. Ind. Ed. 2, 2: 391-394. 1832; Decaisne, Jard. Fruit. 1: 340, pl.1. 1872-74; Rehder, Man., ed. 2: 403. 1940; Terpo and Franco, Fl. Europaea 2: 66. 1968; Fl. Brit. Ind. 2: 374. 1978; Maleev, Fl. USSR. 9: 261. 1985; Fl. China 9: 175. 2003.

Local Names: Common pear, European pear, Pear (Eng.), Bagugosha (Kashmir and U.P.), Tang, Batang, Nak, China Batang (Hindko), Neshpati (Urdu and Pashto).

Trees to 5-15 m tall, some time shrub (5-7m), Branchlets graysh brown to dark brownish or dark brownish red when old, sometime with or with out spines, ascending on young , young twigs stout, reddish-brown, soon becoming glabrous and shining. buds ovoid, apex obtuse, glabrous or subglabrous at maturity. Stipules nonpersistant, linear lanceolate; Calyx 1 cm, membranous, slightly pubescent, margin sparsely glandular denticulate, apex acuminate; Petiole 1.5-5 cm. slender, slightly pubescent when young, soon glabrescent; Leaf blade ovate or subrounded to elliptic, 2-5 (7)x 1.502.5 cm, young pubescent, soon abaxially pubescent along midvein, base broadly cuneate to subrounded, margin obtusely serrate, serrulate, cuneate, creanate rarely entire, apex acute or shortly acuminate or cuspidate, white arachnoid-pubescent below when young, along the veins and leaf margin, lustrous green above and light green below, blackon when become dry. Inflorescences, raceme umbel like, corymb, a few flowered-clusters, 5- 9 flowered, 2.5- 3.0 cm across; Peduncle glabrous or subglabrous; bracts caduceus, linear- lanceolate, 1-1.5 cm , membranous, brown pubescent, margin sparsely glandular serrate, apex acute or acuminate. Pedicel 2-3.5 cm, white hairy, subglabrous and become glabrous at maturity. Flowers 2.5-3 cm in diam. Hypanthium bell shaped, 4-5 mm high, abaxially pubescent. Calyx persistent, Sepals triangular- lanceolate, 5-9 mm, 5- 8m‘2 x 3-4 mm, both surfaces pubescent , margin sparsely glandular denticulate when young, (12- 15 x 120-12 mm, apex acuminate. Petals white or pink, obovate, 1.3- 1.5x 1x1.3 cm, base, 3-5 x 1.5-2 cm, base shortly clawed, suborbicular or obovate, base narrow, short clawed, spreading, glabrous. Stamens 20, filament white, same level when mature, ca. ½ as long as petals. Ovary 5-loculed, style 5 (or 4) nearly as long as stamens, pubescent basally. Fruit, pome, oblong, pyriform, obvoid or subglobose, 3-5 cm, 5- loculed, green or yellow

63 when ripe, rarely redish, dotted; fruiting pedicel 2-3.5 cm, subglabrous; sepals persistant. Fl. Apr., fr. Jul—Aug.

Distribution: Commonly cultivated in N, NE, and Sw China, (Bhutan, Russia, Sikkim, ; Sw Asia, Europ), North Iran, Central Asia, Central and South Europe, Turkey, Russia, Afghanistan and India.

Europe is considered to be the centre of origin of this highly polymorphic species. Many of the cultivated types of P. communis L. probably due to their origin to initial population of natural and artificial hybrids between P. communis var. communis L and P. nivalis Jacq., a native of eastern Europe.

Distribution:

Pakistan: Swat: 1300-1900 m, Matta to Madyan and Mingora to Madyan road on the field borders, common in different Vallies i.e. Shawar, Beha, Rorengar, Sakhara, Madyan, Behran Miandam and Malamjaba and Beykand valley. Shangla: Puran, Alpuri, Chakysar and surrounding areas of Besham. Battagram. Near Peshawar and Charsada along Motarway, Mansehra: Batal, Chatarplane, Shinkiyari, Oggi, Auther sheha, Balakot, Sangar and lower Kaghan valley. Abbottabad, on the way to Galyath and Sherwan. Kashmir (AJK): Dheer Kot, Chikkar, Sudden Gahli, Bagh and Rawalakot.

Geographical Position:

Swat: Madyan (Chail), Altit. 4816 ft., 340 59. 057‘ N and 720, 18. 642‘ E, Altit. 5149 ft., 350 08.19‘ N and 720 38.96‘ E , Kuz Shawar, Altit. 4818 ft., 340 59.02‘ N and 720 18. 68‘ E . Mansehra, Balakot (Sanger) Altit. 5315 ft., 340 34.670‘ Nand 730 22.515‘ E , Batal (Saloona), Altit. 4560 ft., 340 32.924‘ N and 730 08.713‘ E.

64

A B

C D

Fig. 3.7. Pyrus communis: Out look of the tree (A), inflorescence (B & C), inflorescence

with young fruits (D).

65

3.2.7 Pyrus sinkiangensis T. T. Yu. Acta. Phytotax. Sin. 8: 233. 1963; Cuizhi, Fl. of China 9: 175. 2003.

Local Name: Parawoo Tango (Pushto), Kado Batang (Kb), Glass Batang (Gb) (Hindko).

Tree to 6-25 m tall with pyramid or conical shape. Branchlets purplish brown or grayish brown, terete, glabrous, white lenticellate; buds ovoid, apex acute; scale white pubescent at margin. Stipules caducuous linear- lanceolate, 8-10 mm, membranous, white tomentose, margins sparsely glandular denticulate, apex acuminate; petiole 3-5 cm, white tomentose when young, soon glabrous; leaf blade ovate, elliptic, or broadly ovate, 6-8x 3.5.5 cm, glabrous or white tomentose when young, base rounded, rarely broadly cuneate, margin crenate or subentire basally, serrulate apically, apex shortly acuminate. Inforescence, raceme umbel-like, 4-7- flowdered, peduncle tomentose when young, glabrescent, bract caduceus, linear-lanceolate, 1-1.3 cm, membranous, margin sparsely glandular denticulate and long tomentose, apex acuminate. Pedicel 1.5-4 cm, tomentose, whitish wooly when young, glabrous. Sepals triangular-ovate, 6-7 mm, abaxally brown tomentose, margin grandular denticulate, apex acuminate. Hypanthum cupular, abaxially glabrous. Petals white, obovate, 1.2- 1.5x 0.8-1 cm in diam, shortly clawed at base, apex obtusely rounded. Stamens 20 as long as petals. Ovary 5-loculed,with 2 ovules per locule; style 5, not exceeding stamens, pubescent basally. Pome yellowish green, ovoid or obovoid, swollen at the top and narrow at the bottom, narrow grove on the length wise, 2.5-5 cm in diam., 5-loculed; fruiting pedicel 4-5 cm, thickened distally, glabrescent, sepals persistent. Fl. Apr, fr. Aug- Sep. 2n= 34, 51.

Commonly cultivated on field border, uncultivated area and grafted in P. pashia because of its large fruit size and good quality and quantity. Fruit is hard and long shelf life therefore, after flucking it is kept in dry grasses for 10-15 days before it become soft and delicious.

Geographical Position:

66

Swat: Kuz Shawar, Altit. 4825 ft., 350 08. 364‘ N and 720, 33. 086‘ E. Mahsehra: Batal, Altit. 4541 ft., 340 32. 946‘ N and 730, 08. 750‘ E . Balakot: Sangar, Altit. 4773 ft., 340 34.992‘ N and 730 22.202‘ E . A JK: Dheer Kot, Altit. 5090 ft., 340 01.653‘ N and 730 35.345‘ E. Native in Xinjiang, cultivated in Gansu, 200-1100 m., Qinghai, Shaanxi. This species might be a natural hybrid between Pyrus communis and P. brestschneideri.

Pakistan: Swat: 1300-1900 m, Matta to Madyan and Mingora to Madyan roads on the field borders, Common in different Vallies i.e. Beykand, Shawar, Beha, Rorengar, Sakhara, Madyan, Behran Miandam and Malamjaba vallies. Shangla: Puran, Alpuri, Chakysar; Battagram; Mansehra: Batal, Chatarplane, Oggi, Balakot and Sangar. Kashmir (AJK): Dheer Kot, Chikkar, Sudden Gahli, Bagh and Rawalakot.

67

A B

C

D E

Fig. 3.8. Pyrus sinkiangensis: Out look of the tree (A), dorsal view (B) and side view of

inflorescence, side view of fruits (D & E).

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3.2.8 Pyrus hopeienses T. T. Yu., Acta Phytotax. Sin 8: 232. 1963; Cuizhi, Fl. of China 9: 174. 2003.

Local Name: Pykhawary Tango (Pashto)

Trees to 6-8 m tall. Branchlets dark purple or purplish brown, sparsely white lenticellate, apices often becoming thornlike, branches are flexible and have capacity to bear load: buds oblong ovoid or triangular-ovoid, glabrous or scales slightly tomentose at margin and apex acute. Stipules caduceus, linear-lanceolate, 7-11 mm , herbaceous, margin sparsely serrate, apex acuminate; petiole 2-4.5 cm, sparsely pubescent or glabrous; leaf blade ovate, or sub orbicular, 4-7x4-5 cm , lateral veins 8-10 pairs, glabrous, base rounded or subcordate, margin shortly spinulose-serrate, apex long or shortly acuminate. Raceme umbel-like, 6-8 flowered; peduncle subglabrous; bracts caduceus, linear- lanceolate,6-9 mm, membranous, villous, margin serrate, apex acuminate. Pedicel 1.2- 1.5 cm, abaxially thickly tomentose and sparsely pubescent toward axis sub- glabrous. Flower 2.5-3 cm in diam. Hypanthium copular or plate like, subglabrous. Sepals triangular- ovate, elongated, abaxially sparsely pubescent, adaxally densely pubescent, margins denticulate, apex acuminate. Petals white, elliptic-obovate, ca 8x6 mm, narrow and shortly clawed at base. Stamens 20, (rarely 17-19) less than ½ as long as petals, filament white while anthers are pinkish, bending to the center Ovary 4 or 5- loculed, with 2 ovules per locus; styles 5 (or 4), nearly as long as stamens, glabrous, some time three short and two long . Pome brown, spotted, globose or ovoid, 1.5- 2.5 cm in diam., 4 (or 5)- loculed; fruiting pedicel 1.5-3 cm, glabrous; sepals persistant. Fl. Apr, fr. Aug- Sep. 2n=34*. Thicket margins on slopes; 100-800 m.

This species is reported for the first time from Kuz Shawar, Swat, Pakistan during this study.

Geographical Location: Swat: Kuz Shawar (Malawooch) Altit. 5125 ft., 350 50. 045‘N and 720 19.561‘ E.

69

A B

C D

Fig. 3.9. P. hopeiensis: front view of inflorescence (A, B), side view (C) and dorsal view

(D) of inflorescence

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3.2.9 Pyrus armeniacaefolia Yu, Sp. Nov. Pl. XXVII, 2; Yu. Acta Phytotax. Sin. 8: 231. 1963; Cuizhi, Fl. of China 9: 176. 2003.

Local Name: Atti Batang (Hindko), Qualperang Tango (Pashto).

Tree pyramidalis, 8-12 m; branches slightly angular, purple-dark brown lenticels, glabrus; Buds oblong-ovoid, acuminatae, young branches outsides wavy with fairly long soft straight hairs except glabrus. Leaves wide ovate or suborbicular, thick, 4-5 cm long and wide, acute and obtuse, base rounded and ending suddenly, margin crenulat-serrate or near to dentate, on the upper sides dark green, below pallidiora, glabrus, veins on bothsides 8-10 curves, in dry on both sides slightly reticulate; petioles 2-3 cm. long, glabrus. Inflorescence, umbel-raceme, 8-10 flowered, pedicel initially tomentose, become glabrous. Hypanthium bell shaped. Sepals triangular, apex acute, white wooly hairs on both sides. Petals white, narrow at the base, lobed-rounded above. Stamens 18-20, in single circle, ½ of the petals. Styles 5 (or 4), Pome depressed-globose, 3-4 in group, 2.5-3 cm dim, pale yellow green, apex toward attenuatum, Calyx persistent crown, Sepals ovate oblong with pome, 5-locules; Peduncle thick slender, 2.5- 3 cm. long; Seeds obovat, 6 mm. long and 4 mm wide, chestnut. When fruit ripe it appear like maize flour and spread in mouth. Therefore it was known as Atti Batang.

Species similar to P. xerophilae Yu but appearance pyramidal leaves sub orbicular or almost orbicular or broadly ovate, margin crenate (having rounded teeth); fruit depressed-globose fresh state, young.

Distribution: Pakistan: Balakot-Sangar; China, N Xinjiang.

Geographical Location: Altit. 4401 ft., 340, 34. 986‘ N and 0730, 22. 201‘ E.

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A B

C D

F

Fig.3.10. P. armenicefolia: front view (A), dorsal view (B), side view (C) of inflorescence,

branches showing front view of fruits (D &E) and close up of the fruits (F).

72

3.2.10 Pyrus serrulata Rehder, Proc. Amer. Acad. Arts and Sci. 50: 234. 1915; Cuizhi, Fl. of China 9: 175. 2003.

Local Name: Khapa Tango (Pashto).

Trees upto 7-10 m tall and umbrella shape, Branchlets slightly wooly across and brown tomentose when young, purplish brown when old, terete. glabrous when old, sparsely white lenticellate; buds ovoid, dark brown, apex acute; scales adaxally tawny tomentose. Stipules caducous, linear-lanceolate, membranous, adaxially brown tomentose, margin sparsely glandular denticulate, apex acuminate; petiole 3.5-7.5 cm, brown tomentose when young, soon glabrescent; leaf blade ovate or narrowely ovate, suddenly and gradually acuminate, base rounded or wide cuneate, broadly cuneate or rounded, margin serrulate-dentate, most part apprised and slightly incurve acute or slightly acuminate, 5-11x3.5-7.5 cm, lateral veins 7-13 pairs, abaxially brown tomentose when young, soon glabrescent, margin serrulate, apex acuminate. Petiole slender, 3.5- 7.5 long, initially slightly hairy as soon as glabrous Raceme unbel- like, 6-11-flowered; peduncle peduncle brown tomentose, soon glabrescent; bracts caducous, linear-lanceolate, 5-10 mm, membranous, adaxially brown lanate,glandular denticulate, apex acuminate. Pedicel 3-5 cm in diam. Hypanthium sparsely tomentose abaxially.Sepals persistent or caducous, tiangular-ovate, abaxially sparsdely tomentose, adaxially tomentose, margin glandular denticulate, apex acuminate or acute. Petals white, broadly ovate, 1-1.2 cm, shortly clawed at base, apex rounded. Stamens 20-22 ca.1/2 as long as petals. Ovary (3 or 4)- loculed, with 2-ovules per locule; styles 5- (or 4), as long as stamens, sparsely pubescent basally. Pome dark brown, subglobose or obovoid, 1.5-2.2 cm in diam., 3- or 4-loculed, with persistent sepals or sometimes a few caducous; fruiting pedicel 3-4 cm, subglobose. Seeds obovate, 7 mm long and 4 mm wide, chestnut.

The mature fresh fruit game throat therefore they are kept in dry grasses for about 10 days, After this process, they become soft, tasty and delacious.

Fl. Apr, fr. Jun- Aug. 2n=34. Among shrubs, forest margins, thickests; 100-1600 m. Fujian, Guangdong, Guangxi,Guizhou, Hubei, Hunan, Jiangxi, Sichuan, Zhejiang. This

73 species is similar to Pyrus pyrifolia, which differs by its spinulose- serrate leaves, larger, 5- styled flowers, long acuminate sepals and larger, brownish fruit.

Distribution: Pakistan, Swat; Chian, Western Hupeh, Hsing- Shan (1300- 600 m).

Geographical Position:

Pakistan: Swat, Kuz Shawar (Bagh), Altit. 4812 ft., 340 49. 033‘ N and 720 18. 650‘ E.

C

Fig.3.11. Pyrus serrulata: Front view of inflorescence (A,B), dorsal side of inflorescence(C )

74

3.2.11 Pyrus ovoidea Rehder, Proc. Am. Acad. Arts and Sci. 50: 228. 1915, Nakai, The Botanical Magazie. XXX: 349. 26-27. 1916;

Synonymous: Pyrus chinensis Roxburgh, Hort. Bengal. 38. 1814, Pyrusis sinensis Poiret; Fl. Ind. ed. 2, II. 511. 1832, Pyrus simonii Hort., non Carriere. Local Name: Nak Batang (Pashto) Tree, 10-15 m, pyramidal in shape, shining; young branches when mature become purple dark via dark yellow, older, chestnut lenticels small few tempered; buds ovoid conical, chestnut and glabrous; leaves, oblong-ovate, rarely ovate, acuminate, base rounded to subcordate, neat, seteso serrate, dentate, erect dentate via some time ccumbentibus, 7-12 cm long and 4-6.5 cm. wide, initially on margin only and under on ribs tomentose quickly dark yellow vanishing endowed with , as soon glabrous, above yellow green, some time after under surface bright paler, mature paper and with autumn color fear purple-scalletform and orang, utrincus nerve 9-10 above and below slightly lifting. Petiole slender, 2.5-5 cm. long, initially sparsely fulco wooly, as soon as glabrous. Flower 3 cm. in diam. In cluster umbelliform, 3-7 flowers, glabrous while some time tomentum, covered with fluck of wool quickly vanishing; pedicel 2.5-4 cm. long; calyx as well as receptacle outside glabrous; sepals, triangular, lanceolate, wide at base, denticulate, inside to the base densely wooly; petals wide ovate, wide oval, about 12 mm long, base suddenly and very briefly clawed, glabrous, white; stamens about 18- 22, ½ of the petals, rarely level with petals, styles 5, distinct, base hairy, stamens erect a little while back, pome ovoide, rounded, impressed, apex toward attenuatum, calyx persistent, erect or bend inward, peduncle thin 2-4 cm long, seeds oblong-ovate, compress, 9-10 mm. long and 6 mm wide, chestnut and shining. The fruit shape of P. ovoideae is very unusual and quite distinct from any other pear. The fruit is exactly ovate, broad and rounded at the base and tapering from the middle toward the truncate apex. This species is more closely related to P. ussuriensis which differ in the broader orbicular –ovate or ovate leaves, in the lighter colored branches and the short-stalked sub-globose fruit with the persistent calyx in spreading form. Distribution: China, Pakistan. In Pakistan, Swat, Mansehra (Balakot).

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Geographical Position: Swat, Madyan (Chail) Altit. 5160 ft. 720 32.356‘E and 350 08. 191‘ N, Kuz Shawr (Bagh), Altit. 4828 ft., 720 18.656‘E and 340 59. 191‘ N.

A

Fig.3.12. Pyrus ovoidea: Front view of inflorescence (A), young fruit (B), branches showing

fruits (C & D).

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3. 2.12 P. turcomanica Maleev, Acta. Inst. Bot. Ac. Sc. USSR, Ser. 1, 3, 196. 1936; Maleev, Fl. of U.S.S.R. 9: 262. 1985.

Tree 10-15 m tall, with out spines, with broad, irregular crown; buds and other parts of the plant densely and softly white tomentose, later glabrous; shoots initially pubescent, later glabrous, initially lustrous red brown, later smooth and gray with small lenticels; petioles 3.5-5 cm long; thickly white tomentose, leaves initially silvery white below with a very dense pubescence, later glabrescent pubescent only in angles between veins, lustrous green, entire or slightly sinuate and obtusely dentate, mainly near the apex, sub orbicular or broadly oval, 4-5 cm long, 3-5 cm broad, rounded or even slightly emarginated at base at the apex, some time acutely short tapering, more or less cuneate at base; inflorescence, corymb, umbel raceme, 6-8 flowers; sepals triangular, tomentose; petals white lobed above and narrow at the base, stamens 20-22, flower unknown; pedicels 2-4 cm long, thickening upward; fruit broady pyriform, ca. 2.5 cm broad, 2 cm long, gradually passing into pedicel below, flat at apex, with short, broad, white- pubescent scale lobes spreading and appressed to fruit, thick skinned, with numerous grit cells, fl. April. Dry stoney slopes, rarely in valleys, on deep alluvial soils. Distribution: Central Asia: Mtn. Turkm. West Kopet Dagh). Iran, Pakistan: Balakot (Sangar). Different species are prominently varied by its snow white thickly hairy and very characteristics scale lobes oppressed to poem. Geographical Position: Pakistan: Balakot (Sangar), Altit. 4881ft., 340 34. 881‘N and 730 22. 313‘ E.

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A B

C

D E

Fig. 3.13: P. turcomanica: Out look of the tree (A), inflorescence (B), hypanthium

showing calyx and gynacium (C), upper side of fruits (D), side and bottom

views of fruits (E).

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3.2.13 Pyrus ussuriensis Maximowicz, Bull. Acad. Imp. Sci. Saint-Petersbourg, Ser. 2, 15: 132. 1856; Proc. Am. Acad. Arts and Sci. 50: 227. 1915, Fl. of U. S. S. R. 9: 265. 1985, Fl. China. 9: 174. 2003.

Synonyms: Pyrus communis Bunge in Mem. Sav. Etr. Acad. Sci. St. Petersb II. 101. 1983. Pyrus sinensis Decaisne, Jard. Fruit. I. t. 5. (1872), Pyrus simonii Carrier in Rev. Hot. 28. 1872

Local Name: Mamothay (Pashto).

Tree 10-15 m height, small young branches yellowish gray to purplish brown, yellowish gray to yellowish brown when mature, hairless or short small hairs are present, sparsely lenticellate, spiny; crown broad, dense; buds egg shaped, tip blunt, scale sparsely short hairy or subglabrous at margin. Stipule caduceus, linear lanceolate, apex acuminate. Young petiole thickly hairy, soon hairless; leaf blade ovate to broadly ovate, glabrous or thickly hairy when young, soon glabrescent, base rounded or subcordate or sometime cordate, lustrous green above, margin long spinulose serrate, apex shortly acuminate or long tapering acute apex. Inflorescence corymb, 5-7 flowered; peduncle tomentose in young condition, quickly hairless; Hypanthium campanulate, abaxially hairless or rarely tomentose. Sepals triangular-lanceolate, tapering at the apex, sparsely pubescent above, adaxially tomentose, margin initially glandular, denticulate, apex acuminate. Petals white, obovate or broadly ovate and glabrous. Stamens 20, shorter than petals. Ovary 5 loculed, 2 ovules per locule. Styles 5, same level with stamens. Fruit, pome, yellow, subglobose and 5-loculed, fruiting pedicel 1-3 cm glabrous, short stalked, sepals persistant. Fl. April. Fr. Aug- Oct. Fruit, produce red colour and less tasty.

This species varies in size, shape, color and taste of fruit, on the bases of which skvortzov has described several varieties of this species.

Distribution: China, Russia, Korea and Pakistan. Pakiatan: Swat (Kuz Shawar), Altit. 4850 ft., 340 40. 014‘N and 720 19. 621‘ E.

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A B

C

D E

Fig. 3.14: Pyrus ussuriensis: Out look of the tree (A), front view of inflorescence (B), vertical view of inflorescence (C), upper view (D) and side view (E) of fruits.

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3. 2. 14 Pyrus xerophila T. T. Yu. Acta. Phytotax. Sin. 8: 233. 1963; Fl. of China. 9: 176. 2003.

Local Name: Shardi Tanchi (Srt) (Hinko).

Eight to ten meter tall tree, young small branches slightly curved, bands, robust, glabrous or sparsely pubescent, brownish gray when old, sparsely white lenticillate; buds ovoid, small, glabrous or scales pubescent at margin and apex acute. Stipules caduceus, linear-lanceolate, membranous, adaxially white lanceolate soon glabrescent and margin glandular denticulate. Leaf blade ovate or narrowly ovate, oblong–ovate, rarely narrowly elliptic, margin obtusely serrate, rarely serrulate toward apex, acuminate or more rarely acute. Inflorescence, raceme umbel, three to six flowered, pedicel slender and sparsely hairy; soon glabrous, bracts lanceolate and caducous. Pedicel 2-3 cm, sparsely hairy, quickly hairless; Hypanthium bell shaped, abaxially hairless or nearly hairless. Sepals glabrous or almost glabrous, triangular- oval, adaxially thickly hairy, margin glandular denticulate, apex acuminate. Petals white, broadly ovate, base suddenly briefly clawed, tip rounded. Stamens 20, 1/3 shorter than the petals. Ovary 5- loculed or rarely four, two ovules per locule, Styles 5 (or4), same level to stamens, sparsely hairy. Fruit, pome, few pale dots, ovoid or ellipsoid, fruiting pedicel 2-3 cm, sepals persistent, Fl. Apr. fr. Aug-Sep.

Geographical position: Pakistan, Balakot (Sangar) Altit. 5344 ft., 340 34. 721‘N and 730 22. 291‘ E.

3.3 DNA Analysis

The DNA analysis involved the optimization of protocols for isolation of DNA from different parts of plants belonged to the genus Pyrus, followed by the amplification of RAPD primers, analysis of PCR generated data and phylogenetic analysis through different computer programmes. The DNA based results obtained for different land races collected from northern Pakistan is summarized below:

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3.3.1 DNA Extraction

Attempts were made to extract DNA from leaves through standard protocols reported by (Jang et al., 1991; John, 1992; Chevreau et al., 1997; Kimura et al., 2003; Li et al., 2010). However quality DNA could not be extracted from the species of the genus Pyrus. Therefoe attempts were made to extract DNA from other tissues including bark, wood, branches, dry leaves and herbarium specimens by modifying the above cited protocols. The results of the experiment have separately published ( Islam et al., 2013 and Fig. 3.15, 3.16B and 3.17). The published paper is attached in the annexture 6.9. Gel electrophoresis and PCR amplification through our modified protocol (Islam et al., 2013) confirmed that high quality DNA was successfully isolated from Pyrus species. Similarly, the amplified DNA sequences were also successfully eluted from agarose gels. Details of the protocol are given in DNA isolation section of the Materials and Methods chapter 2.

Fig. 3.15. Gel documentation images of genomic DNA extracted from Pyrus samples, from bark (L1) and from wood (L 2).

82

Fig. 3.16. PCR product of 500 bp fragment of 18S rRNA of Pyrus land races. 1 showed negative control, 2, 3 and 4 are of the nuclear DNA bands of Pt, 6 of the landrace Sb, 7, Nashpati, 8, Pkt and M is 1kb marker.

Fig. 3.17. PCR product of 18s rRAN of land races of Pyrus,1-Mt, 2-Nashpat, 3-Nb, 4-Kt, 6-

Ht, 8-Kshf, 9-Gzt, 10-At, 11-At, 12-Kb,13-Tt,14-Kb, 16-Fb and M represent 1kb marker.

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Fig. 3.16B Showing the presence of amplified DNA fragments, 1 shows the amplified DNA seqgment from Pt, 2- Sb, 3-Gt, 4- Kt, M 1kb marker and arrowheads showing their respective sequences.

3.3.2 Amplification ofRAPD primers

The genomic DNA samples of 36 land races (Table 3.3) were subjected to RAPD- PCR analysis. A total of 60 RAPD makers selected from Bioneer Oligo Synthesis kits were tested against DNA samples of all the land races. After initial screening, twenty eight 28 RAPD primers were selected for further analysis. The selected 28 RAPD primers generated 304 scorable amplification products from 36 genomic DNA samples. The results were analyzed by using NTSYSpc software (Rohlf, 1987) on the basis of various parameters such as total bands (TB), polymorphic bands (PB), monomorphic bands (MP) and percentage of polymorphism (PP).

Analysis of the results revealed that the average bands and polymorphic bands per primer were 10.85. The number of amplified bands ranged from 02 to 17, with the approximate size ranging from 150 to 2600bp. The maximum no of polymorphic bands (17) were produced by primers F-13 and F-17 while the minimum no of bands were produced by B-09. The RAPD markers produced 304 polymorphic and no monomorphic bands against 36 Pyrus land races and hence showed 100% polymorphism (Table 3.6).

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Table 3. 3. List of Pyrus land races collected from Northern Pakistan

S. Land races Coded Names Locality Botanical Altitu No Origin de (ft) 1 Malyzay Tango Mt Shawar, Swat 5160 2 Khan Tango Kt Shawar, Swat P. pyrifolia 4830 3 Nashpati Nashpati Chail, Swat P. communis 5149 4 Asmasy Tango At Shawar, Swat 5150 5 Nak Tango Nt Swat, Shawar P. Ovoidea 5140 6 Parawoo Tango Pt Swat, Chail P. sinkiangensis 4825 7 Kacha Tora Tangai Ktt Swat, Chail P. pashia 4720 8 Khapa Tango Kt Shawar, Swat P. serrulata 4812 9 Mamosranga Mamosranga Shawar, Swat Mamosranga 5145 10 Guraky Tango Gkt Shawar, Swat P.ovoideae cv. 4916 11 Pekhawry Tango Pkt Shawar, Swat P. hopeiensis 4916 12 Tora Tangai Tt Ghazi abad, A.K P. pashia 4014 13 Kashmiry Batangi Kshb Batal, Mansehra P.pashia 5180 14 Ghata Zira Tangai Gzt Ghazi abad, A.K P.pseudopashia 4030 15 Kala Batang Kb Sangar, Balakot 5166 16 Nak Batang Balakot Nb balakot Sangar, Balakot 4980 17 Kashmiry Batangi Kshb Sangar, Balakot 5320 18 Kashmiry Flexble Kshf Sangar, Balakot 5320 19 Kado Batang Kb-Balakot Sangar, Balakot 5157 Balakot 20 Ghata Zira Tangai Gzt Sangar, Balakot P. pseudopashia 5100 21 Zira Mamoothay Zm Shawar, Swat P.bretschneideri 4850 22 Spina Mamothy Sm Shawar, Swat P.ussuriensis 4850 23 Khapa Tango Kt Shawar, Swat P. serrulata 4812

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24 Momsay Batal Batal, Mansehra 5099 25 Shaker Batang-10 Sb-10 Batal, Mansehra 5079 26 Batangai Batangai Batal, Mansehra P. pashia 5170 27 Batangai Batangai Batal, Mansehra P.pashia 5160 28 Pashakaly batang Psb-10 Mansehra, 5190 29 Shaker Batang Sb-B1 Batal, Mansehra 5195 30 Mamosay Bat-14 Mamosay-B14 Batal, Mansehra 5160 31 Mamosay Bat-8 Mamosay-B8 Batal, Mansehra 5040 32 Hary Tango Batal Ht-B1 Mansehra, Batal 5190 33 Shaker Bat-10 Shaker-B10 Batal, Mansehra 5079 34 Mamosay Bat-12 Mamosay-B12 Batal, Mansehra 5200 35 Mamosay Bat-15 Mamosay-B15 Batal, Mansehra 5180 36 Mamosay Bat- 13 Mamosay-B13 Batal, Mansehra 5191

The polymorphic data generated by 28 RAPD primers across 36 Pyrus land races is presented in Table 3.4 & 3.6. The total numbers of amplified bands were 1983 and showed polymorphism. In all these amplicons, the maximum number of polymorphic bands were 111 which was produced by the land races 9- Mamosranga and Zira Mamoothay (Zm) with an average number of 3.96 bands The minimum number of a single band was produced by the land race Shaker Batang (Sb-B) with an average of 0.03 band per primer.

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Table 3. 4. Allelic distribution and performance of decamers with respect to location of allelic bands from Pyrus land races

S. No Land races Polymorph-ic Avg. Bands Per Bands Primer 1 Malyzay Tango (Mt) 72 2.57 2 Khan Tango (Kt) 45 1.60 3 Nashpati (Nashpai) 38 1.35 4 Asmasy Tango (At) 04 0.14 5 Nak Tango (Nt) 85 3.03 6 Parawoo Tango (Pt) 81 2.89 7 Kacha Tora Tangai (Ktt) 13 0.46 8 Khapa Tango (Kt) 71 2.53 9 Mamosranga (Mamosranga) 111 3.96 10 Guraky Tango (Gkt) 84 3.00 11 Pikhwary Tango (Pkt) 82 2.92 12 Tora Tangai (Tt) 33 1.17 13 Nikki Batangi (Nkb) 97 3.46 14 Ghata Zara Tangai (Gzt) 92 3.28 15 Kala Batang (Kb) 96 3.42 16 Nak Batang Balakot(Nb-Balakot) 63 2.25 17 Kashmiry Batangi (Ksb) 02 0.07 18 Kashmiry Flexble Batangi (Ksfb) 44 1.57 19 Kado Batang (Kb) 11 0.39 20 Ghata Zara Tangai (Gzt) 31 1.10 21 Zara Mamothy (Zm) 111 3.96 22 Spina Mamothy (Sm) 104 3.71 23 Khapa Tango (Kt) 61 2.17 24 Momsay Batal (Mb) 18 0.64 25 Shaker Batang-10 (Shaker-10) 18 0.64 26 Batangai 04 0.14 27 Batangai 0 0.00 28 Pashakaly batang (Psb) 0 0.00 29 Shaker Batang (Sb-B) 01 0.03 30 Mamosy Bat-14(Mamosay-B14) 41 1.46 31 Mamosy Bat-8 (Mamosay-B8) 81;2 2.89 32 Hary Tango Batal (Ht-B) 64 2.28 33 Shaker Batal-10 (Shaker-B10) 86 3.07 34 Mamosay Bat-12(Mamosay-B12) 85 3.03 35 Mamosay Bat-15(Mamosay-B15) 86 3.07 36 Mamosay Bat-13(Mamosay-B13) 68 2.42 S.Total 1983

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Table 3.5. List of primers specificity to land races

S. No Primers Sequences Bands Specific to Land races (bp) 1 A-11 CAATCGCCGT 300 1- Malyzay Tango (Mt) 2 C-09 CTCACCGTCC 1400 35- Mamosay Bat-15 (Mamosay-B15) 3 D-16 GGGCGTAAG 150 05- Nak Tango (Nt) 4 D-16 GGGCGTAAG 200 9- Mamosranga (Mamosranga) 5 D-16 GGGCGTAAG 300 13- Nikki Batangi (Nkb) 6 D-16 GGGCGTAAG 850 8- Khapa Tango (Kt) 7 G-07 GAACCTGCGG 450 9-Mamosranga (Mamosranga) 8 G-07 GAACCTGCGG 1600 9- Mamosranga (Mamosranga) 9 G-06 GTGCCTAACC 500 16- Nak Batng Balakot (Nb-Balakot) 10 G-06 GTGCCTAACC 1400 20- Ghata Zira Tangai (Gzt) 11 F-17 AACCCGGGAA 700 1-Malyzay Tango (Mt) 12 F-13 GGCTGCAGAA 600 10- Guraky Tango (Gkt) 13 F-13 GGCTGCAGAA 650 12- Tora Tangai (Tt) 14 F-13 GGCTGCAGAA 950 6- Parawoo Tango (Pt) 15 F-13 GGCTGCAGAA 350 2- Khan Tango (Kt) 16 F-13 GGCTGCAGAA 1900 25- Shaker Batang Batal-10 (Sb-10) 17 F-13 GGCTGCAGAA 2100 11- Pikhawary Tango (Pkt) 18 F-19 CCTCTAGACC 350 4- Asmasy Tango (At) 19 F-19 CCTCTAGACC 500 26- Batangai (Batangai) 20 I-16 TCTCCGCCCT 300 13- Nikki Batangi (Nkb) 21 I-16 TCTCCGCCCT 800 13- Nikki Batangi (Nkb) 22 J-05 ACGCACAACC 200 1- Malyzay Tango (Mt) 23 J-05 ACGCACAACC 650 22- Spina Mamothy (Sm) 24 J-05 ACGCACAACC 1000 11- Pikhawary Tango (Pkt) 25 J-05 ACGCACAACC 1500 11- Pikhawary Tango (Pkt) 26 J-05 ACGCACAACC 650 25- Shaker Batang Batal-10 (Sb-10) 27 J-05 ACGCACAACC 800 22- Zira Mamoothay (Zm) 28 J-20 AAGCGGCCTC 200 24- Spina Momsoay (Zm) 29 K-09 CCCTACCGAC 1100 13- Nikki Batangi (Nkb) 30 K-09 CCCTACCGAC 700 25- Shaker Batang Batal-10 (Sb-10) 31 K-09 CCCTACCGAC 800 31- Mamosay Bat-8 (Mamosay-B8) 32 K-09 CCCTACCGAC 1100 13- Nikki Batangi (Nkb) 33 K-09 CCCTACCGAC 1300 31- 31- Mamosay Bat-8 (Mamosay-B8) 34 L -08 TTTGCCCGGT 400 03- Nashpati (Nashpati) 35 E-10 ACCAGGTGA 850 03- Nashpati (Nashpati) Total 14 20 Digits before local names of the land races shows serial number used for list of Pyrus land races in table 3.3

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Table 3.6. Performance of the markers producing polymorphic bands of different

molecular size

S. NO Primer Sequences Polymorphic Range of bands names bands size (bp) 1 G-07 GAACCTGCGG 9 450-1600 2 F-17 AACCCGGGAA 17 300-2600 3 G-06 GTGCCTAACC 16 400-1600 4 G-08 TCACGTCCAC 14 400-1600 5 F-13 GGCTGCAGAA 07 600-1500 6 L-08 AGCAGGTGGA 04 400-1400 7 J-05 CTCCATGGGG 16 200-1400 8 I -08 TTTGCCCGGT 12 400-1500 9 L- 18 ACCACCCACC 11 400-1200 10 J-20 AAGCGGCCTC 15 200-1500 11 E-10 ACCAGGTGA 18 250-2400 12 J-05 ACGCACAACC 15 200-1500 13 A-11 CAATCGCCGT 16 300-2000 14 F-02 TGTCATCCCC 03 500-1100 15 A-02 GCCGAGCTG 13 400-1800 16 C-09 CTCACCGTCC 15 500-2300 17 D-16 GGGCGTAAG 12 150-1200 18 D-15 CATCCGTGCT 12 400-1800 19 E-15 CGCACAACC 12 400-2000 20 C-08 GGACCGGTG 11 400-1500 21 F-07 CCGATATCCC 10 300-1500 22 B-09 GGGGGACTC 02 1100-2400 23 F-13 GGCTGCAGAA 17 350-2600 24 L-02 TGGGCGTCAA 05 500-1500 25 F-19 CCTCTAGACC 05 350-1500 26 M-06 CTGGGCAACT 04 400-800 27 I-16 TCTCCGCCCT 05 300-800 28 K-09 CCCTACCGAC 08 650-1800 Total bands 304, Avg=10.85

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4. 3.3 Primers specificity

The 14 RAPD primers generated 35 bands of different sizes, ranging from 150 to 2100bp specific to twenty (20) different Pyrus land races out of the total 36 land races tested. Further observation and analysis showed that some primers were specific to more than one land races. For example, D-16 was specific to four different land races and band sizes amplified varied from 150 to 800bp. Similarly, F-13 was specific to six different land races and the size of amplicons ranged from 350 to 2100bp. Primer, J-05 was found specific to five different land races and amplified fragments of 650 to 1500bp. K-09 was specific to four different land races and the amplified fragments ranging from from 800 to1300bp in length. Among 14 RAPD primers belonging to A, C, D, G, F, I, J, K, L and E families. A- 11 was specific to land race 1-Malyzay Tango (Mt) producing a single band of 300bp. The decamer C-09 was specific to land race 35- Momosy Bat-15 (Mamosy B15) to a single locus for a 1400bp fragment and G-07 was specific to land race 9- Mamosranga at two different loci revealing bands of 450bp and 1600bp. The primer I-16 was specific to land race Nikki Batangi (Nkb) with two loci producing fragments of 300bp and 800bp. Primer J-05 was specific to land race 22- Zira Mamothai (Zm) at two different loci amplifying band sizes of 650bp and 800bp. K- 09 is specific to land race 31-Mamosay Bat-8 (Mamosay B8) and generated two different bands of sizes 800bp and 1300bp. Primers L-08 and E-10 were specific to land race 03-Nashpati with band sizes 400bp and 800bp, respectively. The other primers were highly specific and amplified at a single locus across the 36 land races. The results of these primers are summarized in Table 3.5

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Table 3. 7. Markers associated with the amplification patterns of DNA in different land

races of Pyrus.

S. No Primers Sequences Bands Specific to Land races (bp) 1 A-11 CAATCGCCGT 300 1- Malyzay Tango (Mt) 2 C-09 CTCACCGTCC 1400 35- Mamosay Bat-15 ((Mamosay- B15) 3 D-16 GGGCGTAAG 150 05- Nak Tango (Nt) 4 D-16 GGGCGTAAG 200 9- Mamosranga 5 D-16 GGGCGTAAG 300 13- Nikki Batnagi (Nkb) 6 D-16 GGGCGTAAG 850 8- Khapa Tango (Kt) 7 G-07 GAACCTGCGG 450 9-Mamosranga 8 G-07 GAACCTGCGG 1600 9- Mamosranga 9 G-06 GTGCCTAACC 500 16- Nak Batng Balakot (Nb-Balakot) 10 G-06 GTGCCTAACC 1400 20- Ghata Zira Tangai (Gzt) 11 F-17 AACCCGGGAA 700 1-Malyzay Tango (Mt) 12 F-13 GGCTGCAGAA 600 10- Guraky Tango (Gt) 13 F-13 GGCTGCAGAA 650 12- Tora Tangai (Tt) 14 F-13 GGCTGCAGAA 950 6- Parawoo Tango (Pt) 15 F-13 GGCTGCAGAA 350 2- Khan Tango (Kt) 16 F-13 GGCTGCAGAA 1900 25- Shaker Batang Batal-10 (Sb-10) 17 F-13 GGCTGCAGAA 2100 11- Pikhawary Tango (Pkt) 18 F-19 CCTCTAGACC 350 4- Asmasy Tango (At) 19 F-19 CCTCTAGACC 500 26- Batangai 20 I-16 TCTCCGCCCT 300 13- Nikki Batangi (Nkb) 21 I-16 TCTCCGCCCT 800 13- Kali Choti Batnagi (Nkb) 22 J-05 ACGCACAACC 200 1- Malyzay Tango (Mt) 23 J-05 ACGCACAACC 650 22- Spina Mamothy (Sm) 24 J-05 ACGCACAACC 1000 11- Pikhawary Tango (Pkt) 25 J-05 ACGCACAACC 1500 11- Pikhawary Tango (Pkt) 26 J-05 ACGCACAACC 650 25- Shaker Batang Batal-10 (Sb-10) 27 J-05 ACGCACAACC 800 22- Zira Mamoothay (Zm) 28 J-20 AAGCGGCCTC 200 24- Spina Momsoay (Sm) 29 K-09 CCCTACCGAC 1100 13- Nikki Batangi (Nkb) 30 K-09 CCCTACCGAC 700 25- Shaker Batang Batal-10 (Sb-10) 31 K-09 CCCTACCGAC 800 31- Mamosay Bat-8 (Mamosay-B8) 32 K-09 CCCTACCGAC 1100 13- Nikki Batangi (Nkb) 33 K-09 CCCTACCGAC 1300 31- Mamosay Bat-8 34 L -08 TTTGCCCGGT 400 03- Nashpati 35 E-10 ACCAGGTGA 850 03- Nashpati Total 14 20

Digits before local names of the land races shows serial number used for list of Pyrus land races in table 3.3.

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3.3.4 Similarities among the land races

The homology tree (Fig. 3.17) constructed on the basis of similarity index which splits the 36 Pyrus land races into different groups. Based on Nei‘s (1978) measures of genetic variance and neighbor joining algorithm of Saitou and Nei (1987), discriminated the land races into six major groups, represented by roman alphabits i.e I, II, III, IV, V and VI (Fig. 3.17). Group-I was further subdivided into 3 sub-group i.e IA, IB and IC. Sub-group IA consists of land races 5-Nak Tango (Nk) and 6-Parawo Tango (Pt) OTUs (operational taxonomic units) that were interconnected to each other with an interior node and possess 82% similarity with other sub-groups. Sub-group IB consists of land races 8- Khapa Tango (Kt) and 9- Mamosranga (OTUs) which are interconnected to each other with an interior node having 100 similarity with each other. Similarly sub-group IC consists of land races 10-Guraky Tango (Gkt) and 11-Pekhawry tango (OTUs), connected through a single interior node showing 100% similarity with each other. Further, group-I also consist of a single operational taxonomic unit (OTU), land race-1, in an odd manner and connected to a sub-group IA through an interior node showing 79% similarity with each other. All of the 3 sub-groups and a single OTU (Mt) were placed in a major group-I showing 74% identity/similarity (Fig. 3.17).

Group-II comprisedof nine (9) sub-groups, sub-group II A consisted of land races 2-Khan Tango (Kt) and 3- Neshpati and connected to each other through an interior node, which showed 87% similarity with each other. Sub-group IIB consisted of land races 4-Asmasy Tango (At), 17- Kashmiry Batangi (Kshb), 27- Batangi, 28- Pashakaly batang (Psb-10), 29- Shaker Batang (Sb-B) and 26-Batangi which showed 98% similarity with each other. The other sub-groups, IIC, IID, IIE, IIF, IIG, IIH, III comprised of land races 19-Kado Batang (Kb), 7-Kacha Tora Tangai (Ktt), 25-Shaker

Batang (Sb-B10), 24-Momsay Batal (Mb), 20-Ghata Zera Tangai (Gzt), 12- Tora Tangai (Tt) and 30- Mamosay Bat-14 (Mamosay B13) showed 96%, 93%, 92%, 88%, 87% and 82% similarity respectively.

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Group-III consists of three sub-groups i.e IIIA, IIIB and IIIC. One each land race included in the groups are 18- Kashmiry Flexble (Kshf), 23-Khapa Tango (Kt) and 16- Nak Batang Balakot (Nk-Balakot) respectively with percent of similarity of 82%,77% and 76% respectively with each other.

Group-IV consists of 3 sub-groups.Sub-group IVA consists of land races 31-

Mamosay Bat-8 (Mamosay-B8) and 32- Hary Tango Batal (Ht-B) which are connected to each other through an interior node with 94% similarity with each other. Sub- group IVB consists of land races 33- Shaker Bat-10 (Shaker –B10) and 34- Mamosay Bat-12 (Mamosay-B12) connected to an interior node showing 94% similarity with each other. Similarly Sub-group IVC consists of land races, 35- Mamosay Bat-15 (Mamosay B-15) and 36-Mamosay Bat- 13 (Mamosay B-13) which are interconnected by an interior node of 94% similarity. Further sub-group IVA and IVB are interconnected by an interior node of 92% similarity. Similarly, both sub-groups IVA and IVB are connected with sub-group IVC through an interior node showing 89% similarity.

Group-V consists of two sub-groups that is VA and VB. On the basis of 85% identity land races (OTUs) 13-Nikki Batangi (Nkb) and 14-Ghata Zira Tangai (Gzt) fall in Subgroup VA while sub-group VA is connected to sub-group VB through an interior node of 80% similarity.

Group-VI comprises of land races (OTUs) 21- Ziara Mamothai (Zm) and 22-Spina Mamothay (Sm) and interconnected with each other through an interior node showing 84% similarity. Group-VI is showing 62% homology with other groups.

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Fig. 3. 17. Homology tree of DNAMAN 5.2.2.0 showing the genetic similarity among 36 Pyrus land races based on Nei's (1978) identities/distances. Detail of the Pyrus land races used are given in table 3.3.

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3.3.5 Phylogenetic Relationship of Pyrus Land races

The phylogenetic relationship of 36 Pyrus land races is given in Fig. 3.18. On the bases of branch length and interdistances among land races, the phylogenetic tree divided thirty six (36) Pyrus land races into six (6) major groups.

Group-I is further sub-divided into three sub-groups, sub-group IA consists of land races 5 –Nak Tango (Nt) and 6-Parawoo Tango (Pt) with branch length of 0.121 and 0.116, respectively. Sub-group IB consist of land races 8-Khapa Tango (Kp) and 9-Mamosranga with branch length of 0.062 and 0.062, respectively. Similarly sub-group IC comprises of land races 10- Guraky Tango (Gkt) and 11- Pikhawary Tango (Pkt) having branch lengths of 0.133 and 0.136, respectively. Group-I also consists of a single cluster having land race 1-Malyzay Tango (Mt) showing minimum branch length of 0.087 as compared to other 4 land races belonging to sub-group A and C.

Group-II is divided into 5 sub-groups, sub-group IIA consist of land races 28- Pashakaly batang (Psb-10) and 27-Batangai having branch lengths of 0.007 and 0.007, respectively while sub-group IIB consists of land races 4-Asmasy Tango (At), 26- Batangai, 29- Shaker Batang (Sb-B) and 17- Kashmiry Batangi (Kshb) having branch lengths of 0.019, 0.018, 0.009 and 0.013, respectively. Similarly sub-group IIC consist of land races 7- Kacha Tora Tangai (Ktt), 12- Tora Tangai (Tt), and 19- Kado batang (Kb) having branch length is 0.035, 0.094 and 0.035 respectively. Sub-group IID consists of 3 single clusters i.e 24- Momsay (Mb), 25- Shaker Batal (Sb-10) and 20- Ghata Zira Tangai (Gzt) having branch lengths of 0.054, 0.048 and 0.080 respectively. Sub-group IIE comprises of land races 31- Mamosay Bat-8 (Mamosay- B8), 33- Shaker Bat-10 (Shaker B10)and 34- Mamosay-B12 (Mamosay-B12) having branch lengths of 0.065, 0.061 and 0.062, respectively. Group-II also consists of single cluster of land races such as 18-Kashmiry Flexble (Kshf), 30- Mamosay Bat-14

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(Mamosay-B14) and 32- Hary Tango ( Ht-B1) having branch lengths as 0.097, 0.039 and 0.050, respectively.

Group-III consists of two land races i.e 2- Khan Tango (Kt) and 3- Nashpati having branch lengths as 0.088 and 0.073, respectively. Group-IV comprises of two single clusters of land races i.e 23- Khapa Tango (Kp) and 16-Nak Batang Balakot (Nb- Balakot) having 0.133 and 0.102, branch lengths. Group-V consists of land races, 13-

Nikki Batangi (Nkb), 14- Ghata Zira Tangai (Gzt) and 15-Kala Batang (Kb) having branch lengths of 0.107, 0.103 and 0.120 respectively. Similarly group VI consists of land races 21- Zira Mamoothay (Zm) and 22- Spina Mamothy (Sm) having branch lengths as 0.150 and 0.135, respectively.

On the basis of branch lengths in the phylogenetic tree, land race 29- Shaker Batang (Sb-B) having branch lengths of 0.009 in sub-group IIB is more closely related to sub-group IIA having land races 28- Pashakaly batang (Psb-10) and 27- Batangai with branch lengths of 0.007 and 0.007, respectively. Similarly, in sub-group IIC land races 7- Kacha Tora Tangai (Ktt) and 19-Kado batang (Kb) having branch lengths of 0.035 and 0.035 respectively and support the topology of homology tree. Further, land races 12- Batangai and 18- Kashmiry Flexble (Kshf) are closely related to each other having 0.094 and 0.097 branch lengths, respectively but they are placed in different sub-groups, sub-group IIB and single cluster. Interestingly, there is 95% match between the phylogenetic and homology trees with respect to tree topology.

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Fig. 3. 18. Phylogenenetic tree of 36 Pyrus land races constituted with DNAMAN 5.2.2.0 showing the genetic relationship among Pyrus land races based on Nei's (1978) identities/distances

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Table 3.8. Distance matrix of 36 sequences

1 0 2 0.174 0 3 0.184 0.130 0 4 0.254 0.163 0.140 0 5 0.191 0.239 0.229 0.297 0 6 0.227 0.239 0.249 0.283 0.179 0 7 0.234 0.187 0.163 0.057 0.283 0.307 0 8 0.261 0.266 0.249 0.243 0.259 0.226 0.230 0 9 0.261 0.266 0.249 0.243 0.259 0.226 0.230 0.000 0 10 0.254 0.277 0.277 0.287 0.260 0.307 0.267 0.233 0.233 0 11 0.274 0.296 0.286 0.280 0.269 0.236 0.297 0.269 0.269 0.227 0 12 0.284 0.240 0.203 0.123 0.327 0.367 0.087 0.267 0.267 0.310 0.370 0 13 0.312 0.346 0.326 0.289 0.332 0.346 0.282 0.285 0.285 0.309 0.279 0.346 0 14 0.326 0.342 0.319 0.315 0.319 0.326 0.309 0.292 0.292 0.329 0.305 0.359 0.153 0 15 0.346 0.350 0.320 0.328 0.353 0.340 0.334 0.323 0.323 0.368 0.323 0.398 0.235 0.164 0 16 0.279 0.267 0.237 0.217 0.317 0.320 0.240 0.277 0.277 0.341 0.330 0.274 0.309 0.218 0.233 0 17 0.241 0.157 0.127 0.020 0.283 0.270 0.050 0.237 0.237 0.280 0.267 0.116 0.282 0.302 0.314 0.211 0 18 0.309 0.243 0.240 0.161 0.330 0.310 0.184 0.323 0.323 0.344 0.333 0.234 0.312 0.305 0.320 0.230 0.147 0 19 0.251 0.159 0.143 0.050 0.292 0.279 0.080 0.272 0.272 0.263 0.256 0.133 0.268 0.292 0.320 0.227 0.043 0.163 0 20 0.278 0.179 0.189 0.117 0.312 0.286 0.147 0.279 0.279 0.313 0.289 0.187 0.305 0.309 0.317 0.227 0.110 0.203 0.113 0 21 0.399 0.390 0.373 0.388 0.390 0.417 0.381 0.410 0.410 0.445 0.440 0.415 0.369 0.336 0.327 0.357 0.381 0.337 0.370 0.317 0 22 0.372 0.387 0.357 0.365 0.413 0.413 0.358 0.413 0.413 0.428 0.410 0.411 0.342 0.322 0.290 0.327 0.358 0.313 0.347 0.333 0.156 0 23 0.308 0.269 0.259 0.220 0.362 0.342 0.210 0.316 0.316 0.320 0.312 0.243 0.312 0.339 0.323 0.277 0.213 0.267 0.196 0.249 0.357 0.333 0 24 0.268 0.163 0.173 0.073 0.302 0.262 0.097 0.236 0.236 0.293 0.306 0.163 0.319 0.332 0.333 0.237 0.060 0.167 0.083 0.130 0.373 0.343 0.239 0 25 0.254 0.163 0.159 0.073 0.322 0.289 0.097 0.276 0.276 0.293 0.292 0.157 0.302 0.339 0.340 0.243 0.067 0.173 0.083 0.130 0.377 0.360 0.199 0.1060 26 0.247 0.157 0.140 0.020 0.290 0.277 0.057 0.243 0.243 0.293 0.287 0.123 0.295 0.315 0.334 0.217 0.020 0.161 0.050 0.117 0.381 0.365 0.220 0.073 0.067 0 27 0.241 0.150 0.127 0.013 0.283 0.270 0.043 0.237 0.237 0.280 0.273 0.110 0.289 0.309 0.321 0.211 0.007 0.147 0.037 0.103 0.375 0.351 0.207 0.060 0.060 0.013 0 28 0.241 0.150 0.127 0.013 0.283 0.270 0.043 0.237 0.237 0.280 0.273 0.110 0.289 0.309 0.321 0.211 0.007 0.147 0.037 0.103 0.375 0.351 0.207 0.060 0.060 0.013 0.000 0 29 0.244 0.153 0.130 0.017 0.286 0.272 0.047 0.239 0.239 0.283 0.276 0.113 0.282 0.309 0.323 0.213 0.010 0.150 0.040 0.106 0.377 0.353 0.209 0.063 0.056 0.010 0.003 0.003 0 30 0.261 0.209 0.193 0.147 0.302 0.296 0.160 0.309 0.309 0.290 0.326 0.217 0.302 0.339 0.347 0.260 0.133 0.187 0.143 0.183 0.387 0.377 0.272 0.166 0.159 0.147 0.133 0.133 0.136 0 31 0.331 0.306 0.296 0.277 0.379 0.385 0.277 0.379 0.379 0.347 0.402 0.300 0.359 0.389 0.403 0.337 0.263 0.250 0.272 0.306 0.437 0.433 0.342 0.276 0.269 0.263 0.263 0.263 0.259 0.143 0 32 0.314 0.256 0.252 0.220 0.349 0.342 0.233 0.349 0.349 0.343 0.379 0.277 0.349 0.379 0.380 0.293 0.207 0.213 0.216 0.249 0.420 0.417 0.299 0.219 0.226 0.207 0.207 0.207 0.203 0.093 0.063 0 33 0.328 0.306 0.309 0.290 0.379 0.399 0.283 0.392 0.392 0.347 0.402 0.320 0.359 0.389 0.403 0.343 0.277 0.270 0.279 0.312 0.437 0.420 0.336 0.282 0.296 0.277 0.277 0.277 0.272 0.169 0.073 0.090 0 34 0.334 0.316 0.312 0.280 0.382 0.395 0.280 0.395 0.395 0.350 0.399 0.317 0.369 0.399 0.420 0.340 0.273 0.280 0.276 0.316 0.447 0.430 0.339 0.279 0.292 0.273 0.273 0.273 0.269 0.166 0.076 0.086 0.037 0 35 0.361 0.306 0.302 0.283 0.385 0.392 0.320 0.419 0.419 0.380 0.402 0.353 0.389 0.406 0.400 0.333 0.283 0.270 0.272 0.319 0.463 0.453 0.362 0.289 0.302 0.277 0.283 0.283 0.279 0.196 0.120 0.123 0.086 0.090 0 36 0.328 0.276 0.266 0.233 0.369 0.369 0.263 0.389 0.389 0.353 0.365 0.303 0.366 0.386 0.387 0.323 0.227 0.240 0.223 0.296 0.447 0.437 0.332 0.252 0.246 0.220 0.227 0.227 0.223 0.166 0.130 0.113 0.103 0.100 0.063 0

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Tabl 3.9 Homology matrix of 36 sequences 1 100% 2 82.6% 100% 3 81.6% 87.0% 100% 4 74.6% 83.7% 86.0% 100% 5 80.9% 76.1% 77.1% 70.3% 100% 6 77.3% 76.1% 75.1% 71.7% 82.1% 100% 7 76.6% 81.3% 83.7% 94.3% 71.7% 69.3% 100% 8 73.9% 73.4% 75.1% 75.7% 74.1% 77.4% 77.0% 100% 9 73.9% 73.4% 75.1% 75.7% 74.1% 77.4% 77.0% 100.0% 100% 10 74.6% 72.3% 72.3% 71.3% 74.0% 69.3% 73.3% 76.7% 76.7% 100% 11 72.6% 70.4% 71.4% 72.0% 73.1% 76.4% 70.3% 73.1% 73.1% 77.3% 100% 12 71.6% 76.0% 79.7% 87.7% 67.3% 63.3% 91.3% 73.3% 73.3% 69.0% 63.0% 100% 13 68.8% 65.4% 67.4% 71.1% 66.8% 65.4% 71.8% 71.5% 71.5% 69.1% 72.1% 65.4% 100% 14 67.4% 65.8% 68.1% 68.5% 68.1% 67.4% 69.1% 70.8% 70.8% 67.1% 69.5% 64.1% 84.7% 100% 15 65.4% 65.0% 68.0% 67.2% 64.7% 66.0% 66.6% 67.7% 67.7% 63.2% 67.7% 60.2% 76.5% 83.6% 100% 16 72.1% 73.3% 76.3% 78.3% 68.3% 68.0% 76.0% 72.3% 72.3% 65.9% 67.0% 72.6% 69.1% 78.2% 76.7%100% 17 75.9% 84.3% 87.3% 98.0% 71.7% 73.0% 95.0% 76.3% 76.3% 72.0% 73.3% 88.4% 71.8% 69.8% 68.6% 78.9% 100% 18 69.1% 75.7% 76.0% 83.9% 67.0% 69.0% 81.6% 67.7% 67.7% 65.6% 66.7% 76.6% 68.8% 69.5% 68.0% 77.0% 85.3% 100% 19 74.9% 84.1% 85.7% 95.0% 70.8% 72.1% 92.0% 72.8% 72.8% 73.7% 74.4% 86.7% 73.2% 70.8% 68.0% 77.3% 95.7% 83.7%100% 20 72.2% 82.1% 81.1% 88.3% 68.8% 71.4% 85.3% 72.1% 72.1% 68.7% 71.1% 81.3% 69.5% 69.1% 68.3% 77.3% 89.0% 79.7% 88.7% 100% 21 60.1% 61.0% 62.7% 61.2% 61.0% 58.3% 61.9% 59.0% 59.0% 55.5% 56.0% 58.5% 63.1% 66.4% 67.3% 64.3% 61.9% 66.3% 63.0% 68.3% 100% 22 62.8% 61.3% 64.3% 63.5% 58.7% 58.7% 64.2% 58.7% 58.7% 57.2% 59.0% 58.9% 65.8% 67.8% 71.0% 67.3% 64.2% 68.7% 65.3% 66.7% 84.4% 100% 23 69.2% 73.1% 74.1% 78.0% 63.8% 65.8% 79.0% 68.4% 68.4% 68.0% 68.8% 75.7% 68.8% 66.1% 67.7% 72.3% 78.7% 73.3% 80.4% 75.1% 64.3% 66.7% 100% 24 73.2% 83.7% 82.7% 92.7% 69.8% 73.8% 90.3% 76.4% 76.4% 70.7% 69.4% 83.7% 68.1% 66.8% 66.7% 76.3% 94.0% 83.3% 91.7% 87.0% 62.7% 65.7% 76.1% 100% 25 74.6% 83.7% 84.1% 92.7% 67.8% 71.1% 90.3% 72.4% 72.4% 70.7% 70.8% 84.3% 69.8% 66.1% 66.0% 75.7% 93.3% 82.7% 91.7% 87.0% 62.3% 64.0% 80.1% 89.4% 100% 26 75.3% 84.3% 86.0% 98.0% 71.0% 72.3% 94.3% 75.7% 75.7% 70.7% 71.3% 87.7% 70.5% 68.5% 66.6% 78.3% 98.0% 83.9% 95.0% 88.3% 61.9% 63.5% 78.0% 92.7% 93.3% 100% 27 75.9% 85.0% 87.3% 98.7% 71.7% 73.0% 95.7% 76.3% 76.3% 72.0% 72.7% 89.0% 71.1% 69.1% 67.9% 78.9% 99.3% 85.3% 96.3% 89.7% 62.5% 64.9% 79.3% 94.0% 94.0% 98.7% 100% 28 75.9% 85.0% 87.3% 98.7% 71.7% 73.0% 95.7% 76.3% 76.3% 72.0% 72.7% 89.0% 71.1% 69.1% 67.9% 78.9% 99.3% 85.3% 96.3% 89.7% 62.5% 64.9% 79.3% 94.0% 94.0% 98.7% 100.0% 100% 29 75.6% 84.7% 87.0% 98.3% 71.4% 72.8% 95.3% 76.1% 76.1% 71.7% 72.4% 88.7% 71.8% 69.1% 67.7% 78.7% 99.0% 85.0% 96.0% 89.4% 62.3% 64.7% 79.1% 93.7% 94.4% 99.0% 99.7% 99.7% 100% 30 73.9% 79.1% 80.7% 85.3% 69.8% 70.4% 84.0% 69.1% 69.1% 71.0% 67.4% 78.3% 69.8% 66.1% 65.3% 74.0% 86.7% 81.3% 85.7% 81.7% 61.3% 62.3% 72.8% 83.4% 84.1% 85.3% 86.7% 86.7% 86.4% 100% 31 66.9% 69.4% 70.4% 72.3% 62.1% 61.5% 72.3% 62.1% 62.1% 65.3% 59.8% 70.0% 64.1% 61.1% 59.7% 66.3% 73.7% 75.0% 72.8% 69.4% 56.3% 56.7% 65.8% 72.4% 73.1% 73.7% 73.7% 73.7% 74.1% 85.7% 100% 32 68.6% 74.4% 74.8% 78.0% 65.1% 65.8% 76.7% 65.1% 65.1% 65.7% 62.1% 72.3% 65.1% 62.1% 62.0% 70.7% 79.3% 78.7% 78.4% 75.1% 58.0% 58.3% 70.1% 78.1% 77.4% 79.3% 79.3% 79.3% 79.7% 90.7% 93.7% 100% 33 67.2% 69.4% 69.1% 71.0% 62.1% 60.1% 71.7% 60.8% 60.8% 65.3% 59.8% 68.0% 64.1% 61.1% 59.7% 65.7% 72.3% 73.0% 72.1% 68.8% 56.3% 58.0% 66.4% 71.8% 70.4% 72.3% 72.3% 72.3% 72.8% 83.1% 92.7% 91.0% 100% 34 66.6% 68.4% 68.8% 72.0% 61.8% 60.5% 72.0% 60.5% 60.5% 65.0% 60.1% 68.3% 63.1% 60.1% 58.0% 66.0% 72.7% 72.0% 72.4% 68.4% 55.3% 57.0% 66.1% 72.1% 70.8% 72.7% 72.7% 72.7% 73.1% 83.4% 92.4% 91.4% 96.3% 100% 35 63.9% 69.4% 69.8% 71.7% 61.5% 60.8% 68.0% 58.1% 58.1% 62.0% 59.8% 64.7% 61.1% 59.4% 60.0% 66.7% 71.7% 73.0% 72.8% 68.1% 53.7% 54.7% 63.8% 71.1% 69.8% 72.3% 71.7% 71.7% 72.1% 80.4% 88.0% 87.7% 91.4% 91.0% 100% 36 67.2% 72.4% 73.4% 76.7% 63.1% 63.1% 73.7% 61.1% 61.1% 64.7% 63.5% 69.7% 63.4% 61.4% 61.3% 67.7% 77.3% 76.0% 77.7% 70.4% 55.3% 56.3% 66.8% 74.8% 75.4% 78.0% 77.3% 77.3% 77.7% 83.4% 87.0% 88.7% 89.7% 90.0%93.7% 100%

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3. 4. Nucleotide Sequencing On the basis of morphological and geographical features, pears have been divided into two groups, i.e the Oriential and Occidential pears (Zhukovsky and Zeelinski, 1965) but their identification to the species level is difficult, because of (i) poor wild population (ii) overlapping and poorly differentiable morphological characteristics and (iii) wide spread crossability and interspecific hybridization. During the past few years many attempts have been made to resolve the disputes and unidentify organisms to the genus, species and cultivar levels. Aomong various approaches, the 18s rRNA gene and its respective nucleotide sequences have successfully been used for identification and establishing phylogenetic relationships among a variety of groups. The nucleotide sequence information obtained from 18s rRNA of various pear land races have been compared with the database sequences with the help of BLAST, Basic Local Alignment Search Tool (Altschul et al., 1990). The results are given below.

3. 4.1 Kacha Tora Tangai

The 18s rRNA specimen of Kacha Tora Tangai (Ktt) collected from differnet areas are provided in appendix II. The information obtained from BLAST (Altschul et al., 1990) with NCBI refrence database showed that Ktt has maximum (99%) identity and query cover with cultivars Sunwhang, Miwhang, Imamuraaki, Soowhang and Okusankichi of Pyrus pyrifolia.

For further identification, phylogenetic relationship of the land races Ktt with other related species and genera was inferred by using Maximum Likelihood method of Jukes et al., (1969). For this, 41 sequences of 18S rRNA of related species of five different genera were retrieved from GenBank and were subjected to the organization of maximum likelihood tree. The aligned datasheet had 1850 genetic characters, which was trimmed for extra and ambiguously aligned fragments both from the 5‘ and 3‘ ends of alignment data sheet, thus resulting in 1222 genetic traits which were used for further analysis.

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Out of the analysed traits, 259 were conserved, 232 were variable, 209 were parsimony- informative and 23 were singleton sites. None of the characters were excluded from the final analysis and multistate taxa were interpreted as uncertain.

The phylogenetic tree (Fig. 3.19) divided the Pyrus land races into two major clades. Clade I was further divided into two sub clades i.e I and II. Our local landrace (Ktt) clustred with sub clade I of the clade I of the cladogram with unresolved bootstrap support. The sub clade I consisted of 19 Pyrus species/cultivars viz Pyrus. pyrifolia cv. Shinil, Kacha Tora Tangai (Ktt), P. pyrifolia cultivars Yeoungsanbae, Kimitsukawase, Wonwhang, Soowhang, P. communis cvs. Cascade, Beurre, P. pyrifolia cvs. Nijisseiki, Kosui, Chojuro, P. communis cvs. Conference, Pachkan's Triumph, P. pyrifolia cvs. Imamuraaki, Miwhang, Sunwhang, Okusankichi, Eoungsanbae and Minibae, respectively.

Sub clade II comprised 6 species/cultivars belonging to the genus Prunus while the sub clade III, IV and V comprised of other rosacious genera related to the genus Pyrus.

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Pyrus _pyrifolia _cultivar _Shinil _ AF179396.1 0.00000 0.00000 Kacha Tora Tangai (Ktt) 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Yeoungsanbae _ AF179400.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Kimitsukawase _ AF179388.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Wonwhang _ AY168822.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Soowhang _ AY168824.1 0.00000 0.00000 Pyrus _communis _cultivar _Cascade _ AF195618.1 0.00000 0.00000 Pyrus _communis _cultivar _Beurre _ AF195617.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Nijisseiki _ AF179394.1 0.00489 0.00000 Pyrus _pyrifolia _cultivar _Kosui _ AF179389.1 Sub_clade_I 0.00000 0.00489 0.00000 Pyrus _pyrifolia _cultivar _Chojuro _ AF179383.1 0.00489 0.00000 Pyrus _communis _cultivar _Conference _ AF195619.1 0.00000 Pyrus _communis _cultivar _Pachkan's _Triumph _ AF195621.1 Clade_I 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Imamuraaki _ AF179387.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Miwhang _ AF179392.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Sunwhang _ AF179398.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Okusankichi _ AF179395.1 0.00000 0.00489 Pyrus _pyrifolia _cultivar _Whangkeumbae - AF179399.1 0.00000 0.00489 Pyrus _pyrifolia _cultivar _Minibae _ AF179391.1 0.00489 0.00489 Prunus_persica_cultivar_Kuemjeokbaekdo_AY179519.1 83 0.00000 Prunus_persica_cultivar_Youngseonghwangdo_AY179522.1 0.00982 0.00000 Prunus_persica_cultivar_Changhowonhwangdo_AY179514.1 0.00000 0.00000 Sub_clade_II 0.52744 Prunus_persica_cultivar_Baekmijosaeng_AY179513.1 0.00000 0.00000 0.00000 Prunus_persica_cultivar_Hakuho_AY179529.1 0.00000 0.00000 Prunus_persica_cultivar_Cheonhong_AY179515.1 0.00000 Pyrus _pyrifolia _cultivar _Mansoo _ AF179390 0.00000 Cydonia_oblonga_AF186531.1 0.00000 0.00000 Cydonia_oblonga_voucher829-84D_JQ392421.1 0.00000 0.00000 Cydonia_oblonga_JQ392420.1 Sub_clade_III 0.00000 0.00000 100 Cydonia_oblonga_AB636344.1 0.09489 0.00000 Cydonia_oblonga_JQ392422.1 0.00000 Rubus_caesius_Rubus_idaeus_AF362709.1 0.00000 100 0.00000 Rubus_caesius_Rubus_idaeus_AF362708.1 0.00000 0.35803 0.00000 Rubus_crataegifolius_clone_KC10-2_GU980798.1 Sub_clade_IV 0.00000 0.01478 Clade_II 72 Rubus_coreanus_isolateP677_JF980336.1 0.04468 0.00982 65 Rubus_picticaulis_clone_29_AF362726.1 0.00000 0.08274 Fragaria_moschata_AF163505.1 99 0.00000 Fragaria_vesca_AF163510.1 0.09648 0.00489 Fragaria_virginiana_AF163479.1 0.00000 0.00000 Sub_clade_V Fragaria_chiloensis_AF163482.1 0.00000 0.00000 0.00000 Fragaria_nubicola_AF163517.1 0.00489 0.00000 Fragaria_chiloensis_AF163511.1 0.00000

Fig. 3.19: Phylogenetic analysis of “Kacha Tora Tangai (Ktt)‖ based upon the Maximum Likelihood Method (Jukes et al., 1969) at 1000 bootstrap replications of the data obtained from 18s rRNA. The analysis concludes that Kacha Tora Tangai ―Ktt‖sorted into the sub clade I of clade I.

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3.4.2 Zira Mamoothay

For molecular characterizations, the nucleotide sequence obtained from 18S rRNA of Zira Mamoothay (Zm) was initially BLAST in NCBI. The blast result showed maximum (99%) identity and query cover with Pyrus communis cultivars Pachkan's Triumph, Conference, Beurre and Cascade. Similar results showed up for species/cultivars belonging to the genus Pyrus.

Further confirmation and phylogenetic relationship of the land race Zira Mamoothay (Zm) with other related species and genera was inferred by using Maximum Likelihood (ML) method (Jukes et al., 1969). A total of 37 sequences of 18S rRNA of related species of five different genera were retrieved from GenBank and were subjected for construction of maximum likelihood tree. The aligned datasheet had maximum 1850 genetic characters and after trimming extra and ambiguously aligned fragments from both 5‘ and 3‘ ends of alignment data sheet, only 1213 characters were used for further analysis. Out of these characters, 258 were conserved, 234 were variable, 209 were parsimony-informative and 25 were singleton sites. None of the characters were excluded from the final analysis and multistate taxa were interpreted as uncertainty.

The evolutionary history was derived by using the Maximum Likelihood (ML) based on the Jukes-Cantor model (Jukes et al., 1969). The tree with the highest log likelihood (- 885.7129) is shown in Fig. 3.20. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Joining and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. The analysis comprises 38 nucleotide sequences and all positions containing gaps and missing data were eliminated.

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Phylogenetic tree (Fig. 3.20) thus organized was divided into two major clades. Clade I was further divided into three sub clades, sub clade I, II and III.

The sub clade I contained 12 Pyrus species/cultivars viz. P. pyrifolia cultivars Sunwhang, Miwhang, Imamuraaki, P. communis cultivars Pachkan's Triumph, Conference, Beurre, cascade, P. pyrifolia cultivars Shinil, Chuwhangbae, Shinsui and P. communis cv. Clapp's Favorite, respectively.

Our land race of Pyrus Zira Mamoothay (Zm) was sorted out into the sub clade I of the major clade I and showed close relationship with its similar species/cultivars of P. communis cv. Clapp's Favorite. While Sub clade II comprised of 7 species/cultivarsof the genus Prunus. The clade II was sub divided into three sub clades, III, IV and VI comprising of Cydonia, Rubus and Frageria, the related genera of family Rosaceae.

104

Pyrus_pyrifolia_cultivar_Sunwhang_AF179398.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Miwhang_AF179392.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Imamuraaki_AF179387.1 0.00000 0.00000 Pyrus_communis_cultivar_Pachkan's_Triumph_AF195621.1 0.00000 0.00000 Pyrus_communis_cultivar_Conference_AF195619.1 0.00000 0.00000 Pyrus_communis_cultivar_Beurre_AF195617.1 0.00000 0.00000 Pyrus_communis_cultivar_Cascade_AF195618.1 0.00000 0.00000 Sub_clade_I Pyrus_pyrifolia_cultivar_Shinil_AF179396.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Chuwhangbae_AF179384.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Shinsui_AF179397.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Niitaka_AF179393.1 Clade_I 0.00492 0.00000 Pyrus_communis_cultivar_Clapp's_Favorite_AF195620.1 0.00000 0.00000 0.00000 Zira Mamothy_Zm 0.01485 Pyrus_pyrifolia_cultivar_Gamcheonbae_AF179385.1 0.00492 0.00492 Prunus_persica_cultivar_Wolmiboksunga_AY179521.1 87 0.00000 Prunus_persica_cultivar_Kuemjeokbaekdo_AY179519.1 0.00987 0.00000 Prunus_persica_cultivar_Youngseonghwangdo_AY179522.1 0.00000 0.00000 Prunus_persica_cultivar_Changhowonhwangdo_AY179514.1 Sub_clade_III 0.50536 0.00000 0.00000 Prunus_persica_cultivar_Baekmijosaeng_AY179513.1 0.00000 0.00000 Prunus_persica_cultivar_Hakuho_AY179529.1 0.00000 0.00000 0.00000 Prunus_persica_cultivar_Cheonhong_AY179515.1 0.00000 Pyrus_pyrifolia_cultivar_Choju_AF179382.1 0.00000 Cydonia_oblonga_AB636344.1 0.00000 0.00000 Cydonia_oblonga_JQ392422.1 0.00000 0.00000 Cydonia_oblonga_AF186531.1 Sub_clade_III 99 0.00000 0.00000 Cydonia_oblonga_voucher829-84D_JQ392421.1 0.08525 0.00000 Cydonia_oblonga_JQ392420.1 0.00000 Rubus_caesius_Rubus_idaeus_AF362709.1 0.00000 99 630.00000 Rubus_caesius_Rubus_idaeus_AF362708.1 0.00000 0.34545 720.00000 Rubus_picticaulis_clone_29_AF362726.1 Sub_clade_IV 0.00000 0.00000 Clade_II 72 Rubus_coreanus_isolateP677_JF980336.1 0.04285 0.00987 Rubus_crataegifolius_clone_KC10-2_GU980798.1 66 0.01485 0.08185 Fragaria_moschata_AF163505.1 0.00000 99 Fragaria_nubicola_AF163517.1 0.00492 0.09291 0.00000 Fragaria_vesca_AF163510.1 0.00492 Sub_clade_V 0.00000 Fragaria_virginiana_AF163479.1 0.00000 Fragaria_chiloensis_AF163482.1 0.00000 0.00000 0.00000 Fragaria_chiloensis_AF163511.1 0.00000

Fig. 3.20: Phylogenetic analysis of “Zira Mamoothay‖ (Zm) based upon the Maximum Likelihood Method (Jukes et al., 1969) at 1000 bootstrap replications of the data obtained from 18s rRNA.

105

3. 4. 3. Khan Tango (Kt)

For molecular characterizations, the nucleotide sequence obtained from the 18S rRNA of the local land race ―Khan Tango (Kt)‖ (Kt-1) was initially BLAST searched in NCBI. The blast result showed 97% maximum identity and query cover with Pyrus communis cultivars Pachkan's Triumph, Beurre, Cascade, Conference and P. pyrifolia cv. Imamuraaki

For further identification and discrimination, phylogenetic relationship of the land race ―Khan Tango (Kt)‖ with other related species and genera was inferred by using Maximum Likelihood method. A total of 41 sequences of 18S rRNA of related species belonging to five different genera were retrieved from GenBank and were subjected for development of maximum likelihood tree. The aligned datasheet has a maximum of 1850 characters. After trimming the extra and ambiguously aligned fragments from both 5‘ and 3‘ ends of alignment data sheet, a total of 1170 genetic characters were used for further analysis. Out of these 258 characters were conserved, 254 were variable, 227 were parsimony-informative and 27 were singleton sites. None of the characters were excluded from the final analysis and multistate taxa were interpreted as uncertainty

The evolutionary history was derived by using the Maximum Likelihood (ML) method based on the Jukes-Cantor model (Jukes et al., 1969). The tree with the highest log likelihood (-1010.9077) is shown in Fig. 3.21. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. The analysis comprises 41 nucleotide sequences and all positions containing gaps and missing data were eliminated. The phylogenetic tree (Fig.3.21) thus constructed was divided into two major clades, Clade I and II. Clade I was further sub divided into two sub clades, sub clade I and II. Sub clade I comprised of 19 Pyrus species/cultivars consisted of P. pyrifolia cultivars

106

Yeoungsanbae, Shinil, Kimitsukawase, Soowhang and Wonwhang, P. communis cultivars Cascade, Beurre and Pachkan's Triumph, P. pyrifolia cultivars Chojuro, Sunwhang, Miwhang and Imamuraaki, P. communis cv. Conference, P. pyrifolia cv. Nijisseiki, local landrace Khan Tango (Kt), P. pyrifolia cultivars Okusankichi, Minibae, Whangkeumbae and Kosui, respectively. The local land race, Khan Tango (Kt) does not make a group to any of the database species/cultivars and fell in the Pyrus clade. This shows that it may be a separate species and require further study for its identification.

The sub clades II, III, IV and V comprised other rosaceous genera related to the genus Pyrus.

107

5 Pyrus _pyrifolia _cultivar _Yeoungsanbae _ AF179400.1 0.00000 0.000000 Pyrus _pyrifolia _cultivar _Shinil _ AF179396.1 0.00000 0.000000 Pyrus _pyrifolia _cultivar _Kimitsukawase _ AF179388.1 0 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Wonwhang _ AY168822.1 0 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Soowhang _ AY168824.1 0 0.00000 0.00000 Pyrus _communis _cultivar _Cascade _ AF195618.1 0 0.00000 0.00000 Pyrus _communis _cultivar _Beurre _ AF195617.1 0.000000 0.00000 Pyrus _communis _cultivar _Pachkan's _Triumph _ AF195621.1 0.000000 0.00000 Pyrus _pyrifolia _cultivar _Chojuro _ AF179383.1 0.00000 0.00429 0 Pyrus _pyrifolia _cultivar _Sunwhang _ AF179398.1 Sub_clade_I 0.00429 0.00000 Pyrus _pyrifolia _cultivar _Miwhang _ AF179392.1 0 0.00000 9 Pyrus _pyrifolia _cultivar _Imamuraaki _ AF179387.1 0.00000 00.00000 7 Pyrus _communis _cultivar _Conference _ AF195619.1 Clade_I 0.00000 0.000000.00429 14 0.00000 Pyrus _pyrifolia _cultivar _Nijisseiki _ AF179394.1 0.00429 0.000009 Khan Tango _Kt-1 0.00000 Pyrus _pyrifolia _cultivar _Okusankichi _ AF179395.1 100.00000 0.00429 Pyrus _pyrifolia _cultivar _Minibae _ AF179391.1 220.00000 0.00429 Pyrus _pyrifolia _cultivar _Whangkeumbae - AF179399.1 0.00000 0.00429 Pyrus _pyrifolia _cultivar _Kosui _ AF179389.1 30 0.00860 9 Prunus_persica_cultivar_Kuemjeokbaekdo_AY179519.1 0.00429 0.00429 85 0.00000 Prunus_persica_cultivar_Cheonhong_AY179515.1 0.00000 Prunus_persica_cultivar_Youngseonghwangdo_AY179522.1 0.00860 4 0.00000 Sub_clade_II Prunus_persica_cultivar_Changhowonhwangdo_AY179514.1 0.47748 0.0000050.00000 12 Prunus_persica_cultivar_Baekmijosaeng_AY179513.1 0.000000.00000 0.00000 Prunus_persica_cultivar_Hakuho_AY179529.1 0.00000 Pyrus _pyrifolia _cultivar _Mansoo _ AF179390 0.00000 16 Cydonia_oblonga_AF186531.1 0.00000 0.000009 Cydonia_oblonga_voucher829-84D_JQ392421.1 0.00000 990.00000 Cydonia_oblonga_JQ392420.1 Sub_clade_III 0.00000 0.08150 16 Cydonia_oblonga_AB636344.1 0.00000 0.00000 Cydonia_oblonga_JQ392422.1 0.00000 30 Rubus_caesius_Rubus_idaeus_AF362709.1 100 0.00000 790.00000 Rubus_caesius_Rubus_idaeus_AF362708.1 0.33575 0.00000 750.00000 Rubus_picticaulis_clone_29_AF362726.1 Sub_clade_IV 0.00000 Clade_II 0.03885 1 Rubus_coreanus_isolateP677_JF980336.1 0.00860 67 0.00000 Rubus_crataegifolius_clone_KC10-2_GU980798.1 0.01293 0.07106 Fragaria_virginiana_AF163479.1 0.00000 100 6 Fragaria_moschata_AF163505.1 0.00000 0.0835520.00000 Fragaria_vesca_AF163510.1 0.00429 Sub_clade_IV 0.00000 Fragaria_nubicola_AF163517.1 60.00429 14 Fragaria_chiloensis_AF163482.1 0.000000.00000 0.00000 Fragaria_chiloensis_AF163511.1 0.00000

Fig. 3.21 Phylogenetic analysis of ―Khan Tango (Kt)‖ by Maximum Likelihood Method (Jukes et al., 1969) at1000 bootstrap replications of the data obtained from 18s rRNA.

108

3. 4. 4. Ghata Zira Tangai

The nucleotide sequence obtained from 18S rRNA of the local land race―Ghata Zira Tangai (Gzt)‖ Gzt-1 was BLAST in NCBI for molecular characterizations. The blast result showed 97% maximum identity and query cover with Pyrus communis cv. Pachkan's Triumph, P. communis cv. Beurre, P. communis cv. Conference, P. pyrifolia cv. Okusankichi. With other related genera i.e Malus domestica and Prunus persica cv. Nunomewase has 97% maximum identity and query cover. For further identification, differentiation and phylogenetic relationship of the land race of ―Ghata Zira Tangai‖ with other related species and genera was inferred by using Maximum Likelihood method (Jukes et al., 1969). A total of 41 sequences of 18S rRNA of related species of five different genera were retrieved from GenBank and were subjected for construction of maximum likelihood tree. The aligned datasheet had maximum 1850 characters which were trimmed for extra and ambiguously aligned fragments both from the 5‘ and 3‘ ends of alignment data sheet. As a result only 1170 characters were used for further analysis. Out of the analysed characters, 235 were conserved, 258 were variable, 225 were parsimony-informative and 33 were singleton sites. None of the characters were excluded from the final analysis and multistate taxa were interpreted as uncertain. The phylogenetic tree (Fig. 3.22) segregated the data into two major clades, Clade I and II. Clade I was further divided into two sub clades, sub clade I and II. Sub clade I consisted of 19 Pyrus species/cultivars viz. Pyrus communis cultivars Cascade, Beurre and Pachkan's Triumph, P. pyrifolia cultivars Soowhang, Sunwhang, Miwhang, Imamuraaki, P. communis cv. Conference, P. pyrifolia cultivars Kimitsukawase, Yeoungsanbae, Shinil, Nijisseiki, Chojuro, Nijisseiki, Okusankichi, Whangkeumbae, Minibae, Kosui and Mansoo and our local land race Ghata Zira Tangai (Gzt-1), respectively. The sub clades II, III, IV and V comprised the related genera of the genus Pyrus i.e Prunus, Cydonia, Rubus and Frageria belonging to the family Rosaceae.

109

Pyrus _communis _cultivar _Cascade _ AF195618.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Soowhang _ AY168824.1 0.00000 0.00000 Pyrus _communis _cultivar _Beurre _ AF195617.1 0.00000 0.00000 Pyrus _communis _cultivar _Pachkan's _Triumph _ AF195621.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Sunwhang _ AF179398.1 0.00434 0.00000 Pyrus _pyrifolia _cultivar _Miwhang _ AF179392.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Imamuraaki _ AF179387.1 0.00000 0.00000 Pyrus _communis _cultivar _Conference _ AF195619.1 0.00000 0.00434 Pyrus _pyrifolia _cultivar _Wonwhang _ AY168822.1 0.00000 Pyrus _pyrifolia _cultivar _Kimitsukawase _ AF179388.1 0.00000 Sub_clade_I 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Yeoungsanbae _ AF179400.1 0.00000 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Shinil _ AF179396.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Nijisseiki _ AF179394.1 0.00434 Clade_I 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Chojuro _ AF179383.1 0.00434 Pyrus _pyrifolia _cultivar _Okusankichi _ AF179395.1 0.00434 0.00000 Pyrus _pyrifolia _cultivar _Whangkeumbae - AF179399.1 0.00434 0.00000 Pyrus _pyrifolia _cultivar _Minibae _ AF179391.1 0.00434 Pyrus _pyrifolia _cultivar _Kosui _ AF179389.1 0.00000 0.00871 Pyrus _pyrifolia _cultivar _Mansoo _ AF179390 0.00435 0.00434 Ghata Zira Tangai (Gzt-1) 0.04459 0.47097 Prunus_persica_cultivar_Changhowonhwangdo_AY179514.1 0.00000 84 Prunus_persica_cultivar_Baekmijosaeng_AY179513.1 0.00000 0.00436 0.00000 Prunus_persica_cultivar_Hakuho_AY179529.1 0.00000 Sub_clade_II 0.00000 Prunus_persica_cultivar_Youngseonghwangdo_AY179522.1 0.00000 0.00000 Prunus_persica_cultivar_Kuemjeokbaekdo_AY179519.1 0.00434 0.00000 Prunus_persica_cultivar_Cheonhong_AY179515.1 0.00000 Cydonia_oblonga_AF186531.1 0.00000 0.00000 Cydonia_oblonga_voucher829-84D_JQ392421.1 0.00000 0.00000 100 Cydonia_oblonga_JQ392420.1 Sub_clade_III 0.00000 0.08312 Cydonia_oblonga_AB636344.1 0.00000 0.00000 Cydonia_oblonga_JQ392422.1 0.00000 Rubus_caesius_Rubus_idaeus_AF362709.1 100 0.00000 800.00000 Rubus_caesius_Rubus_idaeus_AF362708.1 0.35740 0.00000 0.00000 69 Rubus_picticaulis_clone_29_AF362726.1 Sub_clade_IV 0.00000 Clade_II 0.03841 Rubus_coreanus_isolateP677_JF980336.1 0.00871 61 0.00000 Rubus_crataegifolius_clone_KC10-2_GU980798.1 0.07250 0.01310 Fragaria_vesca_AF163510.1 100 0.00434 Fragaria_moschata_AF163505.1 0.08567 64 0.00000 Fragaria_virginiana_AF163479.1 0.00000 0.00000 Sub_clade_V Fragaria_nubicola_AF163517.1 0.00000 0.00434 0.00000 Fragaria_chiloensis_AF163482.1 0.00000 0.00000 Fragaria_chiloensis_AF163511.1 0.00000

Fig. 3.22. Phylogenetic analysis of ―Ghata Zira Tangai (Gzt-1)‖ by Maximum

Likelihood Method (Jukes et al., 1969) at1000 bootstrap replications.

110

3. 4. 5. Nashpati

For molecular evaluation, the 18s rRNA nucleotide sequence of the candidate land race ―Nashpati‖ was initially BLAST searched in NCBI. The BLAST result showed 99% maximum identity and query cover with Pyrus. pyrifolia cultivars Sunwhang, Miwhang Miwhang and Imamuraaki.

For further confirmation and identification, phylogenetic relationship of the land race ―Nashpati‖ with other related species and genera was inferred on the bases of Maximum Likelihood method (Jukes et al., 1969). A total of 41 sequences of 18S rRNA of related species were retrieved from GenBank and were subjected for construction of maximum likelihood tree. The aligned datasheet had maximum 1850 characters, which were trimmed for extra and ambiguously aligned fragments both from 5‘ and 3‘ ends of alignment data sheet. Finally, a total of 1223 characters were used for further analysis. Out of the analysed characters, 245 were conserved, 231 were variable, 208 were parsimony-informative and 23 were singleton sites. None of the characters were excluded from the final analysis and multistate taxa were interpreted as uncertainty.

The evolutionary history was derived by using the Maximum Likelihood based on the Jukes-Cantor model (Jukes et al., 1969). The tree with the highest log likelihood (- 885.1550) shown in Fig. 3.23. The percentage of trees in which the associated taxa clustered together was shown next to the branches. Initial tree‘s for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. The analysis comprises 40 nucleotide sequences and all positions containing gaps and missing data were eliminated.

Phylogenetic tree (Fig.3.23) was grouped into two major clades, Clade I and clade II. Clade I was further subdivided into two sub clades, sub clade I and sub clade II.

111

Sub clade I comprised 19 Pyrus species/cultivars belong ing to P. communis cultivars Cascade, Conference and Pachkan's Triumph, P. pyrifolia cultivars Imamuraaki, Soowhang, Miwhang, Wonwhang, Sunwhang, Chojuro, Kimitsukawase, Yeoungsanbae, Shinil, Nijisseiki, Whangkeumba, Okusankichi, Chojuro,Minibae, and our local landrace Nashpati, respectively.

Our local land race “Nashpati”, fall in Clade I and showed close relationship with its similar species/cultivars of P. pyrifolia cv. Minibae.While Sub clade II comprised 6 species/cultivars of the genus Prunus. The clade II was sub divided into three sub clades, sub clade III, IV and V which comprisesd related genera belonging to family Rosaceae.

112

Pyrus _communis _cultivar _Cascade _ AF195618.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Soowhang _ AY168824.1 0.00000 0.00000 Pyrus _communis _cultivar _Beurre _ AF195617.1 0.00000 0.00000 Pyrus _communis _cultivar _Conference _ AF195619.1 0.00000 0.00000 Pyrus _communis _cultivar _Pachkan's _Triumph _ AF195621.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Imamuraaki _ AF179387.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Miwhang _ AF179392.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Sunwhang _ AF179398.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Wonwhang _ AY168822.1 0.00000 Pyrus _pyrifolia _cultivar _Chojuro _ AF179383.1 Sub_clade_I 0.00000 0.00504 Pyrus _pyrifolia _cultivar _Kimitsukawase _ AF179388.1 0.00000 0.00000 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Yeoungsanbae _ AF179400.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Shinil _ AF179396.1 Clade_I 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Nijisseiki _ AF179394.1 0.00504 0.00000 Pyrus _pyrifolia _cultivar _Kosui _ AF179389.1 0.00504 Pyrus _pyrifolia _cultivar _Okusankichi _ AF179395.1 0.00000 0.00504 Pyrus _pyrifolia _cultivar _Whangkeumbae - AF179399.1 0.00000 0.00504 Pyrus _pyrifolia _cultivar _Minibae _ AF179391.1 0.00000 0.00504 Nashpati 0.00504 0.00504 Prunus_persica_cultivar_Kuemjeokbaekdo_AY179519.1 0.00000 86 Prunus_persica_cultivar_Youngseonghwangdo_AY179522.1 0.01012 0.00000 Prunus_persica_cultivar_Changhowonhwangdo_AY179514.1 0.00000 0.00000 Sub_clade_II 0.54655 Prunus_persica_cultivar_Baekmijosaeng_AY179513.1 0.00000 0.00000 0.00000 Prunus_persica_cultivar_Hakuho_AY179529.1 0.00000 0.00000 Prunus_persica_cultivar_Cheonhong_AY179515.1 0.00000 Pyrus _pyrifolia _cultivar _Mansoo _ AF179390 0.00000 Cydonia_oblonga_AF186531.1 0.00000 0.00000 Cydonia_oblonga_voucher829-84D_JQ392421.1 0.00000 0.00000 99 Cydonia_oblonga_JQ392420.1 Sub_clade_III 0.00000 0.08627 Cydonia_oblonga_AB636344.1 0.00000 0.00000 Cydonia_oblonga_JQ392422.1 0.00000 Rubus_caesius_Rubus_idaeus_AF362709.1 100 0.00000 790.00000 Rubus_caesius_Rubus_idaeus_AF362708.1 0.37149 0.00000 0.00000 76 Rubus_picticaulis_clone_29_AF362726.1 Sub_clade_IV 0.00000 Clade_II 0.04547 Rubus_coreanus_isolateP677_JF980336.1 0.01012 74 0.00000 Rubus_crataegifolius_clone_KC10-2_GU980798.1 0.01523 0.09124 Fragaria_virginiana_AF163479.1 0.00000 100 Fragaria_moschata_AF163505.1 0.00000 0.09394 0.00000 Fragaria_vesca_AF163510.1 0.00504 Sub_clade_V 0.00000 Fragaria_nubicola_AF163517.1 0.00504 0.00000 Fragaria_chiloensis_AF163482.1 0.00000 0.00000 Fragaria_chiloensis_AF163511.1 0.00000

Fig.3.23. Phylogenetic analysis of “Nashpati”, by Maximum Likelihood Method (ML)

(Jukes et al., 1969) with 1000 bootstrap replications.

113

3. 4. 6 Parawoo Tango

For molecular characterizations, the nucleotide sequence obtained from the 18S rRNA of the local land race ― Parawoo Tango‖ (Acc. No. Pt-1) was BLAST searched in NCBI. The BLAST result showed 99% maximum identity and query cover with Pyrus comamunis cultivars Pachkan's Triumph, Beurre, cascade, Clapp's Favorite and P. pyrifolia cv. Sunwhang. Similarly with other related genera such as with Malus domestica showed 99% maximum identity and 99% query cover and with Prunus persica cv. sungold showed 99% maximum identity and 99% query cover. For further confirmation, the phylogenetic relationship of the local land race Parawoo Tango Pt with other related species and genera was inferred by Maximum Likelihood method (Jukes et al., 1969). A total of 41 sequences of 18S rRNA of related species were retrived from GenBank and subjected to the organization of maximum likelihood tree. The aligned data sheet had maximum 1850 characters and after trimming the extra and ambiguously aligned fragments from both 5‘ and 3‘ ends of alignment data sheet. A total of 1159 characters were used for further analysis. Out of these traits, 258 were conserved, 259 were variable, 230 were parsimony-informative and 29 were singleton sites. None of the characters were excluded from the final analysis and multistate taxa were interpreted as uncertainty.

The evolutionary history was inferred by using the Maximum Likelihood (ML) method based on the Jukes-Cantor model (Jukes et al., 1969). The tree with the highest log likelihood (-1010.9077) is shown in Fig. 3.24. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. The analysis comprised 38 nucleotide sequences and all positions containing gaps and missing data were eliminated. The phylogenetic tree (Fig.3.24) thus organized was divided into two major clades, Clade I and II. Clade I is further sub divided into two sub clades, sub clade I, sub clade II.

114

The sub clade I consist of 19 Pyrus species/cultivars viz. P. pyrifolia cv. Sunwhang, our ―Parawoo Tango‖, P. pyrifolia cultivars Shinil, Yeoungsanbae, Chojuro, Kimitsukawase, Kosui, Minibae, Nijisseiki, Okusankichi, Wonwhang, Soowhang, P. communis cultivars Beurre and Pachkan's Triumph, P. pyrifolia cultivars Miwhang, Imamuraaki, communis Conference and Whangkeumbae, respectively.

Our local pear land race ―Parawoo Tango‖ fall in Clade I and showed close relationship with its similar species/cultivars of P. pyrifolia cvs. Sunwhang and Shinil, while Sub clade II consist of 6 species/cultivars of the genus Prunus. Clade II consist of three sub clades, sub clade III, sub clade IV and sub clade V comprised related genera belonging to family Rosaceae

115

84 Pyrus pyrifolia cultivar Sunwhang AF179398.1 0.0043 850.0000 Parawoo Tango 0.0000 850.0000 Pyrus pyrifolia cultivar Shinil AF179396.1 840.0000 0.0000 Pyrus pyrifolia cultivar Yeoungsanbae AF179400.1 840.0000 0.0000 Pyrus pyrifolia cultivar Chojuro AF179383.1 500.0000 0.0043 Pyrus pyrifolia cultivar Kimitsukawase AF179388.1 700.0000 0.0000 Pyrus pyrifolia cultivar Kosui AF179389.1 710.0000 0.0086 Pyrus pyrifolia cultivar Mansoo AF179390 660.0000 0.0043 Pyrus pyrifolia cultivar Minibae AF179391.1 500.0000 0.0043 Pyrus pyrifolia cultivar Nijisseiki AF179394.1 710.0000 0.0043 Sub_Clade_I Pyrus pyrifolia cultivar Okusankichi AF179395.1 710.0000 0.0043 Pyrus pyrifolia cultivar Whangkeumbae - AF179399.1 710.0000 0.0043 Pyrus pyrifolia cultivar Wonwhang AY168822.1 710.0000 0.0000 Clade_I Pyrus pyrifolia cultivar Soowhang AY168824.1 710.0000 0.0000 Pyrus communis cultivar Cascade AF195618.1 710.0000 0.0000 Pyrus communis cultivar Beurre AF195617.1 710.0000 0.0000 Pyrus communis cultivar Conference AF195619.1 00.0000 0.0043 Pyrus communis cultivar Pachkan's Triumph AF195621.1 720.0000 0.0000 Pyrus pyrifolia cultivar Miwhang AF179392.1 0.0043 0.0000 Pyrus pyrifolia cultivar Imamuraaki AF179387.1 0.0000 0.5148 Prunus persica cultivar Youngseonghwangdo AY179522.1 55 0.0000 Prunus persica cultivar Cheonhong AY179515.1 0.0043 98 0.0000 Prunus persica cultivar Hakuho AY179529.1 0.000099 0.0000 Sub_Clade_II Prunus persica cultivar Baekmijosaeng AY179513.1 0.0000100 0.0000 0.000099 Prunus persica cultivar Kuemjeokbaekdo AY179519.1 0.0043 0.0000 Prunus persica cultivar Changhowonhwangdo AY179514.1 0.0000 Rubus coreanus isolateP677 JF980336.1 0.0043 53 Rubus picticaulis clone 29 AF362726.1 0.0000 530.0000 Rubus crataegifolius clone KC10-2 GU980798.1 0.0129 Sub_Clade_IV 100 500.0000 Rubus caesius Rubus idaeus AF362708.1 0.3287 0.0000 0.0000 Rubus caesius Rubus idaeus AF362709.1 0.0000 99 Cydonia oblonga AB636344.1 0.0000 14 980.0000 Cydonia oblonga voucher829-84D JQ392421.1 0.0000 0.0043 970.0000 Cydonia oblonga AF186531.1 Sub_Clade_III 1000.0000 0.0000 Clade_II Cydonia oblonga JQ392420.1 0.1494 0.0000 37 Cydonia oblonga JQ392422.1 0.0000 0.0454 Fragaria virginiana AF163479.1 100 0.0000 Fragaria nubicola AF163517.1 0.078038 0.0043 Fragaria chiloensis AF163482.1 0.000099 0.0000 Sub_Clade_V Fragaria vesca AF163510.1 0.0000100 0.0043 0.0000100 Fragaria moschata AF163505.1 0.0000 0.0000 Fragaria chiloensis AF163511.1 0.0000

Fig.3.24. Phylogenetic analysis of Parawoo Tango by Maximum Likelihood Method

(ML) (Jukes et al., 1969) with 1000 bootstrap replications.

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3. 4. 7 Pekhawry Tango

The nucleotide sequence of 18S rRNA of the local pear land race ―Pekhawry Tango‖ (Acc. No. Pkt-1) was initially BLAST searched in NCBI for molecular evaluation. The BLAST result showed 97% maximum identity and query cover with Pyrus communis cv. Pachkans Triumph, P. communis cv. Beurre. The blast result also showed 97% maximum identity and 94% query cover with P. pyrifolia cv. Imamuraaki. Similarly the maximum identity was 96% and query cover was 94% with P. pyrifolia cultivars Sunwhang and Okusankichi, P. communis cv. Clapp's Favorite while with other related genera, the BLAST result showed that the maximum identity was 96% and query cover was 94% with Malus domestica and similar result showed for other species/cultivars of the same genus. For further identification, phylogenetic relationship of the local pear land race ―Pekhawry Tango‖ with other related species and genera was inferred by using Maximum Likelihood method (Jukes et al., 1969). A total of 41 sequences of 18S rRNA of related species of five different genera were retrieved from GenBank and subjected for construction of maximum likelihood tree. The aligned datasheet had maximum 1850 characters which were trimmed for extra and ambiguously aligned fragments both from 5‘ and 3‘ ends of alignment data sheet. As a result only 1207 characters were used for further analysis. Out of the analysed traits, 259 characters were conserved, 236 were variable, 210 were parsimony-informative and 26 were singleton sites. None of the characters were excluded from the final analysis and multistate taxa were interpreted as uncertainty.

The evolutionary history was derived by using the ML method based on the Jukes- Cantor model (Jukes et al., 1969). The tree with the highest log likelihood (-868.3724) is shown in Fig. 3.25. The percentage of trees in which the associated taxa clustered together was shown next to the branches. Initial tree‘s for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. The

117

analysis comprised 40 nucleotide sequences and all positions containing gaps and missing data were eliminated.

The phylogenetic tree (Fig. 3.25) was divided into two major clades, Clade I and clade II. Clade I is further divided into two sub clades, sub clade I and sub clade II. The sub clade I contained 16 Pyrus species/cultivars belong to P. pyrifolia cultivars Kosui,. Chojuro, Kimitsukawase, Nijisseiki, Yeoungsanbae, Wonwhang and Soowhang, P. communis cultivars Cascade, Cascade, Conference and Pachkan's Triumph, P. pyrifolia cultivars Imamuraaki, Miwhang, Sunwhang, Shinil and Okusankichi, respectively.

Our local pear land race ―Pekhawry Tango” developing a separate sub clade III with closely related species/cultivars such as Pyrus pyrifolia cv. Mansoo. Similarly Sub clade II consist of 6 species/cultivars of the genus Prunus. Clade II consist of three sub clades, sub clade IV, sub clade V and sub clade VI comprised related genera of the family Rosaceae.

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Pyrus pyrifolia cultivar Sunwhang AF179398.1 0.0000 0.0000 Pyrus pyrifolia cultivar Shinil AF179396.1 0.0000 0.0000 Pyrus pyrifolia cultivar Yeoungsanbae AF179400.1 0.0000 0.0000 Pyrus pyrifolia cultivar Chojuro AF179383.1 0.0000 0.0051 Pyrus pyrifolia cultivar Kimitsukawase AF179388.1 0.0000 0.0000 Pyrus pyrifolia cultivar Minibae AF179391.1 0.0051 0.0000 0.0000 Pyrus pyrifolia cultivar Kosui AF179389.1 0.0051 Pyrus pyrifolia cultivar Nijisseiki AF179394.1 0.0000 0.0051 Pyrus pyrifolia cultivar Okusankichi AF179395.1 0.0000 0.0051 Sub_Clade_I Pyrus pyrifolia cultivar Whangkeumbae - AF179399.1 0.0000 0.0051 Pyrus pyrifolia cultivar Wonwhang AY168822.1 0.0000 0.0000 Pyrus pyrifolia cultivar Soowhang AY168824.1 0.0000 0.0000 Clade_I Pyrus communis cultivar Cascade AF195618.1 0.0000 0.0000 Pyrus communis cultivar Beurre AF195617.1 0.0000 0.0000 Pyrus communis cultivar Conference AF195619.1 0.0000 0.0000 Pyrus communis cultivar Pachkan's Triumph AF195621.1 0.0000 0.0000 Pyrus pyrifolia cultivar Imamuraaki AF179387.1 0.0000 0.0000 Pyrus pyrifolia cultivar Miwhang AF179392.1 0.0000 0.0000 Prunus persica cultivar Youngseonghwangdo AY179522.1 0.0000 Prunus persica cultivar Cheonhong AY179515.1 0.0051 0.0000 Prunus persica cultivar Hakuho AY179529.1 0.0000 0.0000 Sub_Clade_II 0.0130 Prunus persica cultivar Baekmijosaeng AY179513.1 0.0000 0.0000 0.0000 Prunus persica cultivar Kuemjeokbaekdo AY179519.1 0.0000 0.0000 Prunus persica cultivar Changhowonhwangdo AY179514.1 0.5319 0.0000 Pyrus pyrifolia cultivar Mansoo AF179390 Sub_Clade_III 0.0051 Pekhawary Tango 0.0130 Rubus coreanus isolateP677 JF980336.1 0.0051 0.0000 Rubus crataegifolius clone KC10-2 GU980798.1 0.0155 0.0000 Rubus caesius Rubus idaeus AF362708.1 Sub_Clade_IV 0.0000 0.0000 Rubus caesius Rubus idaeus AF362709.1 0.0000 0.0000 Rubus picticaulis clone 29 AF362726.1 0.0000 Cydonia oblonga AB636344.1 0.0000 0.0000 Cydonia oblonga voucher829-84D JQ392421.1 0.0000 0.3321 0.0000 Cydonia oblonga AF186531.1 Sub_Clade_V 0.0000 0.0000 Clade_II Cydonia oblonga JQ392420.1 0.1650 0.0000 Cydonia oblonga JQ392422.1 0.0000 0.0530 Fragaria nubicola AF163517.1 0.0051 Fragaria virginiana AF163479.1 0.0844 0.0000 Fragaria chiloensis AF163482.1 0.0000 0.0000 Sub_Clade_VI Fragaria vesca AF163510.1 0.0000 0.0051 0.0000 Fragaria moschata AF163505.1 0.0000 0.0000 Fragaria chiloensis AF163511.1 0.0000

Fig.3.25. Phylogenetic analysis of “Pekhawry Tango‖ by Maximum Likelihood Method, ML (Jukes et al., 1969) with 1000 bootstrap replications.

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3.4. 8 Nak Tango

For molecular characterizations, the nucleotide sequence obtained from 18S rRNA gene of the pear land race ―Nak Tango‖ (Acc. No. Nt-1) was initiallyBLAST in NCBI. The blast result showed 97% maximum identity and 87% query cover with Pyrus communis cultivars Pachkan's Triumph, Conference, Beurre, Cascade and P. pyrifolia cv. Okusankichi.

The phylogenetic relationship of the local pear land race ―Nak Tango (Nk)‖ with other related species and genera was obtained from Maximum Likelihood method (Jukes et al., 1969). A total of 41 nucleotide sequences of 18S rRNA gene of related species were retrieved from GenBank and subjected for generation of maximum likelihood tree. The aligned datasheet had maximum 1850 characters and after trimming the extra and ambiguously aligned fragments from both 5‘ and 3‘ ends of alignment data sheet. A total of 1207 characters were considered for analysis. Out of the analysed characters, 248 were conserved, 241 were variable, 209 were parsimony-informative and 32 were singleton sites. None of the characters were excluded from the final analysis and multistate taxa were interpreted as uncertainty.

The evolutionary history was derived by using the Maximum Likelihood (ML) method based on the Jukes-Cantor model (Jukes et al., 1969). The tree with the highest log likelihood (-910.4194) is shown in Fig.3.26. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree‘s for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. The analysis comprises 41 nucleotide sequences and all positions containing gaps and missing data were eliminated. The phylogenetic tree (Fig.3.26) thus organized was divided into two major clades, Clade I and II. The clade I was further sub divided into three sub clades, sub clade I, II and III. The sub clade I consist of 16 P. species/cultivars belong to P. pyrifolia cultivars Wonwhang, Nijisseiki, Soowhang, P. communis cultivars Cascade, Beurre, Conference,

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Pachkan's Triumph, P. pyrifolia cultivars Imamuraaki, Miwhang,. Sunwhang, Kimitsukawase, Yeoungsanbae, Shinil, Kosui, Chojuro, Okusankichi, Mansoo and Minibae, respectively.

The sub clade II comprised 6 species/cultivars belonging to the genus Prunus. The local pear land race Nak Tango (Nk) was fall independently in between the sub clade II (Prunus species) and sub clade IV (Cydonia species), while the sub clade V and sub clade VI consists of Rubus and Frageria species.

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Pyrus _pyrifolia _cultivar _Wonwhang _ AY168822.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Nijisseiki _ AF179394.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Soowhang _ AY168824.1 0.00000 0.00000 Pyrus _communis _cultivar _Cascade _ AF195618.1 0.00000 0.00000 Pyrus _communis _cultivar _Beurre _ AF195617.1 0.00000 0.00000 Pyrus _communis _cultivar _Conference _ AF195619.1 0.00000 0.00000 Pyrus _communis _cultivar _Pachkan's _Triumph _ AF195621.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Imamuraaki _ AF179387.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Miwhang _ AF179392.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Sunwhang _ AF179398.1 Sub_clade_I 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Kimitsukawase _ AF179388.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Yeoungsanbae _ AF179400.1 0.00000 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Shinil _ AF179396.1 Clade_I 0.00000 Pyrus _pyrifolia _cultivar _Kosui _ AF179389.1 0.00534 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Chojuro _ AF179383.1 0.00534 Pyrus _pyrifolia _cultivar _Whangkeumbae - AF179399.1 0.00000 0.00534 0.00000 Pyrus _pyrifolia _cultivar _Okusankichi _ AF179395.1 0.00534 0.00000 Pyrus _pyrifolia _cultivar _Minibae _ AF179391.1 0.00534 64 Pyrus _pyrifolia _cultivar _Mansoo _ AF179390 0.00534 0.02273 Prunus_persica_cultivar_Kuemjeokbaekdo_AY179519.1 86 0.00000 Prunus_persica_cultivar_Youngseonghwangdo_AY179522.1 0.01071 0.00000 Prunus_persica_cultivar_Changhowonhwangdo_AY179514.1 0.00000 0.00000 Sub_clade_II 0.47194 Prunus_persica_cultivar_Baekmijosaeng_AY179513.1 0.00000 0.00000 0.00000 Prunus_persica_cultivar_Hakuho_AY179529.1 0.00000 0.00000 Prunus_persica_cultivar_Cheonhong_AY179515.1 0.00000 Nak Tango Sub_clade_III 0.09823 Cydonia_oblonga_AF186531.1 0.00000 0.00000 Cydonia_oblonga_voucher829-84D_JQ392421.1 0.00000 0.00000 Cydonia_oblonga_JQ392420.1 Sub_clade_IV 0.00000 0.00000 99 Cydonia_oblonga_JQ392422.1 0.07451 0.00000 Cydonia_oblonga_AB636344.1 0.00000 Rubus_caesius_Rubus_idaeus_AF362709.1 0.00000 100 810.00000 Rubus_caesius_Rubus_idaeus_AF362708.1 0.00000 0.37796 770.00000 Rubus_picticaulis_clone_29_AF362726.1 Sub_clade_V 0.00000 0.00000 Clade_II 80 Rubus_coreanus_isolateP677_JF980336.1 0.04972 0.01071 72 Rubus_crataegifolius_clone_KC10-2_GU980798.1 0.01613 0.10071 Fragaria_vesca_AF163510.1 100 0.00534 Fragaria_moschata_AF163505.1 0.0861660 0.00000 Fragaria_virginiana_AF163479.1 0.00000 0.00000 Sub_clade_VI Fragaria_nubicola_AF163517.1 0.00000 0.00534 0.00000 Fragaria_chiloensis_AF163482.1 0.00000 0.00000 Fragaria_chiloensis_AF163511.1 0.00000

Fig.3.26 Phylogenetic analysis of “Nak Tango‖, by Maximum Likelihood Method

(Jukes et al., 1969) at 1000 bootstrap replications.

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3. 4. 9 Spina Mamothy

The nucleotide sequence of 18S rRNA of local pear land race ―Spina Mamothy‖ (Acc. No. Sm-1) was obtained and BLAST searched in NCBI for molecular characterizations. The BLAST result showed 98% maximum identity and 100% query cover with Pyrus communis cultivars Pachkan's Triumph, Conference, Beurre, Beurre, P. pyrifolia cultivars Sunwhang and Miwhang.

For further conformation and identification, phylogenetic relationship of the local land race ―Spina Mamothy‖ (Sm-1) with other related species and genera was inferred by using Maximum Likelihood method (Jukes et al., 1969). A total of 37 sequences of 18S rRNA of related species and genera were retrieved from GenBank and were subjected for construction of maximum likelihood tree. The aligned datasheet had maximum 1850 characters which were trimmed for extra and ambiguously aligned fragments both from 5‘ and 3‘ ends of alignment data sheet. A total of 1209 genetic characters were used for further analysis. Out of the analysed characters, 257 characters were conserved, 236 were variable, 209 were parsimony-informative and 27 were singleton sites. None of the characters were excluded from the final analysis and multistate taxa were interpreted as uncertainty.

The evolutionary history was inferred by using the ML method based on the Jukes- Cantor model (Jukes et al., 1969). The bootstrap consensus tree inferred from 1000 replicates (Felsenstein J. 1985) was taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Initial tree‘s for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value.

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Phylogenetic tree (Fig.3.27) thus organized was divided into two major clades, Clade I and clade II. The clade I was further sub divided into two sub clades, sub clade I and sub clade II.

The sub clade I consists of 14 P. species/ cvultivars viz. P. pyrifolia cultivars Sunwhang, Miwhang, Imamuraaki, P. communis cultivars Pachkan's Triumph, Conference, Beurre, ascade, P. pyrifolia cultivars Shinil, Chuwhangbae Shinsui, Niitaka, P. communis cv. Clapp's Favorite and our local pear land race, ―Spina Mamothy‖, respectively.

Our local pear land race ―Spina Mamothy‖ (Sm-1) fall in Clade I and showed close relationship with its similar species/cultivars of P. communis cv. Clapp's Favorite while Sub clade II consist of 7 species/cultivars of the genus Prunus. Clade II consist of three sub clades, sub clade III, sub clade IV and sub clade V comprised of related genera belonging to family Rosaceae.

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Pyrus_pyrifolia_cultivar_Sunwhang_AF179398.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Miwhang_AF179392.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Imamuraaki_AF179387.1 0.00000 0.00000 Pyrus_communis_cultivar_Pachkan's_Triumph_AF195621.1 0.00000 0.00000 Pyrus_communis_cultivar_Conference_AF195619.1 0.00000 0.00000 Pyrus_communis_cultivar_Beurre_AF195617.1 0.00000 0.00000 Sub_clade_I Pyrus_communis_cultivar_Cascade_AF195618.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Shinil_AF179396.1 0.00000 Pyrus_pyrifolia_cultivar_Chuwhangbae_AF179384.1 0.00000 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Shinsui_AF179397.1 0.00000 0.00000 0.00000 Pyrus_communis_cultivar_Clapp's_Favorite_AF195620.1 Clade_I 0.00000 Spina Mamothy_Sm 0.00000 0.01485 Prunus_persica_cultivar_Wolmiboksunga_AY179521.1 87 0.00000 Prunus_persica_cultivar_Kuemjeokbaekdo_AY179519.1 0.00987 0.00000 Prunus_persica_cultivar_Youngseonghwangdo_AY179522.1 0.00000 0.00000 Prunus_persica_cultivar_Changhowonhwangdo_AY179514.1 Sub_clade_II 0.00000 0.00000 0.00000 Prunus_persica_cultivar_Baekmijosaeng_AY179513.1 0.00000 0.00000 Prunus_persica_cultivar_Hakuho_AY179529.1 0.00000 0.00000 0.00492 0.00000 Prunus_persica_cultivar_Cheonhong_AY179515.1 0.00000 Pyrus_pyrifolia_cultivar_Niitaka_AF179393.1 0.00492 0.51794 Pyrus_pyrifolia_cultivar_Gamcheonbae_AF179385.1 0.00492 Pyrus_pyrifolia_cultivar_Choju_AF179382.1 0.00000 Cydonia_oblonga_AF186531.1 0.00000 0.00000 Cydonia_oblonga_voucher829-84D_JQ392421.1 0.00000 0.00000 Cydonia_oblonga_JQ392420.1 Sub_clade_III 0.00000 0.00000 99 Cydonia_oblonga_JQ392422.1 0.10008 0.00000 Cydonia_oblonga_AB636344.1 0.00000 Rubus_caesius_Rubus_idaeus_AF362709.1 0.00000 99 64 0.00000 Rubus_caesius_Rubus_idaeus_AF362708.1 0.00000 0.35410 70 0.00000 Rubus_picticaulis_clone_29_AF362726.1 Sub_clade_IV 0.00000 0.00000 Clade_II 68 Rubus_coreanus_isolateP677_JF980336.1 0.04314 0.00987 61 Rubus_crataegifolius_clone_KC10-2_GU980798.1 0.01485 0.08000 Fragaria_vesca_AF163510.1 99 0.00492 Fragaria_moschata_AF163505.1 0.09869 61 0.00000 Fragaria_virginiana_AF163479.1 0.00000 0.00000 Sub_clade_V Fragaria_chiloensis_AF163482.1 0.00000 0.00000 Fragaria_nubicola_AF163517.1 0.00000 0.00492 0.00000 Fragaria_chiloensis_AF163511.1 0.00000

Fig.3.27. Phylogenetic analysis of Spina Mamothy by Maximum Likelihood Method (ML) (Jukes et al., 1969) at 1000 bootstrap replications.

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3. 4. 10 Asmasy Tango

For molecular characterization, the nucleotide sequence obtained from 18S rRNA of the local Pear land race ―Asmasy Tango‖ having Acc. No At was initially BLAST (Altschul et al., 1990) searched in NCBI. The BLAST result showed 99% maximum identity and 100% query cover with Pyrus communis cultivars Pachkans Triumph, Beurre and

Cascade. Further identification and plogenetic relationship of the local pear land race ―Asmasy Tango‖ with other related species and genera was inferred by using maximum likelihood method (Jukes et al., 1969). A total of 36 sequences of 18S rRNA of related species and genera were retrieved from GenBank and used for maximum likelihood tree. The aligned datasheet has a 1185 characters which were trimmed for extra, durtty and ambiguously aligned fragments both from 5‘ and 3‘ ends of alignment data sheet. As a result only 438 characters were considered for further analysis. Out of the analyzed characters, 59 were conserved, 462 were variable, 290 were parsimony- informative and 171 were singleton sites. None of the characters were excluded from the final analysis and multistate taxa were interpreted as uncertainty.

The evolutionary history was concluded by using the Maximum Likelihood (ML) method based on the Jukes-Cantor model (Jukes et al., 1969). The tree with the highest log likelihood (-970.8643) is shown in Fig. 3.28. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The analysis involved 37 nucleotide sequences. All positions containing gaps and missing data were eliminated. The phylogenetic tree (Fig.3.28) thus obtained was segregated the data into three major clades, clade I comprised of Pyrus+Prunus, clade II consisted of Cydonia and clade III constained Rusbus+Frageria. Clade I, Pyrus+Prunus was further sub divided into two sub clades, sub clade Pyrus and sub clade Prunus. Sub clade Pyrus consists of 14 cultivars belonging to Pyrus communis and P. pyrifolia. The P. communis cultivars consists

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of Pachkan's Triumph, Beurre, cascade, Clapp's Favorite, Conference, P. pyrifolia cultivars consists of Shinsui, Shinil, Chuwhangbae, Miwhang, Imamuraaki, Sunwhang, Niitaka, Gamcheonbae and Choju and the local land race, Asmasy Tango fall in Pyrus clade with its similar species/cultivars, Pyrus communis cv. Beurre.

The sub clade Prunus comprised of seven species i.e Prunus persica cultivars Kuemjeokbaekdo, Wolmiboksunga, Youngseonghwangdo, Changhowonhwangdo, Baekmijosaeng, Hakuho and Cheonhong, respectively. The sub clade II Cydonia consisted of five species such as Cydonia oblonga, C. oblonga,C. oblonga, C.oblonga and C. oblonga voucher while the clade III Rubus+Frageria consisted of two generia, Rubus and Frageria. The genus Rubus comprised of five species viz. R. coreanus, R. picticauli, R. caesius,R. caesius, R. idaeus,R. crataegifolius, and S. aucupari while the genus Frageria consist of Fragaria moschata, F. virginiana, F. nubicola, F. chiloensis, F. vesca, and F. chiloensis,respectively.

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Pyrus_pyrifolia_cultivar_Shinsui_AF179397.1 0.00000 0.00000 Pyrus_communis_cultivar_Clapp's_Favorite_AF195620.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Chuwhangbae_AF179384.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Shinil_AF179396.1 0.00000 0.00000 Pyrus_communis_cultivar_Cascade_AF195618.1 0.00000 0.00000 Asmasy Tango_At 0.00000 0.00000 Pyrus_communis_cultivar_Beurre_AF195617.1 0.00000 0.00000 Sub_Clade_I Pyrus_communis_cultivar_Pachkan's_Triumph_AF195621.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Imamuraaki_AF179387.1 0.00674 0.00000 Pyrus_pyrifolia_cultivar_Miwhang_AF179392.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Sunwhang_AF179398.1 0.01348 Clade_I 0.00000 -0.00674 Pyrus_communis_cultivar_Conference_AF195619.1 0.01348 Pyrus_pyrifolia_cultivar_Niitaka_AF179393.1 0.00000 0.00674 Pyrus_pyrifolia_cultivar_Gamcheonbae_AF179385.1 0.00674 -0.00122 Prunus_persica_cultivar_Wolmiboksunga_AY179521.1 88 0.00075 Prunus_persica_cultivar_Kuemjeokbaekdo_AY179519.1 0.00599 0.00149 Prunus_persica_cultivar_Youngseonghwangdo_AY179522.1 -0.00075 0.00000 Prunus_persica_cultivar_Changhowonhwangdo_AY179514.1 Sub_clade_II 0.00149 0.00000 0.46816 Prunus_persica_cultivar_Baekmijosaeng_AY179513.1 0.00000 0.00000 Prunus_persica_cultivar_Hakuho_AY179529.1 0.000000.00000 0.00000 Prunus_persica_cultivar_Cheonhong_AY179515.1 0.00000 Pyrus_pyrifolia_cultivar_Choju_AF179382.1 0.00552 Cydonia_oblonga_AF186531.1 0.00000 0.00000 Cydonia_oblonga_voucher829-84D_JQ392421.1 0.00000 0.00000 Cydonia_oblonga_JQ392420.1 Sub_clade_III 100 0.00000 0.00000 Cydonia_oblonga_JQ392422.1 0.00000 0.11053 Cydonia_oblonga_AB636344.1 0.00000 Rubus_coreanus_isolateP677_JF980336.1 100 0.01813 73-0.00906 Rubus_picticaulis_clone_29_AF362726.1 0.01813 0.36315 0.00000 Rubus_caesius_Rubus_idaeus_AF362709.1 Sub_clade_IV Clade_II 74 0.00000 0.00906 Rubus_caesius_Rubus_idaeus_AF362708.1 0.05541 0.00000 64 Rubus_crataegifolius_clone_KC10-2_GU980798.1 0.00906 0.04606 Fragaria_vesca_AF163510.1 100 0.00149 Fragaria_virginiana_AF163479.1 0.0629866 0.00149 Fragaria_chiloensis_AF163482.1 Sub_clade_V 0.00000 0.00299 Fragaria_nubicola_AF163517.1 -0.001490.00598 -0.00299 Fragaria_chiloensis_AF163511.1 0.00598

Fig. 3.28: Phylogenetic analysis of “Asmasy Tango‖ (Acc. No. At) by Maximum Likelihood Method (Jukes et al., 1969) at 1000 bootstrap replications.

128

3. 4. 11 Mamosay Batal-8

The nucleotide sequence of 18S rRNA of local pear land race ―Mamosay Batal-8‖ having Acc. No. Mamosay B8 was BLAST (Altschul et al., 1990) searched in NCBI for molecular characterization. The blast result showed 97% maximum identity and 100% query cover with Pyrus communis cv. Pachkans Triumph. Similarly, local land race also showed 97% maximum identity and 100% query cover with P. pyrifolia cultivars Imamuraaki and Wonwhang while 96% maximum identity and 100% quary cover with P. pyrifolia cv. Yeoungsanbae. Maximum Likelihood (ML) method (Jukes et al., 1969) was applied to infer the phylogenetic relationship of the local land race ―Mamosay Batal-8‖ with other related species and genera. A total of 37 nucleotide sequences of 18S rRNA of related species belonging to five different genera were retrieved from GenBank and subjected for construction of maximum likelihood cladogram. The aligned datasheet had 1287 genetic characters and after trimming the extra tails and ambiguously aligned fragments from both 5‘ and 3‘ ends of alignment data sheet, a total of 315 characters were used for further analysis. Out of which, 210 characters were conserved, 314 were variable, 289 were parsimony-informative and 25 were singleton sites. None of the characters were excluded from the final analysis and multistate taxa were interpreted as uncertain.

The evolutionary history was inferred by using the Maximum Likelihood (ML) method based on the Jukes-Cantor model (Jukes et al., 1969). The tree with the highest log likelihood (-927.6052) is shown in Fig. 3.29. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree‘s for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The analysis involved 37 nucleotide sequences. All positions containing gaps and missing data were eliminated.

129

The phylogenetic tree (Fig.3.29) thus orgaized was divided into two major clades, Clade I and II. The clade I was further sub divided into two sub clades, sub-Clade I and II. Sub clade I comprisesd of 14 Pyrus species/cultivars viz. P.pyrifolia cultivars Shinsui,. Gamcheonbae, Chuwhangbae, Shinil, P.communis cultivars cascade, Beurre, P. Conference, Pachkan's Triumph, P. pyrifolia cultivars Imamuraaki, Sunwhang, Miwhang, Niitaka, P. communis cv. Clapp's Favorite, respectively. The candidate pear land race ―Mamosay Batal-8‖ fall in clade I with its similar species/cultivar, P. pyrifolia cv. Shinsui.

The sub clade II comprised of seven species/cultivars of Prunus i.e Prunus persica cultivars Kuemjeokbaekdo, Wolmiboksunga, Youngseonghwangdo, Changhowonhwangdo, Baekmijosaeng, Hakuho and Cheonhong, respectively.

The sub clade III comprised of five species/cultivars of Cydonia such as Cydonia oblonga,, C. oblonga, C. oblonga, C.oblonga, and C. oblonga voucher while the sub clade IV consisted of five species of Rubus viz. R. coreanus isolate R. picticaulis, R. caesius, R. caesius, R. idaeus, R. crataegifolius, and R. aucuparia, while the sub clade V consist of Fragaria moschata, F. virginiana, F. nubicola, F. chiloensis, F. vesca and F. chiloensis.

The sub clade IV comprised of five species of Rubus viz. R. coreanus, R. picticaulis, R. caesius, R. caesius, R. idaeus, R. crataegifolius and R. aucuparia while the sub clade V consist of Fragaria moschata, F. virginiana, F. nubicola, F. chiloensis, F. vesca and F. chiloensis.

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Pyrus_pyrifolia_cultivar_Shinsui_AF179397.1 Mamosay Batal-8 Pyrus_pyrifolia_cultivar_Gamcheonbae_AF179385.1 Pyrus_pyrifolia_cultivar_Chuwhangbae_AF179384.1 Pyrus_pyrifolia_cultivar_Shinil_AF179396.1 Pyrus_communis_cultivar_Cascade_AF195618.1 Pyrus_communis_cultivar_Beurre_AF195617.1 Sub_clade_I Pyrus_communis_cultivar_Conference_AF195619.1 Pyrus_communis_cultivar_Pachkan's_Triumph_AF195621.1 Pyrus_pyrifolia_cultivar_Imamuraaki_AF179387.1 Pyrus_pyrifolia_cultivar_Sunwhang_AF179398.1 Clade_I Pyrus_pyrifolia_cultivar_Miwhang_AF179392.1 Pyrus_pyrifolia_cultivar_Niitaka_AF179393.1 Pyrus_communis_cultivar_Clapp's_Favorite_AF195620.1 Prunus_persica_cultivar_Wolmiboksunga_AY179521.1 Prunus_persica_cultivar_Kuemjeokbaekdo_AY179519.1 98 Prunus_persica_cultivar_Youngseonghwangdo_AY179522.1 Prunus_persica_cultivar_Changhowonhwangdo_AY179514.1 Sub_clade_II Prunus_persica_cultivar_Baekmijosaeng_AY179513.1 Prunus_persica_cultivar_Hakuho_AY179529.1 Prunus_persica_cultivar_Cheonhong_AY179515.1 Pyrus_pyrifolia_cultivar_Choju_AF179382.1 Cydonia_oblonga_AF186531.1 Cydonia_oblonga_voucher82984D_JQ392421.1 Cydonia_oblonga_JQ392420.1 Sub_clade_III 96 Cydonia_oblonga_JQ392422.1 Cydonia_oblonga_AB636344.1 Rubus_caesius_Rubus_idaeus_AF362709.1 83 Rubus_caesius_Rubus_idaeus_AF362708.1 100 87 Rubus_picticaulis_clone_29_AF362726.1 Sub_clade_IV Clade_II 94 Rubus_coreanus_isolateP677_JF980336.1 Rubus_crataegifolius_clone_KC102_GU980798.1 99 Fragaria_vesca_AF163510.1 Fragaria_virginiana_AF163479.1 100 Fragaria_nubicola_AF163517.1 Sub_clade_V 65 Fragaria_chiloensis_AF163482.1 Fragaria_chiloensis_AF163511.1

Fig 3.29. Phylogenetic analysis of Mamosy Batal_8 by Maximum Likelihood Method at 1000 bootstrap replications by adopting Jukes_Cantor model

131

3. 4. 12 Mamosay Bat_12

For molecular characterization, the nucleotide sequence obtained from18S rRNA of the local pear land race ―Mamosay Bat_12‖ having accession no (Mamosay B12) was compared to the sequences of the reference library with the help of BLAST (Altschul et al., 1990) in NCBI. The BLAST result showed 97% maximum identity and 100% query cover with Pyrus pyrifolia cultivars Imamuraaki, Soowhang, Wonwhang and Yeoungsanbae while ;2with Malus domestica showed 96% maximum identity and 100% quary cover.

For further confirmation and identification, phylogenetic relationship of the local land race ―Mamosay Batal-12‖ with other related species and genera was inferred by using Maximum Likelihood method (Jukes et al., 1969). A total of 37 nucleotide sequences of 18S rRNA of related species and genera were retrieved from GenBank and subjected for development of maximum likelihood tree. The aligned datasheet had 1850 genetic characters and after trimming the extra and ambiguously aligned fragments from both 5‘ and 3‘ ends of alignment data sheet, a total of 385 characters were used for further analysis. Out of these characters, 279 were conserved, 249 were variable, 223 were parsimony-informative and 26 were singleton sites. None of the characters were excluded from the final analysis and multistate taxa were interpreted as uncertainty.

The evolutionary history was inferred by using the Maximum Likelihood (ML) method based on the Jukes-Cantor model (Jukes et al., 1969). The tree with the highest log likelihood (-946.6432) is shown in Fig.3.30. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree‘s for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The analysis involved 38 nucleotide sequences. All positions containing gaps and missing data were eliminated.

132

The phylogenetic tree (Fig.3.30) thus achived was divided into two major clades, Clade I and clade II. The clade I was further sub divided into two sub clades, sub-Clade I and sub clade II. The sub clade I comprised of 14 P. species/cultivars belong to Pyrus communis cultivars Clapp's Favorite, cascade, Beurre, Pachkan's Triumph, Conference, and P.pyrifolia cultivars Shinsui, Chuwhangbae, Shinil, Imamuraaki, Sunwhang, Miwhang, Niitaka, Gamcheonbae and candidate landrace, Mamosay Batal_12. The local pear land race, Mamosay Batal-8 fall in clade I in between with its similar species/cultivars of P. pyrifolia cv. Miwhang and P. communis cv. Conference. The results confirming that it might be a hybrid of P. pyrifolia cv. Miwhang P. communis cv. Conference.

The sub clade II consisted of seven species/cultivars of Prunus i.e P. persica cultivars Wolmiboksunga, Kuemjeokbaekdo, Youngseonghwangdo, Changhowonhwangdo, Baekmijosaeng, Hakuho and Cheonhong, respectively.

The sub clade III comprised of five species/cultivars of Cydonia viz. C. oblonga, C. oblonga, C. oblonga, C. oblonga and Cydonia oblonga. The sub clade IV/consisted of five species of Rubus viz. R. caesius, R. caesius, R. idaeus, R. picticaulis, R. coreanus, R. crataegifolius, and R. aucuparia. While the sub clade V consist of Fragaria. vesca, F. virginiana, F. chiloensis, F. moschata, F. nubicola and F. chiloensis.

133

Pyrus_pyrifolia_cultivar_Shinsui_AF179397.1 Pyrus_communis_cultivar_Clapp's_Favorite_AF195620.1 Pyrus_pyrifolia_cultivar_Chuwhangbae_AF179384.1 Pyrus_pyrifolia_cultivar_Shinil_AF179396.1 Pyrus_communis_cultivar_Cascade_AF195618.1 Pyrus_communis_cultivar_Beurre_AF195617.1 Pyrus_communis_cultivar_Pachkan's_Triumph_AF195621.1 Sub_clade_I Pyrus_pyrifolia_cultivar_Imamuraaki_AF179387.1 Pyrus_pyrifolia_cultivar_Sunwhang_AF179398.1 Pyrus_pyrifolia_cultivar_Miwhang_AF179392.1 Mamosay Bat_12 Clade_I Pyrus_communis_cultivar_Conference_AF195619.1 Pyrus_pyrifolia_cultivar_Niitaka_AF179393.1 Pyrus_pyrifolia_cultivar_Gamcheonbae_AF179385.1 Prunus_persica_cultivar_Wolmiboksunga_AY179521.1 Prunus_persica_cultivar_Kuemjeokbaekdo_AY179519.1 85 Prunus_persica_cultivar_Youngseonghwangdo_AY179522.1 Prunus_persica_cultivar_Changhowonhwangdo_AY179514.1 Sub_clade_II Prunus_persica_cultivar_Baekmijosaeng_AY179513.1 Prunus_persica_cultivar_Hakuho_AY179529.1 Prunus_persica_cultivar_Cheonhong_AY179515.1 Pyrus_pyrifolia_cultivar_Choju_AF179382.1 Cydonia_oblonga_AF186531.1 Cydonia_oblonga_voucher829-84D_JQ392421.1 Cydonia_oblonga_JQ392420.1 Sub_clade_III 99 Cydonia_oblonga_JQ392422.1 Cydonia_oblonga_AB636344.1 Rubus_caesius_Rubus_idaeus_AF362709.1 69 Rubus_caesius_Rubus_idaeus_AF362708.1 99 71 Rubus_picticaulis_clone_29_AF362726.1 Sub_clade_IV 70 Clade_II Rubus_coreanus_isolateP677_JF980336.1 Rubus_crataegifolius_clone_KC10-2_GU980798.1 62 Fragaria_vesca_AF163510.1 Fragaria_moschata_AF163505.1 99 Fragaria_virginiana_AF163479.1 61 Sub_clade_V Fragaria_chiloensis_AF163482.1 Fragaria_nubicola_AF163517.1 Fragaria_chiloensis_AF163511.1

Fig.3.30. Molecular phylogenetic analysis of ―Mamosay B12 by Maximum Likelihood Method (Jukes et al., 1969)replications by adopting at 1000 bootstrap replications.

134

3. 4. 13 Mamoosay Bat_14

For molecular analysis, the nucleotide sequence obtained from 18S rRNA of the local pear land race ―Mamosay Batal-14‖ was initially BLAST searched in NCBI. The blast result showed 97% maximum identity and 100% query cover with Pyrus communis cultivars Pachkan's Triumph, Beurre, Pachkans Triumph, Beurre, Conference and P. pyrifolia cv. Imamuraaki. It also showed 96% maximum identity and 100% query cover with P. communis cv. Conference.

For further conformation and identification, the phylogenetic relationship of the local pear land race ―Mamosay Bat-14‖ with other related species and genera was inferred by using Maximum Likelihood method (Jukes et al., 1969). A total of 37 sequences of 18S rRNA of related species and genera were retrieved from GenBank and were subjected for organization of maximum likelihood tree. The aligned datasheet had 1850 characters which were trimmed for the extra and ambiguously aligned fragments both from 5‘ and 3‘ ends of alignment data sheet and resultantly only 384 characters were used for further analysis. Out of the analysed traits, 280 were conserved, 247 variable, 221 parsimony-informative and 26 were singleton sites. None of the genetic characters were excluded from the final analysis and multistate taxa were interpreted as uncertain. The evolutionary history was determined by applying the Maximum Likelihood (ML) method based on the Jukes-Cantor model (Jukes et al., 1969). The tree with the highest log likelihood (-938.6893) is shown in Fig.3.31. The percentage of trees in which the associated taxa clustered together shown next to the branches. Initial tree‘s for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The analysis comprises of 38 nucleotide sequences and all positions containing gaps and missing data were deleted. The phylogenetic cladogram (Fig.3.31) thus organized was divided into two major clades, Clade I and II. The clade I was further sub divided into two sub clades, sub- Clade I and clade II.

135

The sub clade I comprised of 14 Pyrus species/cultivars belong to P.pyrifolia and P.communis Viz. P. pyrifolia cultivars Miwhang, Imamuraaki, Sunwhang, Chuwhangbae, Shinsui, Shinil, Niitaka, Gamcheonbae P.communis cultivars Conference, Clapp's Favorite, Pachkan's Triumph, Beurre, cascade and local landrace Mamosay Batal-14. The local land race ―Mamosay Batal-14‖ fall in Pyrus clade in between with its similar species of P. pyrifolia cv. Shinil and P. pyrifolia cv. Niitaka. The results showing that it may be a hybrid of P. pyrifolia cv. Shinil and P. pyrifolia cv. Niitaka.

The sub clade II comprised of seven species/cutivar of Prunus i.e P. persica cultivars Wolmiboksunga, Kuemjeokbaekdo,Youngseonghwangdo, Changhowonhwangdo, Baekmijosaeng, Hakuho and Cheonhong.

The sub clade III consisted of five species of Rubus viz. R. caesius, R. caesius, R. idaeus,R. picticaulis, R. coreanus isolate R. crataegifolius and R. aucuparia.The sub clade IV consisted of five species/cultivars of Cydonia such as C. oblonga, C. oblonga, C. oblonga, C. oblonga and Cydonia oblonga while the sub clade V consist of Fragaria vesca, F. moschata, F. virginiana, F. chiloensis,F. nubicola and F. chiloensis.

136

Pyrus_pyrifolia_cultivar_Sunwhang_AF179398.1 Pyrus_communis_cultivar_Conference_AF195619.1 Pyrus_communis_cultivar_Clapp's_Favorite_AF195620.1 Pyrus_pyrifolia_cultivar_Miwhang_AF179392.1 Pyrus_pyrifolia_cultivar_Imamuraaki_AF179387.1 Pyrus_communis_cultivar_Pachkan's_Triumph_AF195621.1 Pyrus_communis_cultivar_Beurre_AF195617.1 Sub_clade_I Pyrus_communis_cultivar_Cascade_AF195618.1 Pyrus_pyrifolia_cultivar_Shinil_AF179396.1 Pyrus_pyrifolia_cultivar_Chuwhangbae_AF179384.1 Pyrus_pyrifolia_cultivar_Shinsui_AF179397.1 Clade_I Mamosay Bat-14 Pyrus_pyrifolia_cultivar_Niitaka_AF179393.1 Pyrus_pyrifolia_cultivar_Gamcheonbae_AF179385.1 Prunus_persica_cultivar_Wolmiboksunga_AY179521.1 Prunus_persica_cultivar_Kuemjeokbaekdo_AY179519.1 85 Prunus_persica_cultivar_Youngseonghwangdo_AY179522.1 Prunus_persica_cultivar_Changhowonhwangdo_AY179514.1 Sub_clade_II Prunus_persica_cultivar_Baekmijosaeng_AY179513.1 Prunus_persica_cultivar_Hakuho_AY179529.1 Prunus_persica_cultivar_Cheonhong_AY179515.1 Pyrus_pyrifolia_cultivar_Choju_AF179382.1 Rubus_caesius_Rubus_idaeus_AF362709.1 67 Rubus_caesius_Rubus_idaeus_AF362708.1 68 Rubus_picticaulis_clone_29_AF362726.1 Sub_clade_III 68 Rubus_coreanus_isolateP677_JF980336.1 Rubus_crataegifolius_clone_KC10-2_GU980798.1 Cydonia_oblonga_AF186531.1 Cydonia_oblonga_voucher829-84D_JQ392421.1 99 Cydonia_oblonga_JQ392420.1 Sub_clade_IV 99 Clade_II Cydonia_oblonga_JQ392422.1 Cydonia_oblonga_AB636344.1 Fragaria_vesca_AF163510.1 Fragaria_moschata_AF163505.1 99 Fragaria_virginiana_AF163479.1 65 Sub_clade_V Fragaria_chiloensis_AF163482.1 Fragaria_nubicola_AF163517.1 Fragaria_chiloensis_AF163511.1

Fig. 3.31. Phylogenetic analysis of ―Mamosy Batal-14‖ by Maximum Likelihood Method (Jukes et al., 1969) at 1000 bootstrap replications.

137

3. 4. 14 Mamoosay B15

For molecular characterizations the nucleotides sequence obtained from 18S rRNA of the local pear land race ―Mamosoay B15‖ having accession no ―Mamoosay-B15‖ was initially BLAST in NCBI. The BLAST result showed 97% maximum identity and query cover with Pyrus communis cultivars Pachkan's Triumph, Conference and Beurre.

Maximum Likelihood method was used to infer the phylogenetic relationship of the land race ―Mamosay Batal_15‖ with other related species and genera further conformation and identification. A total of 37 nucleotide sequences of 18S rRNA of related species belonging to five different genera were retrived from GenBank and were subjected fororganization of maximum likelihood tree. The aligned datasheet had a total of 1850 characters and after trimming the unrelated and ambiguously aligned fragments from both 5‘ and 3‘ ends of alignment data sheet. The remaining 414 characters were used for further analysis. Out of the analysed characters, 288 were conserved, 255 were variable, 223 were parsimony-informative and 32 were singleton sites. None of the characters were excluded from the final analysis and multistate taxa were interpreted as uncertainty.

The evolutionary history was determined by applying the Maximum Likelihood (ML) method based on the Jukes-Cantor model (Jukes et al., 1969). The tree with the highest log likelihood (-906.0039) is shown in Fig. 3.32. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The analysis comprises of 38 nucleotide sequences and all positions containing gaps and missing data were eliminated.

The phylogenetic tree (Fig.3.32) was obtained and the analysed data were organized into two major clades, Clade I and II. Clade I was further sub divided into two sub clades, sub-Clade I and II.

138

The sub clade I comprised of 14 Pyrus species/cultivars beloned to Pyrus communis cv. cascade, P. pyrifolia cv. Shinil, P. communis cultivars Beurre, Pachkan's Triumph, P. pyrifolia cultivars Imamuraaki, Miwhang, Sunwhang, Chuwhangbae, Shinsui, P. communis cv. Clapp's Favorite, local land race, Mamosay Batal 15, P. pyrifollia cultivars Niitaka, Gamcheonbae and P.communis cv. Conference, P. pyrifolia cv. Niitaka, respectively. The local pear land race, Mamosay Batal 15 fall in clade I in between P. communis cv. Clapp's Favorite and P. pyrifolia cv. Niitaka. This showing that, it is a hybrid of P. communis cv. Clapp's Favorite and P. pyrifolia cv. Niitaka.

The sub clade II consisted of seven species/cultivars of Prunus i.e P. persica cv. Wolmiboksunga, P. persica cv. Kuemjeokbaekdo, P. persica cv. Youngseonghwangdo, P. persica cv. Changhowonhwangdo, P. persica cv. Baekmijosaeng, P. persica cv. Hakuho and P.persica cv. Cheonhong.

The sub clade III comprised of five species/cultivarsof Cydonia such as C. oblonga, C. oblonga, C. oblonga, C. oblonga and Cydonia oblonga. Sub clade IVconsisted of five species of Rubus viz. R. caesius,R. caesius, R. idaeus, R. picticaulis, R. coreanus, R. crataegifolius and R. aucuparia while the sub clade V comprised of Fragerai virginiana, F. moschata, F. vesca, F. nubicola, F. chiloensis and F. chiloensis.

139

Pyrus_communis_cultivar_Cascade_AF195618.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Shinil_AF179396.1 0.00000 0.00000 Pyrus_communis_cultivar_Beurre_AF195617.1 0.00000 0.00000 Pyrus_communis_cultivar_Pachkan's_Triumph_AF195621.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Imamuraaki_AF179387.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Miwhang_AF179392.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Sunwhang_AF179398.1 0.00473 Sub_clade_I 0.00000 0.00000 Pyrus_communis_cultivar_Conference_AF195619.1 0.00473 Pyrus_pyrifolia_cultivar_Chuwhangbae_AF179384.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Shinsui_AF179397.1 0.00000 0.00000 Pyrus_communis_cultivar_Clapp's_Favorite_AF195620.1 Clade_I 0.00000 0.00000 Mamoosay_B15 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Niitaka_AF179393.1 0.00000 0.00473 Pyrus_pyrifolia_cultivar_Gamcheonbae_AF179385.1 0.00473 0.00473 Prunus_persica_cultivar_Wolmiboksunga_AY179521.1 86 0.00000 Prunus_persica_cultivar_Kuemjeokbaekdo_AY179519.1 0.00949 0.00473 Prunus_persica_cultivar_Youngseonghwangdo_AY179522.1 0.00000 0.00000 Prunus_persica_cultivar_Changhowonhwangdo_AY179514.1 Sub_clade_II 0.00000 0.00000 0.48087 Prunus_persica_cultivar_Baekmijosaeng_AY179513.1 0.00000 0.00000 Prunus_persica_cultivar_Hakuho_AY179529.1 0.000000.00000 0.00000 Prunus_persica_cultivar_Cheonhong_AY179515.1 0.00000 Pyrus_pyrifolia_cultivar_Choju_AF179382.1 0.00000 Cydonia_oblonga_AF186531.1 0.00000 0.00000 Cydonia_oblonga_voucher829-84D_JQ392421.1 0.00000 990.00000 Cydonia_oblonga_JQ392420.1 Sub_clade_III 0.00000 0.09237 Cydonia_oblonga_AB636344.1 0.00000 0.00000 Cydonia_oblonga_JQ392422.1 0.00000 Rubus_coreanus_isolateP677_JF980336.1 99 0.00949 0.00000 Rubus_crataegifolius_clone_KC10-2_GU980798.1 0.32656 0.01429 690.00000 Rubus_picticaulis_clone_29_AF362726.1 Sub_clade_IV 0.00000 Clade_II 0.04127 Rubus_caesius_Rubus_idaeus_AF362709.1 0.00000 62 0.00000 Rubus_caesius_Rubus_idaeus_AF362708.1 0.00000 0.07368 Fragaria_virginiana_AF163479.1 0.00000 99 Fragaria_moschata_AF163505.1 0.00000 0.09486 0.00000 Fragaria_vesca_AF163510.1 0.00473 Sub_clade_V 0.00000 Fragaria_nubicola_AF163517.1 0.00473 Fragaria_chiloensis_AF163482.1 0.000000.00000 0.00000 Fragaria_chiloensis_AF163511.1 0.00000

Fig.3.32. Phylogenetic analysis of ―Mamosy B15‖by Maximum Likelihood Method Method (Jukes et al., 1969) at 1000 bootstrap replications.

140

3. 4. 15 Gultar Tango

The nucleotide sequence obtained from the 18s rRNA of the local pear land race ―Gultar Tango‖, accession number Gt-1 was compared with the database referecnces with the help of BLAST(Altschul et al., 1990) in NCBI for molecular characterizations. The BLAST result showed 100% maximum identity and query cover with Pyrus communis cultivars Pachkan's Triumph, conference, Beurre and P. pyrifolia cv. Wonwhang.

For further conformation and identification, phylogenetic relationship of the land race ―Gultar Tango‖ with other related species and genera was inferred by using the Maximum Likelihood method. A total of 37 sequences of 18S rRNA of related species and genera were retrived from GenBank and subjected for construction of maximum likelihood tree. The aligned datasheet had maximum 1850 characters which was trimmed for extra and ambiguously aligned fragments both from 5‘ and 3‘ ends of alignment data sheet. A total of 280 characters were used for analysis. Out of the analysed characters, 229 characters were conserved, 251 were variable, 226 were parsimony-informative and 25 were singleton sites. None of the characters were excluded from the final analysis and multistate taxa were interpreted as uncertainty

The evolutionary history was determined by applying the Maximum Likelihood (ML) method based on the Jukes-Cantor model (Jukes et al., 1969). The tree with the highest log likelihood (-974.3893) is shown in Fig. 3.33. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The analysis comprises of 38 nucleotide sequences and all positions containing gaps and missing data were eliminated.

The phylogenetic tree (Fig.3.33) thus obtained was divided into two major clades, Clade I and II. Clade I was further sub divided into two, sub-Clade I and sub clade II.

141

The sub clade I consisted of 14 Pyrus species/cultivars belong to Pyrus pyrifolia cultivars Sunwhang, Miwhang, Imamuraaki, Shinil, Chuwhangbae, Shinsui, Niitaka, Gamcheonbae and P. communis cultivars Conference, Pachkan's Triumph, Beurre,. Cascade, Clapp's Favorite and the local land race, Gultar Tango. The local land race, Gultar Tango fall in sub-Clade I and showed close relationship with P. communis cv. Clapp's Favorite.

The sub clade II comprised of seven species/cultivars of Prunus i.e P. persica cultivars Wolmiboksunga, Kuemjeokbaekdo, Cheonhong, Youngseonghwangdo, Changhowonhwangdo, Baekmijosaeng and Hakuho and the sub clade III consisted of five species/cultivarsof Cydonia such as C. oblonga, C. oblonga, C. oblonga, C. oblonga and Cydonia oblonga.

The sub clade IV consisted of five species of Rubus viz. R. caesius, R. caesius, R. idaeus, R. picticaulis, R. coreanus, R. crataegifolius R. aucuparia while the sub clade V consist of Fragaria vesca, F. moschata, F. virginiana, F. nubicola, F. chiloensis and F. chiloensis.

142

Pyrus_pyrifolia_cultivar_Sunwhang_AF179398.1 0.00429 0.00000 Pyrus_pyrifolia_cultivar_Miwhang_AF179392.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Imamuraaki_AF179387.1 0.00000 0.00000 Pyrus_communis_cultivar_Conference_AF195619.1 0.00429 0.00000 Pyrus_communis_cultivar_Pachkan's_Triumph_AF195621.1 0.00000 0.00000 Pyrus_communis_cultivar_Beurre_AF195617.1 0.00000 0.00000 Pyrus_communis_cultivar_Cascade_AF195618.1 0.00000 0.00000 Sub_clade_I Pyrus_pyrifolia_cultivar_Shinil_AF179396.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Chuwhangbae_AF179384.1 0.00000 Pyrus_pyrifolia_cultivar_Shinsui_AF179397.1 0.00000 0.00000 Pyrus_communis_cultivar_Clapp's_Favorite_AF195620.1 Clade_I 0.00000 0.00000 0.00000 0.00000 Gultar_Tango_Gt 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Niitaka_AF179393.1 0.00429 Pyrus_pyrifolia_cultivar_Gamcheonbae_AF179385.1 0.00429 0.00429 Prunus_persica_cultivar_Wolmiboksunga_AY179521.1 0.00000 87 Prunus_persica_cultivar_Kuemjeokbaekdo_AY179519.1 0.00429 0.00860 0.00000 Prunus_persica_cultivar_Cheonhong_AY179515.1 0.00000 Prunus_persica_cultivar_Youngseonghwangdo_AY179522.1 Sub_clade_II 0.00000 0.00000 0.47536 Prunus_persica_cultivar_Changhowonhwangdo_AY179514.1 0.00000 0.00000 Prunus_persica_cultivar_Baekmijosaeng_AY179513.1 0.00000 0.00000 0.00000 Prunus_persica_cultivar_Hakuho_AY179529.1 0.00000 Pyrus_pyrifolia_cultivar_Choju_AF179382.1 0.00000 Cydonia_oblonga_AF186531.1 0.00000 0.00000 Cydonia_oblonga_voucher829-84D_JQ392421.1 0.00000 0.00000 Cydonia_oblonga_JQ392420.1 Sub_clade_III 99 0.00000 0.00000 Cydonia_oblonga_JQ392422.1 0.00000 0.08468 Cydonia_oblonga_AB636344.1 0.00000 Rubus_caesius_Rubus_idaeus_AF362709.1 99 0.00000 610.00000 Rubus_caesius_Rubus_idaeus_AF362708.1 0.00000 0.33456 700.00000 Rubus_picticaulis_clone_29_AF362726.1 Sub_clade_IV 0.00000 Clade_II 0.03759 Rubus_coreanus_isolateP677_JF980336.1 63 0.00860 0.00000 Rubus_crataegifolius_clone_KC10-2_GU980798.1 0.01293 0.06892 Fragaria_vesca_AF163510.1 99 0.00429 Fragaria_moschata_AF163505.1 0.0847664 0.00000 Fragaria_virginiana_AF163479.1 0.00000 0.00000 Sub_clade_V Fragaria_nubicola_AF163517.1 0.00000 0.00429 Fragaria_chiloensis_AF163482.1 0.00000 0.00000 0.00000 Fragaria_chiloensis_AF163511.1 0.00000

Fig.3.33. Phylogenetic analysis of “Gultar_Tango” by Maximum Likelihood Method (Jukes et al., 1969) at 1000 bootstrap replications.

143

3. 4. 16 Hary Tango Batal

For molecular characterizations, the nucleotide sequence of 18S rRNA of the local pear land race ‖Hary Tango-Batal‖ (Ht-B1) was initially BLAST in NCBI. The BLAST result showed 95% maximum identity and 100% query cover with Pyrus communis cultivars Pachkan's Triumph, Beurre and P. pyrifolia cv. Imamuraaki and 94% maximum identity and 100% query cover with P. pyrifolia cultivars Soowhang and Wonwhang.

For further identification and conformation, the phylogenetic relationship of the local land race ―Hary Tango Batal‖ with other related species and genera was inferred by using Maximum Likelihood method (Jukes et al., 1969). A total of 37 nucleotides sequences of 18S rRNA of related species and genera were retrived from GenBank and used for construction of maximum likelihood tree. The aligned datasheet had maximum 1850 traits which were trimmed for extra and ambiguously aligned fragments from both 5‘ and 3‘ ends of alignment data sheet and resultantlyonly 1173 characteers were used for further analysis. Out of the analysed characters 280 were conserved, 249 were variable, 223 were parsimony-informative and 26 were singleton sites. None of the characters were excluded from the final analysis and multistate taxa were interpreted as uncertain.

The evolutionary history was inferred by applying the Maximum Likelihood (ML) method based on the Jukes-Cantor model (Jukes et al., 1969). The tree with the highest log likelihood (-931.1918) is shown in Fig. 16. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree‘s for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The analysis comprises of 38 nucleotide sequences and all positions containing gaps and missing data were eliminated.

The phylogenetic tree (Fig.3.34) thus organized was divided into two major clades, Clade I and clade II. Clade I was further sub divided into two sub clades, sub-Clade I

144

and II. Sub clade I consisted of 14 P. species/cultivars belong to Pyrus pyrifolia cultivars Sunwhang, Miwhang, Imamuraaki, Shinil, Chuwhangbae, Shinsui, Niitaka and Gamcheonbae, and P. communis cultivars Beurre, Pachkan's Triumph, Conference, cascade, Clapp's Favorite and candidate landrace, Hary Tango Batal. Our local land race, Hary Tango Batal, fall in sub-Clade I and showed close relationship with P. communis cv. Clapp's Favorite. The sub clade II consisted of seven species/cultivars of Prunus species while the sub clade III comprises of five species/cultivars of the genus Cydonia. Similarly the sub clade IV ‗and V consist of five and six species/cv. of the genus Rubus and Frageria respectively.

145

Pyrus_pyrifolia_cultivar_Sunwhang_AF179398.1 0.00454 0.00000 Pyrus_communis_cultivar_Beurre_AF195617.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Miwhang_AF179392.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Imamuraaki_AF179387.1 0.00000 0.00000 Pyrus_communis_cultivar_Pachkan's_Triumph_AF195621.1 0.00000 0.00000 Pyrus_communis_cultivar_Conference_AF195619.1 0.00000 0.00454 Pyrus_communis_cultivar_Cascade_AF195618.1 0.00000 0.00000 Sub_clade_I Pyrus_pyrifolia_cultivar_Shinil_AF179396.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Chuwhangbae_AF179384.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Shinsui_AF179397.1 0.00000 0.00000 Pyrus_communis_cultivar_Clapp's_Favorite_AF195620.1 Clade_I 0.00000 0.00000 0.00000 Hary Tango-Batal_Ht-B1 0.00000 Pyrus_pyrifolia_cultivar_Niitaka_AF179393.1 0.00000 0.00454 Pyrus_pyrifolia_cultivar_Gamcheonbae_AF179385.1 0.00454 0.00454 Prunus_persica_cultivar_Wolmiboksunga_AY179521.1 87 0.00000 Prunus_persica_cultivar_Kuemjeokbaekdo_AY179519.1 0.00910 0.00454 Prunus_persica_cultivar_Youngseonghwangdo_AY179522.1 0.00000 0.00000 Prunus_persica_cultivar_Changhowonhwangdo_AY179514.1 Sub_clade_II 0.00000 0.00000 0.46726 Prunus_persica_cultivar_Baekmijosaeng_AY179513.1 0.00000 0.00000 Prunus_persica_cultivar_Hakuho_AY179529.1 0.00000 0.00000 0.00000 Prunus_persica_cultivar_Cheonhong_AY179515.1 0.00000 Pyrus_pyrifolia_cultivar_Choju_AF179382.1 0.00000 Cydonia_oblonga_AF186531.1 0.00000 0.00000 Cydonia_oblonga_voucher829-84D_JQ392421.1 0.00000 990.00000 Cydonia_oblonga_JQ392420.1 Sub_clade_III 0.00000 0.08786 Cydonia_oblonga_AB636344.1 0.00000 0.00000 Cydonia_oblonga_JQ392422.1 0.00000 Rubus_coreanus_isolateP677_JF980336.1 99 0.00910 0.00000 Rubus_crataegifolius_clone_KC10-2_GU980798.1 0.31987 0.01370 710.00000 Rubus_picticaulis_clone_29_AF362726.1 Sub_clade_IV 0.00000 Clade_II 0.03955 Rubus_caesius_Rubus_idaeus_AF362709.1 0.00000 61 0.00000 Rubus_caesius_Rubus_idaeus_AF362708.1 0.00000 0.07042 Fragaria_virginiana_AF163479.1 0.00000 99 Fragaria_moschata_AF163505.1 0.00000 0.09061 0.00000 Fragaria_vesca_AF163510.1 0.00454 Sub_clade_V 0.00000 Fragaria_nubicola_AF163517.1 0.00454 Fragaria_chiloensis_AF163482.1 0.00000 0.00000 0.00000 Fragaria_chiloensis_AF163511.1 0.00000

Fig.3.34. Phylogenetic analysis of ―Hary Tango-Batal‖, by Maximum Likelihood Method (Jukes et al., 1969) with 1000 bootstrap replications.

146

3. 4. 17 Kado Batang (Kb)

For molecular characterizations the nucleotide sequence obtained from18S rRNA of local pear land race ―Kado batang (Kb)‖ having accession nuber Kb -1 was initially compared to the sequences of database reference library in NCBI with the help of BLASTsearch. The BLAST result showed 97% maximum identity and 92% query cover with Pyrus communis cv. Pachkan's Triumph, 96% maximum identity and 95% query cover with P. communis cv. Conference, 99% maximum identity and 100 query cover with P. communis cultivars Beurre and Cascade. For further conformation and identification, the phylogenetic relationship of the local land race Kado batang (Kb) Balakot with other related species and genera was inferred by using Maximum Likelihood method (Jukes et al., 1969). A total of 37 nucleotide sequences of 18S rRNA of related species and genera were retrived from GenBank and subjected for organization of maximum likelihood tree. The aligned datasheet has maximum 1850 characters which was trimmed for extra and ambiguously aligned fragments from both 5‘ and 3‘ ends of alignment data sheet. As a result only 1221 characters were used for analysis and out of these analysed characters, 244 were conserved, 230 were variable, 209 were parsimony-informative and 21 were singleton sites. None of the characters were excluded from the final analysis and multistate taxa were interpreted as uncertainty.

The evolutionary history was derived by using the Maximum Likelihood (ML) method based on the Jukes-Cantor model (Jukes et al., 1969). The tree with the highest log likelihood (864.2906) is shown in Fig. 3.35. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree‘s for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The analysis comprises of 38 nucleotide sequences and all positions containing gaps and missing data were eliminated.

The phylogenetic tree (Fig.3.35) was constructed and the analysed data were segregated into two major clades, Clade I and II. Clade I was further divided into two sub clades, 147

sub-Clade I and sub clade II. The sub clade I consists of 14 Pyrus species/cultivars belong to P. pyrifolia cultivars Sunwhang, Miwhang, Imamuraaki, Chuwhangbae, Shinil, Shinsui, Niitaka, Gamcheonbae and P. communis cultivars Pachkan's Triumph, Conference, Beurre, Cascade, Clapp's Favorite and local landrace, Kado batang (Kb). The laocal land race, Kado batang (Kb) Balakot fall in sub-Clade I in between P. communis cv. Clapp's Favorite and P. pyrifolia cv. Niitaka.

The sub clade II consisted of seven species/cultivars of Prunus species while the sub clade III comprises of five species/cultivars of the genus Cydonia. Similarly the sub clade IV and V consist of five and six species/cultivar of the genus Rubus and Frageria respectively belonging to family rosaceae.

148

Pyrus_pyrifolia_cultivar_Sunwhang_AF179398.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Miwhang_AF179392.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Imamuraaki_AF179387.1 0.00000 0.00000 Pyrus_communis_cultivar_Pachkan's_Triumph_AF195621.1 0.00000 0.00000 Pyrus_communis_cultivar_Conference_AF195619.1 0.00000 0.00000 Pyrus_communis_cultivar_Beurre_AF195617.1 0.00000 0.00000 Pyrus_communis_cultivar_Cascade_AF195618.1 0.00000 0.00000 Sub_clade_I Pyrus_pyrifolia_cultivar_Shinil_AF179396.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Chuwhangbae_AF179384.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Shinsui_AF179397.1 0.00000 0.00000 Pyrus_communis_cultivar_Clapp's_Favorite_AF195620.1 Clade_I 0.00000 0.00000 Kado_Batang_Kb 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Niitaka_AF179393.1 0.00000 0.00494 Pyrus_pyrifolia_cultivar_Gamcheonbae_AF179385.1 0.00494 0.00494 Prunus_persica_cultivar_Wolmiboksunga_AY179521.1 84 0.00000 Prunus_persica_cultivar_Kuemjeokbaekdo_AY179519.1 0.00992 0.00000 Prunus_persica_cultivar_Youngseonghwangdo_AY179522.1 0.00000 0.00000 Prunus_persica_cultivar_Changhowonhwangdo_AY179514.1 Sub_clade_II 0.00000 0.00000 0.52547 Prunus_persica_cultivar_Baekmijosaeng_AY179513.1 0.00000 0.00000 Prunus_persica_cultivar_Hakuho_AY179529.1 0.00000 0.00000 0.00000 Prunus_persica_cultivar_Cheonhong_AY179515.1 0.00000 Pyrus_pyrifolia_cultivar_Choju_AF179382.1 0.00000 Cydonia_oblonga_AF186531.1 0.00000 0.00000 Cydonia_oblonga_voucher829-84D_JQ392421.1 0.00000 990.00000 Cydonia_oblonga_JQ392420.1 Sub_clade_III 0.00000 0.10054 Cydonia_oblonga_AB636344.1 0.00000 0.00000 Cydonia_oblonga_JQ392422.1 0.00000 Rubus_coreanus_isolateP677_JF980336.1 99 0.00992 0.00000 Rubus_crataegifolius_clone_KC10-2_GU980798.1 0.35574 0.01493 710.00000 Rubus_picticaulis_clone_29_AF362726.1 Sub_clade_IV 0.00000 Clade_II 0.04345 Rubus_caesius_Rubus_idaeus_AF362709.1 0.00000 60 0.00000 Rubus_caesius_Rubus_idaeus_AF362708.1 0.00000 0.08050 Fragaria_virginiana_AF163479.1 0.00000 99 Fragaria_moschata_AF163505.1 0.00000 0.09915 0.00000 Fragaria_vesca_AF163510.1 0.00494 Sub_clade_V 0.00000 Fragaria_chiloensis_AF163482.1 0.00000 Fragaria_nubicola_AF163517.1 0.00000 0.00494 0.00000 Fragaria_chiloensis_AF163511.1 0.00000

Fig.3.35. Phylogenetic analysis of “Kado Batang‖ by Maximum Likelihood Method (Jukes et al., 1969) with 1000 bootstrap replications.

149

3. 4. 18 Malyzay Tango

For molecular characterizations the nucleotide sequence of 18S rRNA of the local pear land race ―Malyzay Tango‖ having accession unmber Mt-1 was initially BLAST searched in NCBI. The BLAST result showed 99% maximum identity and 98% query cover with Pyrus communis cv. Pachkan's Triumph, 99% maximum identity and 98% query cover with P. communis cultivars Beurre and Cascade.

The phylogenetic relationship of the local pear land race ―Malyzay Tango‖ with other related species and genera was inferred by using Maximum Likelihood method (Jukes et al., 1969). A total of 37 nucleotied sequences of 18S rRNA of related species and genera were retrived from GenBank and used for construction of maximum likelihood cladogram. The aligned datasheet had a maximum 1850 characters and after trimming extra and ambiguously aligned fragments from both 5‘ and 3‘ ends of alignment data sheet. A total of 1278 characters were used for further analysis. Out of which, 243 genetic characters were conserved, 196 were variable, 170 were parsimony-informative and 26 were singleton sites.

The evolutionary history was derived by using the Maximum Likelihood (ML) method based on (Jukes et al., 1969). The tree with the highest log likelihood (-642.1204) is shown in Fig. 18. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The analysis comprises of 38 nucleotide sequences and all positions containing gaps and missing data were eliminated. The phylogenetic tree (Fig. 3.36) thus organized was divided into two major clades, clade I and II. Clade I is further divided into two sub clades, sub clade I, Clade II and sub clade III.

150

The sub clade I comprised of 07 Pyrus species/cultivars belong to P. pyrifolia cultivars Sunwhang, Miwhang, Imamuraaki and P. communis cultivars Pachkan's Triumph, Conference, Beurre and Cascade while the sub clade II comprises of P. pyrifolia cultivars Shinil, Chuwhangbae, Shinsui, Gamcheonbae, Clapp's Favorite and candidate landrace, Malyzay Tango. The local land race ―Malyzay Tango‖ fall in sub II and showed close relationship with P. communis cv. Clapp's Favorite. The sub clade III comprised of seven species/cultivars of Prunus species while the sub clade IV comprised of five species/cultivars of the genus Cydonia. Similarly the sub clade V and VI consist of five and six species/cultivar of the genus Rubus and Frageria respectively belonging to family Rosaceae.

151

Pyrus_pyrifolia_cultivar_Sunwhang_AF179398.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Miwhang_AF179392.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Imamuraaki_AF179387.1 0.00000 0.00000 Pyrus_communis_cultivar_Pachkan's_Triumph_AF195621.1 Sub_clade_I 0.00000 0.00000 Pyrus_communis_cultivar_Conference_AF195619.1 0.00000 0.00000 Pyrus_communis_cultivar_Beurre_AF195617.1 0.00000 0.00000 Pyrus_communis_cultivar_Cascade_AF195618.1 0.00000 0.00002 Pyrus_pyrifolia_cultivar_Shinil_AF179396.1 0.00000 Pyrus_pyrifolia_cultivar_Chuwhangbae_AF179384.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Shinsui_AF179397.1 0.00000 0.00000 Sub_clade_II 0.00012 Pyrus_pyrifolia_cultivar_Gamcheonbae_AF179385.1 Clade_I 0.00000 0.00000 Pyrus_communis_cultivar_Clapp's_Favorite_AF195620.1 0.00000 0.00000 0.00000 Malyzay Tango_Mt 0.00000 0.00665 Pyrus_pyrifolia_cultivar_Niitaka_AF179393.1 0.00665 Prunus_persica_cultivar_Wolmiboksunga_AY179521.1 85 0.00000 Prunus_persica_cultivar_Kuemjeokbaekdo_AY179519.1 0.01336 0.00000 Prunus_persica_cultivar_Youngseonghwangdo_AY179522.1 0.00000 0.00000 0.47388 Prunus_persica_cultivar_Changhowonhwangdo_AY179514.1 Sub_clade_III 0.00000 0.00000 Prunus_persica_cultivar_Baekmijosaeng_AY179513.1 0.00000 0.00000 Prunus_persica_cultivar_Hakuho_AY179529.1 0.00000 0.00000 0.00000 Prunus_persica_cultivar_Cheonhong_AY179515.1 0.00000 Pyrus_pyrifolia_cultivar_Choju_AF179382.1 0.00000 Cydonia_oblonga_AF186531.1 0.00000 0.00000 Cydonia_oblonga_voucher829-84D_JQ392421.1 0.00000 0.00000 Cydonia_oblonga_JQ392420.1 Sub_clade_IV 0.00000 0.00000 99 Cydonia_oblonga_JQ392422.1 0.10849 0.00000 Cydonia_oblonga_AB636344.1 0.00000 Rubus_coreanus_isolateP677_JF980336.1 0.00665 99 84 0.00000 Rubus_caesius_Rubus_idaeus_AF362708.1 0.31639 0.00000 0.00000 Rubus_picticaulis_clone_29_AF362726.1 Sub_clade_V 73 0.00000 Clade_II 0.00000 Rubus_caesius_Rubus_idaeus_AF362709.1 0.04352 0.00000 65 Rubus_crataegifolius_clone_KC10-2_GU980798.1 0.02014 0.08218 Fragaria_vesca_AF163510.1 99 0.00665 Fragaria_nubicola_AF163517.1 0.08867 62 0.00665 Fragaria_moschata_AF163505.1 0.00000 0.00000 Sub_clade_VI Fragaria_virginiana_AF163479.1 0.00000 0.00000 Fragaria_chiloensis_AF163482.1 0.00000 0.00000 0.00000 Fragaria_chiloensis_AF163511.1 0.00000

Fig.3.36. Phylogenetic analysis of ―Malyzay Tango‖ by Maximum Likelihood Method (Jukes et al., 1969) with 1000 bootstrap replications.

152

3. 4. 19 Mamosranga

The nucleotide sequences of 18S rRNA of the local pear land race ‖Mamosranga‖ was initially compared to the reference sequences in NCBI with the help of BLAST (Altschul et al., 1990) for molecular characterization. The BLAST result showed 99% maximum identity and query cover with Pyrus communis cultvars Pachkan's Triumph, Beurre, Cascade and Conference.

For further identification and phylogenetic relationship of the local land race ―Mamosranga‖ with other related species and genera was inferred by using Maximum Likelihood method (Jukes et al., 1969). A total of 37 sequences of 18S rRNA of related species and genera were retrived from GenBank and subjected for construction of maximum likelihood tree. The aligned datasheet had maximum 1850 characters and after trimming the extra and ambiguously aligned fragments from both 5‘ and 3‘ ends of alignment data sheet. A total of 1208 characters were used for further analysis. Out of these characters, 253 were conserved, 142 were variable, 210 were parsimony- informative and 32 were singleton sites. None of the characters were excluded from the final analysis and multistate taxa were interpreted as uncertainty.

The evolutionary history was derived by using the Maximum Likelihood (ML) method based on the Jukes-Cantor model (Jukes et al., 1969). The tree with the highest log likelihood (-807.5118) is shown in Fig. 3.37. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. The analysis comprises of 38 nucleotide sequences and all positions containing gaps and missing data were eliminated.

The phylogenetic tree (Fig.3.37) divided the pear land races into two major clades, Clade I and clade II. Clade I was further divided into two sub clades, sub clade I and sub clade II. The sub clade I consisted of 11 Pyrus species/cultivars belong to P. pyrifolia 153

cultivars Sunwhang, Miwhang, Imamuraaki, Chuwhangbae, Shinil, Shinsui and P. communis cultivars Pachkan's Triumph, Conference, Beurre, cascade and Clapp's Favorite. The local land race ―Mamosranga‖ fall in sub clade II near to P. pyrifolia cv. Gamcheonbae. The sub_clade II consist of 8 species/cultivars of the genus Prunus. The clade II comprised of three sub clades, sub clade IV, V and VI comprised of related genera belonging to family Rosaceae.

154

Pyrus_pyrifolia_cultivar_Sunwhang_AF179398.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Miwhang_AF179392.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Imamuraaki_AF179387.1 0.00000 0.00000 Pyrus_communis_cultivar_Pachkan's_Triumph_AF195621.1 0.00000 0.00000 Pyrus_communis_cultivar_Conference_AF195619.1 0.00000 0.00000 Pyrus_communis_cultivar_Beurre_AF195617.1 Sub_clade_I 0.00000 0.00000 Pyrus_communis_cultivar_Cascade_AF195618.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Shinil_AF179396.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Chuwhangbae_AF179384.1 0.00000 0.00000 Pyrus_pyrifolia_cultivar_Shinsui_AF179397.1 0.00125 0.00000 Pyrus_communis_cultivar_Clapp's_Favorite_AF195620.1 0.00000 Clade_I 0.00108 Pyrus_pyrifolia_cultivar_Choju_AF179382.1 0.00523 Prunus_persica_cultivar_Wolmiboksunga_AY179521.1 0.00071 64 0.00000 Prunus_persica_cultivar_Kuemjeokbaekdo_AY179519.1 0.00523 0.00000 Prunus_persica_cultivar_Youngseonghwangdo_AY179522.1 62 0.00000 0.00000 Sub_clade_II 0.00523 Prunus_persica_cultivar_Changhowonhwangdo_AY179514.1 0.00000 0.00000 Prunus_persica_cultivar_Baekmijosaeng_AY179513.1 0.00000 0.00000 65 Prunus_persica_cultivar_Hakuho_AY179529.1 0.00000 0.00000 0.00000 0.00000 Prunus_persica_cultivar_Cheonhong_AY179515.1 0.00000 Pyrus_pyrifolia_cultivar_Niitaka_AF179393.1 0.00523 0.49040 Pyrus_pyrifolia_cultivar_Gamcheonbae_AF179385.1 0.00000 Mamosranga 0.10612 Rubus_caesius_Rubus_idaeus_AF362709.1 0.00000 620.00000 Rubus_caesius_Rubus_idaeus_AF362708.1 0.00000 650.00289 Rubus_picticaulis_clone_29_AF362726.1 Sub_clade_IV 0.00000 0.00000 62 Rubus_coreanus_isolateP677_JF980336.1 0.00000 0.00523 Rubus_crataegifolius_clone_KC10-2_GU980798.1 0.01579 Cydonia_oblonga_AF186531.1 0.00000 99 0.00000 Cydonia_oblonga_voucher829-84D_JQ392421.1 0.00000 0.36547 0.00000 Cydonia_oblonga_JQ392420.1 Sub_clade_V 0.00000 0.00000 Clade_II 99 Cydonia_oblonga_JQ392422.1 0.17335 0.00000 51 Cydonia_oblonga_AB636344.1 0.00000 0.05771 Fragaria_moschata_AF163505.1 0.00000 99 Fragaria_virginiana_AF163479.1 0.08279 0.00000 Fragaria_nubicola_AF163517.1 0.00523 Sub_clade_VI 0.00000 0.00000 Fragaria_chiloensis_AF163511.1 0.00000 0.00000 Fragaria_chiloensis_AF163482.1 0.00000 0.00000 Fragaria_vesca_AF163510.1 0.00000

Fig.3.37. Phylogenetic analysis of ― Mamosranga‖by Maximum Likelihood Method (Jukes et al., 1969) with 1000 bootstrap replications.

155

3. 4. 20 Guraky Tango

For molecular characterizations the nucleotide sequence obtained from 18S rRNA of local pear land race ―Guraky Tango‖ having accession number Gkt-1 was initially BLAST searched in NCBI. The BLAST result showed 99% maximum identity and 88% query cover with Pyrus communis cultivars Pachkan's Triumph, Conference and Beurre.

For further conformation and identification, the phylogenetic relationship of the local pear land race ―Guraky Tango‖ with other related species and genera was inferred by using the Maximum Likelihood method (Jukes et al., 1969). A total of 41 nucleotide sequences of 18S rRNA of related species of five different genera were retrived from GenBank and subjected for organization of maximum likelihood tree. The aligned datasheet had maximum 1850 characters which were trimmed for extra and ambiguously aligned fragments from both 5‘ and 3‘ ends of alignment data sheet. As a results only 1217 characters were used for further analysis. Out of analysed characters, 248 were conserved, 242 were variable, 210 were parsimony-informative and 29 were singleton sites. None of the characters were excluded from the final analysis and multistate taxa were interpreted as uncertainty.

The evolutionary history was derived by using the Maximum Likelihood (ML) method based on the Jukes-Cantor model (Jukes et al., 1969). The tree with the highest log likelihood (-724.6311) is shown in Fig. 3.38. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. The analysis comprises of 41 nucleotide sequences and all positions containing gaps and missing data were eliminated.

The phylogenetic tree (Fig.3.38) thus organized was divided into two major clades, Clade I and II. Clade I was further divided into three sub clades, sub clade I, II and III. The sub clade I comprised of 16 Pyrus species/cultivars belong to P. pyrifolia cultivars Chojuro, Shinil, Yeoungsanbae, Kimitsukawase, Wonwhang, Soowhang, Kosui, 156

Sunwhang, Miwhang, Imamuraaki, Okusankichi, Yeoungsanbae, Minibae and P. communis cultivars Cascade, Beurre, Conference and Pachkan's Triumph. The sub clade III coprised of Pyrus pyrifolia cv. Nijisseiki and the local pear land race, Gurakay Tango.

The sub clade II comprised of 6 species/cultivars belonging to the genus Prunus. While the sub clade IV consist of different species and cultivars of the genus Rubus while the sub clade V and VI consisted of species of Cydonia and Frageria.

157

2 Pyrus _pyrifolia _cultivar _Chojuro _ AF179383.1 0.00502 0.000000 Pyrus _pyrifolia _cultivar _Shinil _ AF179396.1 0.00000 0.000000 Pyrus _pyrifolia _cultivar _Yeoungsanbae _ AF179400.1 0 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Kimitsukawase _ AF179388.1 0 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Wonwhang _ AY168822.1 0 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Soowhang _ AY168824.1 0 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Kosui _ AF179389.1 0.000000 0.00502 Pyrus _communis _cultivar _Cascade _ AF195618.1 0.000000 0.00000 Pyrus _communis _cultivar _Beurre _ AF195617.1 Sub_clade_I 0.00000 0.00000 8 Pyrus _communis _cultivar _Conference _ AF195619.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Sunwhang _ AF179398.1 0 0.00000 13 Pyrus _pyrifolia _cultivar _Miwhang _ AF179392.1 0.00000 00.00000 3 Pyrus _pyrifolia _cultivar _Imamuraaki _ AF179387.1 0.00000 0.000000.00000 Clade_I 12 0.00000 Pyrus _communis _cultivar _Pachkan's _Triumph _ AF195621.1 0.00000 100.00000 Pyrus _pyrifolia _cultivar _Okusankichi _ AF179395.1 0.00502 Pyrus _pyrifolia _cultivar _Whangkeumbae - AF179399.1 0.00000 0.00502 7 Pyrus _pyrifolia _cultivar _Minibae _ AF179391.1 0.00502 0.00000 Prunus_persica_cultivar_Kuemjeokbaekdo_AY179519.1 88 0.00000 Prunus_persica_cultivar_Youngseonghwangdo_AY179522.1 0.01007 7 0.00000 36 Prunus_persica_cultivar_Changhowonhwangdo_AY179514.1 0.000003 0.00000 Sub_clade-II 0.00000 Prunus_persica_cultivar_Baekmijosaeng_AY179513.1 0.0000040.00000 10 Prunus_persica_cultivar_Hakuho_AY179529.1 0.000000.00000 0.00000 Prunus_persica_cultivar_Cheonhong_AY179515.1 0.00000 0.50360 Pyrus _pyrifolia _cultivar _Mansoo _ AF179390 0.00502 46 Pyrus _pyrifolia _cultivar _Nijisseiki _ AF179394.1 0.00000 Sub_clade_III 0.00502 Guraky Tango 0.08460 31 Rubus_caesius_Rubus_idaeus_AF362709.1 0.00000 800.00000 Rubus_caesius_Rubus_idaeus_AF362708.1 0.00000 730.00000 Rubus_picticaulis_clone_29_AF362726.1 Sub_clade_IV 72 0.00000 0.00000 Rubus_coreanus_isolateP677_JF980336.1 0.01007 0.00000 Rubus_crataegifolius_clone_KC10-2_GU980798.1 0.01515 17 Cydonia_oblonga_AF186531.1 0.00000 100 0.000008 Cydonia_oblonga_voucher829-84D_JQ392421.1 0.00000 0.36677 100.00000 Cydonia_oblonga_JQ392420.1 Sub_clade_V 99 0.00000 Clade_II 0.00000 Cydonia_oblonga_JQ392422.1 0.17221 0.00000 44 Cydonia_oblonga_AB636344.1 0.00000 0.05423 Fragaria_moschata_AF163505.1 0.00000 100 Fragaria_virginiana_AF163479.1 0.00000 0.085995 12 Fragaria_nubicola_AF163517.1 0.00502 Sub_clade_VI 0.000000.000003 Fragaria_chiloensis_AF163511.1 0.00000 0.0000015 Fragaria_chiloensis_AF163482.1 0.00000 0.00000 Fragaria_vesca_AF163510.1 0.00000

Fig. 3.38. Phylogenetic analysis of “Guraky Tango” by Maximum Likelihood Method (ML)

(Jukes et al., 1969) with 1000 bootstrap replications.

158

3. 4. 21 Ghata Tora Tangai (Gtt)

For molecular characterizations, the nucleotide sequence obtained from 18S rRNA of the local pear land race ―Ghata Tora Tangai (Gtt)‖ was compared to the nucleotide sequences of reference liberary with the help of BLAST searched in NCBI. The BLAST result showed 100% maximum identity and query cover with Pyrus pyrifolia cv. Imamuraaki while the blast result also showed 99% maximum identity and 100% query cover with P. communis cultivars. Pachkans Triumph and Beurre, P. pyrifolia cv. Imamuraaki.

For further conformation and identification, the phylogenetic relationship of the local pear land race ―Ghata Tora Tangai (Gtt)‖ with other related species and genera was inferred by Maximum Likelihood method (Jukes et al., 1969). A total of 41 sequences of 18S rRNA of related species of five different genera were retrived from GenBank and were subjected for construction of maximum likelihood cladogram. The aligned datasheet had maximum 1850 characters which were trimmed for extra and ambiguously aligned fragments both 5‘ and 3‘ ends of alignment data sheet. A total of 1175 characters were used for further analysis. Out of analysed characters, 291 were conserved, 194 were variable, 07 were parsimony-informative and 187 were singleton sites. None of the characters were excluded from the final analysis and multistate taxa were interpreted as uncertain.

The evolutionary history was derived by using the Maximum Likelihood method based on the bases of Jukes-Cantor model (Jukes et al., 1969). The tree with the highest log likelihood (-1010.9077) is shown in Fig.3.39. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree‘s for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. The analysis comprises of 41 nucleotide sequences and all positions containing gaps and missing data were eliminated.

159

The phylogenetic tree (Fig.3.39) segregated the data into two major clades, Clade I and II. Clade I was further divided into two sub clades, sub-Clade I and II. The sub clade I comprised of 19 Pyrus species/cultivars viz. P. pyrifolia cultivars Yeoungsanbae, Shinil, Kimitsukawase, Wonwhang, Soowhang, Chojuro, Sunwhang, Miwhang, Imamuraaki Nijisseiki, Okusankichi, Minibae, Whangkeumbae and Kosui and P. communis cultivars Cascade, Beurre, Pachkan's Triumph, Conference and Ghata Tora Tangai (Gtt). The local landrace, Ghata Tora Tangai (Gtt) fall independently within the Pyrus clade but does not make cluster to any of the Pyrus species/cultivars. This shows that it is a separate species of the genus Pyrus.

The sub clade II comprised of 6 species/cultivars belonging to the genus Prunus. While the sub clade III, IV and V belong to Clade II comprised of related genera of the genus Pyrus belonging to family Rosaceae.

160

Pyrus _pyrifolia _cultivar _Yeoungsanbae _ AF179400.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Shinil _ AF179396.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Kimitsukawase _ AF179388.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Wonwhang _ AY168822.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Soowhang _ AY168824.1 0.00000 0.00000 Pyrus _communis _cultivar _Cascade _ AF195618.1 0.00000 0.00000 Pyrus _communis _cultivar _Beurre _ AF195617.1 0.00000 0.00000 Pyrus _communis _cultivar _Pachkan's _Triumph _ AF195621.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Chojuro _ AF179383.1 0.00000 0.00429 Pyrus _pyrifolia _cultivar _Sunwhang _ AF179398.1 Sub_clade_I 0.00429 0.00000 Pyrus _pyrifolia _cultivar _Miwhang _ AF179392.1 0.00000 Pyrus _pyrifolia _cultivar _Imamuraaki _ AF179387.1 0.00000 0.00000 0.00000 0.00000 Pyrus _communis _cultivar _Conference _ AF195619.1 Clade_I 0.00429 0.00000 Pyrus _pyrifolia _cultivar _Nijisseiki _ AF179394.1 0.00429 0.00000 Ghata Tora Tangai_Gtt 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Okusankichi _ AF179395.1 0.00429 Pyrus _pyrifolia _cultivar _Minibae _ AF179391.1 0.00000 0.00429 Pyrus _pyrifolia _cultivar _Whangkeumbae - AF179399.1 0.00000 0.00429 Pyrus _pyrifolia _cultivar _Kosui _ AF179389.1 0.00860 Prunus_persica_cultivar_Kuemjeokbaekdo_AY179519.1 0.00429 0.00429 0.00000 Prunus_persica_cultivar_Cheonhong_AY179515.1 85 0.00000 0.00860 Prunus_persica_cultivar_Youngseonghwangdo_AY179522.1 0.00000 Sub_clade_II Prunus_persica_cultivar_Changhowonhwangdo_AY179514.1 0.47748 0.00000 0.00000 0.00000 Prunus_persica_cultivar_Baekmijosaeng_AY179513.1 0.00000 0.00000 Prunus_persica_cultivar_Hakuho_AY179529.1 0.00000 Pyrus _pyrifolia _cultivar _Mansoo _ AF179390 0.00000 Cydonia_oblonga_AF186531.1 0.00000 0.00000 Cydonia_oblonga_voucher829-84D_JQ392421.1 0.00000 0.00000 99 Cydonia_oblonga_JQ392420.1 Sub_clade_III 0.00000 0.08150 Cydonia_oblonga_AB636344.1 0.00000 0.00000 Cydonia_oblonga_JQ392422.1 0.00000 Rubus_caesius_Rubus_idaeus_AF362709.1 100 0.00000 790.00000 Rubus_caesius_Rubus_idaeus_AF362708.1 0.33575 0.00000 0.00000 75 Rubus_picticaulis_clone_29_AF362726.1 Sub_clade_IV 0.00000 Clade_II 0.03885 Rubus_coreanus_isolateP677_JF980336.1 0.00860 67 0.00000 Rubus_crataegifolius_clone_KC10-2_GU980798.1 0.01293 0.07106 Fragaria_virginiana_AF163479.1 0.00000 100 Fragaria_moschata_AF163505.1 0.00000 0.08355 0.00000 Fragaria_vesca_AF163510.1 0.00429 Sub_clade_V 0.00000 Fragaria_nubicola_AF163517.1 0.00429 0.00000 Fragaria_chiloensis_AF163482.1 0.00000 0.00000 Fragaria_chiloensis_AF163511.1 0.00000

Fig. 3.39. Molecular phylogenetic analysis of ―Ghata Tora Tangai ‖, by Maximum Likelihood Method (Jukes et al., 1969) with 1000 bootstrap replications.

161

3. 4. 22 Shaker Tango

The nucleotide sequence obtained from 18S rRNA local pear land race ‖Shaker Tango‖ having accession no. St_1 was BLAST in NCBI for molecular characterization. The BLAST result showed 97% maximum identity and 93% query cover with Pyrus communis cultivars Pachkans Triumph, Conference, Beurre, Cascade and P. pyrifolia cv. Sunwhang.

Further identification and phylogenetic relationship of the local pear land race ―ShakerTango‖ with other related species and genera was inferred by using the Maximum Likelihood method (Jukes et al., 1969). For this purpose, a total of 41 sequences of 18S rRNA of related species and genera were retrived from GenBank and were subjected for construction of maximum likelihood tree. The aligned datasheet had maximum 1850 characters which were trimmed for extra and ambiguously aligned fragments from both 5‘ and 3‘ ends of alignment data sheet. As a result, a total of 1169 characters were used for further analysis. Out of the analysed characters, 285 were conserved, 254 were variable, 227 were parsimony-informative and 27 were singleton sites. None of the characters were excluded from the final analysis and multistate taxa were interpreted as uncertainty.

The evolutionary history was derived by using the Maximum Likelihood (ML) method based on the Jukes-Cantor model (Jukes et al., 1969). The tree with the highest log likelihood (-1010.9077) is shown in Fig.3.40. The percentage of trees in which the associated taxa clustered together was shown next to the branches. Initial tree‘s for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. The analysis comprises of 41 nucleotide sequences and all positions containing gaps and missing data were eliminated. The phylogenetic tree (Fig.3.40) thus developed was divided into two major clades, Clade I and Clade II. Clade I was further divided into two sub clades, sub-Clade I and sub clade II. The sub clade I comprised of 19 Pyrus species/cultivars viz. P. pyrifolia

162

cultivars Yeoungsanbae, Shinil, Kimitsukawase, Wonwhang, Soowhang, Cascade, Chojuro, Sunwhang, Miwhang, Imamuraaki, Nijisseiki, Shaker Tango, Okusankichi, Minibae, Whangkeumbae and. Kosui and P. communis cultivars Beurre, Pachkan's Triumph, Conference and Shaker Tango (St). The local landrace, Shaker Tango (St) fall in sub clade I (Pyrus clade) independently and does not make cluster with any of available Pyrus species and cultivar. This shows that, it may be a separate species of the genus Pyrus. Sub clades II, III, IV and V consist of related genera to the genus Pyrus belonging to the family Rosaceae.

163

Pyrus _pyrifolia _cultivar _Yeoungsanbae _ AF179400.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Shinil _ AF179396.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Kimitsukawase _ AF179388.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Wonwhang _ AY168822.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Soowhang _ AY168824.1 0.00000 0.00000 Pyrus _communis _cultivar _Cascade _ AF195618.1 0.00000 0.00000 Pyrus _communis _cultivar _Beurre _ AF195617.1 0.00000 0.00000 Pyrus _communis _cultivar _Pachkan's _Triumph _ AF195621.1 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Chojuro _ AF179383.1 0.00000 0.00429 Pyrus _pyrifolia _cultivar _Sunwhang _ AF179398.1 Sub_clade_I 0.00429 0.00000 Pyrus _pyrifolia _cultivar _Miwhang _ AF179392.1 0.00000 Pyrus _pyrifolia _cultivar _Imamuraaki _ AF179387.1 0.00000 0.00000 0.00000 0.00000 Pyrus _communis _cultivar _Conference _ AF195619.1 Clade_I 0.00429 0.00000 Pyrus _pyrifolia _cultivar _Nijisseiki _ AF179394.1 0.00429 0.00000 Shaker_Tango_St 0.00000 0.00000 Pyrus _pyrifolia _cultivar _Okusankichi _ AF179395.1 0.00429 Pyrus _pyrifolia _cultivar _Minibae _ AF179391.1 0.00000 0.00429 Pyrus _pyrifolia _cultivar _Whangkeumbae - AF179399.1 0.00000 0.00429 Pyrus _pyrifolia _cultivar _Kosui _ AF179389.1 0.00860 Prunus_persica_cultivar_Kuemjeokbaekdo_AY179519.1 0.00429 0.00429 0.00000 Prunus_persica_cultivar_Cheonhong_AY179515.1 85 0.00000 0.00860 Prunus_persica_cultivar_Youngseonghwangdo_AY179522.1 0.00000 Sub_clade_II Prunus_persica_cultivar_Changhowonhwangdo_AY179514.1 0.47748 0.00000 0.00000 0.00000 Prunus_persica_cultivar_Baekmijosaeng_AY179513.1 0.00000 0.00000 Prunus_persica_cultivar_Hakuho_AY179529.1 0.00000 Pyrus _pyrifolia _cultivar _Mansoo _ AF179390 0.00000 Cydonia_oblonga_AF186531.1 0.00000 0.00000 Cydonia_oblonga_voucher829-84D_JQ392421.1 0.00000 0.00000 99 Cydonia_oblonga_JQ392420.1 Sub_clade_III 0.00000 0.08150 Cydonia_oblonga_AB636344.1 0.00000 0.00000 Cydonia_oblonga_JQ392422.1 0.00000 Rubus_caesius_Rubus_idaeus_AF362709.1 100 0.00000 790.00000 Rubus_caesius_Rubus_idaeus_AF362708.1 0.33575 0.00000 0.00000 75 Rubus_picticaulis_clone_29_AF362726.1 Sub_clade_IV 0.00000 Clade_II 0.03885 Rubus_coreanus_isolateP677_JF980336.1 0.00860 67 0.00000 Rubus_crataegifolius_clone_KC10-2_GU980798.1 0.01293 0.07106 Fragaria_virginiana_AF163479.1 0.00000 100 Fragaria_moschata_AF163505.1 0.00000 0.08355 0.00000 Fragaria_vesca_AF163510.1 0.00429 Sub_clade_V 0.00000 Fragaria_nubicola_AF163517.1 0.00429 0.00000 Fragaria_chiloensis_AF163482.1 0.00000 0.00000 Fragaria_chiloensis_AF163511.1 0.00000

Fig. 3.40. Phylogenetic analysis of ―Shaker Tango‖ by Maximum Likelihood Method (ML)

(Jukes et al., 1969) with 1000 bootstrap replications.

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3. 4. 23 “Pak-24”

For molecular characterizations, the nucleotide sequence obtained from 18S rRNA of local pear land race ―Pak-24‖ was compared to the sequences of database library with the help of BLAST searched in NCBI. The BLAST result revealed 100% maximum identity and query cover with Pyrus pyrifolia cultivars Wonwhang and Kimitsuka while the BLAST result also showed 99% maximum identity and 100% query cover with P. pyrifolia cv. Soowhang

For further conformation and identification, the phylogenetic relationship of the local land race ―PAK-24‖ with other related species and genera was inferred by using Maximum Likelihood mehod (Jukes et al., 1969). A total of 42 sequences of 18S rRNA of related species belonged to five different genera were retrived from GenBank and were subjected for construction of maximum likelihood tree. The aligned datasheet had maximum 1850 characters which was trimmed for extra and ambiguously aligned fragments both from 5‘ and 3‘ ends of the alignet data sheet. As a result, only 1168 characters were used for further analysis. Out of the analysed characters, 242 were conserved, 251 were variable, 226 were parsimony informative and 25 were singleton sites. None of the characters were excluded from the final analysis and multistate taxa were interpreted as uncertainty.

The evolutionary history was derived by using the Maximum Likelihood (ML) method based on the Jukes-Cantor model (Jukes et al., 1969). The tree with the highest log likelihood (-1010.9077) is shown in Fig. 3.41. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree‘s for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. The analysis comprises of 41 nucleotide sequences and all positions containing gaps and missing data were eliminated.

The phylogenetic tree (Fig.3.41) divided the Pyrus land races into two major clades, Clade I and II. Clade I was further sub divided into two sub clades, sub-clade I and sub 165

clade II. The Sub clade I consist of 19 Pyrus species/cultivars viz. P. pyrifolia cultivars Yeoungsanbae, Shinil, Kimitsukawase, Wonwhang, Soowhang, Chojuro, Sunwhang, Miwhang, Imamuraaki, Nijisseiki, Okusankichi, Minibae, Whangkeumbae and Kosui. The P. communis cultivars consists of Conference, Cascade, Beurre, Pachkan's Triumph and the local pear land race, PAK-24. The results showed that the local landrace, PAK- 24 was placed in the sub clade I, Pyrus clade but does not make cluster or close resemblance to the available species and cultivars. This shows that it is an independent species of the genus Pyrus. The sub clades II, III, IV and V comprised related genera to the genus Pyrus, belonging to the family Rosaceae.

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Fig. 3.41. Phylogenetic analysis of ―Pak-24‖ by Maximum Likelihood Method (ML)(Jukes et al., 1969) with 1000 bootstrap replications .

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3. 4. 24 Shaker Tango

For molecular characterizations and evaluation the nucleotide sequence of the local land race, Shaker Tango was initially BLAST in NCBI. The BLAST result showed 97% maximum identity and 93% query cover with Pyrus communis cultivars Pachkans Triumph, Conference, Beurre, Cascade and P. pyrifolia cv. Sunwhang.

For further confirmation and identification, the phylogenetic relationship of the local land race, Shaker Tango with other related species and genera was inferred by using the Maximum Likelihood method. A total of 43 nucleotide sequences of 18S rRNA of related species and genera were retrived from GenBank and were subjected for construction of maximum likelihood tree. The aligned datasheet has a maximum of 1783 characters and after trimming the extra and ambiguously aligned fragments from both 5‘ and 3‘ ends of alignment data sheet, a total of 1210 characters were used for further analysis. Out of these characters, 138 were conserved, 360 were variable, 335 were parsimony-informative and 25 were singleton sites. None of the characters were excluded from the final analysis and multistate taxa were interpreted as uncertainty.

Phylogenetic tree (Fig.3.42) was divided into three major clades, Prunus_clade, Rubus_clade and Pyrus_clade. Prunus and Rubus Clades consists of the reference species/cultivars of Prunus and Rubus genera.

Pyrus_clade further divided into Pyrus sub clade_1 and Pyrus sub_clade_2. The local land race, Shaker Tango fall independently adjusent to P. communis cv. Pachkan's Triumph in the Pyrus sub_clade_1 of Pyrus _clade. The local landrace, Shaker Tango does not make cluster to any of the available species/cultivars of the genus Pyrus. This shows that it may be a sparate species of the genus Pyrus and need further work for its evaluation.

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71 Prunus_padus_JQ926626.1 12 Prunus_padus_ JQ926626.1 92 Prunus_virginiana_JQ926625.1

26 Prunus_virginiana_ JQ926625.1 Prunus_ovalis_Q926613.1 Prunus_reflexa_JQ926614.1 98 35 41 Prunus_amplifolia_Q926612.1 85 Prunus_myrtifolia_JQ926611.1 Prunus_scoparia_JQ926604.1 Prunus Clade

59 91 Prunus_spinosissima_JQ926605.1 49 Prunus_mira_18S_JQ926602.1

100 Prunus_buergeriana_JQ926628.1 Prunus_buergeriana_JQ926628.1

99 Prunus_arborea_JQ926621.1 59 Prunus_arborea_JQ926621.1 100 96 Prunus_henryi_JQ926620.1 89 Prunus_henryi_ JQ926620.1 Rubus_corchorifolius_ JF708203.1

33 Rubus_crataegifolius_clone_KC10-4_ GU980800.1 98 Rubus_crataegifolius_clone_KC10-4_ GU980800.1 85 Rubus_crataegifolius_GU980800.1 93 Rubus_crataegifolius_GU980799.1 1 93 Rubus_crataegifolius_clone_KC10-3_ GU980799.1 Rubus_coreanus_isolate_P677_ JF980336.1 Rubus Clade 50 Rubus_reflexus_isolate_ JN407523.1 Rubus_caesius_Rubus_idaeus_ AF362717.1 36 Rubus_corchorifolius_isolate_HLJ_ DQ217770.1 42 Rubus_picticaulis_clone_29_ AF362726.1 7 4 Rubus_maximiformis_clone_9_ AF362722.1 11 Rubus_idaeus_AF055757.2 Pyrus_bretschneideri_cultivar_Dangshan_ JF911820.1 Prunus_persica_(L.)_Batsch._ L28749.1 Pyrus_pyrifolia_cultivar_Tama_ AY168823.1 Pyrus_communis _cultivar _Beurre _ AF195617.1 62 10 4 Pyrus _communis _cultivar _Cascade _ AF195618.1 4 12 Pyrus_communis _cultivar _Conference _ AF195619.1 P. Sub. Clade-1 37 Pyrus_ communis_cultivar_Pachkan's _Triumph_ AF195621.1 Shaker Tango (St) 10 Pyrus _communis _cultivar _Clapp's _Favorite _ AF195620.1 Pyrus Clade Pyrus _pyrifolia _cultivar _Sunwhang_ AF179398.1 0 Pyrus_pyrifolia _cultivar _Miwhang _ AF179392.1 52 Pyrus _pyrifolia _cultivar _Kosui _ AF179389.1 P.Sub.Clade.2 3 Pyrus _pyrifolia _cultivar _Imamuraaki _ AF179387.1 2 3 Pyrus _pyrifolia _cultivar _Soowhang_ AY168824.1 8 Pyrus _pyrifolia _cultivar _Wonwhang _ AY168822.1

Fig. 3.42 Phylogenetic analysis of ―Shaker Tango St‖ by maximum Likelihood Method (ML) with 1000 bootstrap replications by adopting Jukes Cantor Model.

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Chapter 4 DISCUSSION

The information obtained from the numerical, phenotypic and molecular analysis of 110 specimens collected from 70 land races sporadically found in the study area spread over 100565 Km2 of Northern Pakistan revealed that only two species viz. Pyrus communis and P. pashia were previously recognized from the area. Our findings have identified 12 new species prevesiously not documented in the species diversity of Pyrus from Pakistan. The results obtained are logically discussed in this section under the headings numerical variations, taxonomic description of the holotypes, markers assisted assay and finally the 18S rRNA conformation of new taxa. 4.1. NUMERICAL VARIATIONS The taxomonically important numerical traits of leaf and fruit which morphologically differentiated the taxa were analyzed and discussed below:

4.1.1 Petiole length

Analysis of variance (Table 3.2) showed significant differences with respect to petiole length which showed that all land races were genetically different with respect to petiole length. Variation in table of means showed (Table3.1) that the length of petiole ranged from 30.6 – 56.07 mm. The largest petioles were recorded for land race Kushbago Batang (Kbb) which was 56.07 mm, followed by Atti Batang (49.00 mm) and Shardi Tanchi (Srt) (48.47mm). The shortest petioles were observed in Batangi (30.60 mm). Our results with respect to variation in petiole length are in conformity with the available information. For example Boucek (1954) reported that petiole length in Pyrus ranges from 30–50 mm, Roloff (1998) has reported a rang of 20- 50 mm, while Hofmann (1993) and Muller & Litschauer (1994) gave the values up to 60 mm in different cultivars of pear. Similarly Terpo (1960) and Peniastekova (1992) reported a range of 20–70 mm for petiole length of different cultivars of pears. Boratynska (1990) has reported 15–70 mm range of petiole length. Franci batang (42.03 mm) and Ghata Zira Tangai (Gzt) (44.83 mm) have very similar values. In the same way, Kado batang (Kb), Tanchi (Tb) and Nak batang (Nb) have closely related values with respect to petiole length. The land races, China batang (Cb), Kacha Zira Tangai (Kzt) and Klak Nak (Nhs)

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also have variation for petiole length falling in a narrow range of 35 to 37mm. All these land races showed variation with respect to petiole length. Islam & Ahmad (2012) had previously evaluated 14 Pyrus landraces through petiole lengths and other agronomic and yield parameters for assessing variability.

4.1.2 Leaf area

Analysis of variance (Table 3.2) showed non-significant (0.1345) result at 5% level of significance and showed that the land races were genetically similar with respect to leaf area. The highest value of mean with respect to leaf area was for land race, Ghata Zira Tangai (Gzt) (2672A), while all others land races have closely related values and fall in the same group-B, showing resemblance with each other with respect to leaf area. The present result is supported by previous work of Rittershoffer (1998) and Kuhn (1998), having reported the ratio of leaf length to leaf width as 0.9–1.59 mm. Wagner (1995) had concluded the relative ratio of leaf length to its width which was equal to 1.00 in Pyrus land races. Through similar traits, the genotypes of species in other generas were also studied and evaluated successfully (Islam et al., 2004; Mekki, 2013).

4.1.3 Pedicel length

Analysis of variance (Table 3.2) showed that the difference among land races were significant at 5% level of probability (0.0000) with respect to pedicel length. The highest mean value for pedicel length was noted for the land race, Kushbago Batang (65.53 mm) followed by Taunchi batang (Tb) and Glass Batang (50.87 B). Similarly, Terpo (1960) had reported that pedicel length ranged from 15–50 mm in different cultivars of Pyrus. In the current study, the lowest values of pedicel length ranged from 15-17 mm for the land races, Klak Nak (Kn), Shardi Tannchi (Srt) and Batangi. These finding were also supported by Kuhn (1998) who reported pedicel length of 6–16 mm in different land races of Pyrus species. The remaining genotypes in present work showed intermediate values with respect to pedicel length. Islam and Ahmad, 2012 had evaluated fourteen land races of Pyrus in which the pedicel length ranged from 15-65 mm. Accordingly, Muller and Litschauer (1994) conducted a study in which the length of pedicels were longer than fruits. Similary, Hofmann (1993), Boucek (1954) and

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Boratynska (1990) recorded 10–35 mm, 20–40 mm and 20–50 mm pedicel lengths, respectively in their studies.

4.1.4 Fruit length

Analysis of variance (Table 3.2) showed significant (0.0000) variation at 5% level of significance with respect to fruit length while the LSD value at (5%) was noted to be 10.058. The lowest mean value for fruit length was recorded for land races, Batangi (21.47 mm), Shardi taunchi (23.30 mm) and Ghata Zira Tangai (23.80 mm), respectively. while highest mean value was shown by land race, Glass Batang (95.23A), followed by Kado Batang (73.17 mm) and China Batang (72.23 mm). The land races, Klak Nak (Kn) and Atti Batang showed intermediate values, respectively. Guleryuz and Ercisli (1997) reported 61-91 mm fruit length in pear cultivars. According to Terpo (1960) the length of fruits within the taxon were 25-50 mm and depends on the variety. Similarly Peniastekova (1992) observed the range of fruit length from 30–50 mm in different cultivars of Pyrus.

4.1.5 Fruit width

Analysis of variance (Table 3.2) showed significant variation (0.0000) with respect to fruit width at 5% level of probability. The highest value of mean was observed for gennotype, China batang (59.37), followed by land race, Klak Nak (54.97mm). The lowest value was shown by land races, Batangi (23.60 mm), Shardi taunchi (23.40 mm) and Ghata Zira Tangai (25.93 mm), respectively. The land races, Kushbago Batang (Kbb), Kadobatang and Glass Batang showed intermediate values with respect to fruit width. Edizer & Gunes (1997) have reported 59 - 78 mm fruit width. Terpo (1960) has recorded that the values of fruit width ranged from 25–50 mm. Similarly, Peniastekova (1992) observed that fruit width ranged from 30–50 mm in different cultivars of Pyrus species.

4.1.6 Fruit weight

Analysis of variance (Table 3.2) with respect to fruit weight showed statistically significant differences (0.0000) at 5% level of probability. The land races, Glass Batang (163.4 g), and China batang (148.0 g) have the highest mean value followed by land

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races, Kado batang (113.2 g), Kushbago Batang (109.2g) and Klak Nak (108.5 g). Similarly Karadeniz and Sen (1990) reported that fruit weight ranged from 50 – 368 g in pear cultivars. In the current work, land races, Batangi and Shardi Tanchi (Srt) possess lowest values (8.7, 9. 3 g), respectively with respect to fruit weight. The present data is supported by the results of Abe et al., (1993) who concluded that the fruit weight of wild ancestor of the cultivated Japanese pear was probably only a few grams. Similarly, the local land races, Franci Batang (Fb) and Atti Batang (Ab) have 81.27g and 52.67g fruit weight, respectively and hold intermediate values as compared to other land races. Modern cultivars produce excellent fruits and a single fruit may weigh up to 2 kg (2000 g), showing a 100-fold increase in weight over wild ancestors (Abe et al., 1993). Previously, fourteen land races of Pyrus were evaluated for yield parmeters in which the fruit weight ranged from 8-113 g (Islam & Ahmad, 2012). The above parameter have also been used to evaluate several other cultivars successfully (Ahmad et al., 2008; Bendhifi et al., 2013).

Before the discovery of molecular markers, morphological markers were used to study the genetic variability to obtain phylogenetic trees (Raven et al., 2004). Morphological markers correspond to the visualy scoring of qualitative traits and are influenced by environmental factors, plant biology and the plant developmental stage (Paul et al., 2010; Ali et al., 2011). In the present investigation, the data generated from morphological markers, were considered for cluster analysis. The dendrogram thus constructed (Fig.3.1), segregated all the local land races into two main groups, A and B. Group-A was further divided into two subgroups A1 and A2. Subgroup A1 consisted of 4 land races while sub group A2 consisted 5 of land races. Group-B comprised of 4 land races of pears (Islam & Ahmad, 2012).

4.2 Markers assisted assay 4.2.1. DNA Isolation

The modified protocol is used for DNA extraction in present study can be utilized throughout the year for any part of plant while no sophisticated equipment‘s, high grade chemicals, liquid nitrogen, RNase were used. The extracted DNA was intact, had

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high quantity, quality and was found highly suitable for several molecular biological applications such as PCR; nucleotides sequencing, RAPD and SSR etc. DNA isolation from mature trees of high altitude is difficult job due to the presence of a large number of phenolic compounds (Gupta et al., 2011). The co-precipitation of impurities such as terpenes and polyphenolics present additional complications (Aganga & Tshwenyane, 2003). Similarl, highly viscous polysaccharides (Adams & Do, 1991; Shepherd et al., 2002) and rigid polysaccharide cell wall, tannin, secondary metabolites and pigments make DNA isolation difficult (Varma et al., 2007). However, isolation of genomic DNA from mature tree species is an important issue in plant molecular biology (Barzegari et al., 2010; Li et al., 2010; Smyth et al., 2010). To overcome the above hindrances and resolve this issue, a protocol was developed in this study. The DNA extracted using this protocol was suitable for PCR and other downstream applications in molecular biology. It is evident from the figure (Fig. 3.15) that all the samples had approximately similar amount of best quality DNA. In this protocol no sophisticated equipments, no costly chemicals, no RNase treatment even during grinding no use of liquid nitrogen or incubation in oven were used. For incubation, common gas heater and steel pot were used. Due to frequent power failures, the plant materials were not stored in -20ºC but it were grinded and stored at room temperature as stock materials. By using the modified protocol (Islam et al., 2013), high quality genomic DNA were extracted (Fig.3.15), suitable for PCR reaction, other biological tools and applications. Furthermore to counter check the quality of the extracted DNA, the PCR amplified products of the Pyrus samples were eluted and sequenced (Fig.3.16). The sequence data obtained were BLAST against the available nucleotide database using online tool at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequence data confirmed that the amplified fragments were indeed 18S rRNA of pears. The protocol developed in the present study, applied as a useful tool for molecular biology of woody trees, herbarium specimens, mushrooms, cultivated wild plants etc. The protocol is economical, less time consuming and handy in application.

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4.2.2 The RAPD Assay

Molecular tools have the ability to detect variation at the DNA level and generate valuable data on diversity. On the basis of these data, individuals can be identified and classified accurately. A number of molecular techniques are available for studying genetic variation among individuals. The selection and application of these techniques for any specific use depend upon on the nature of materials and problem. With the invention of new technologies, DNA polymorphisms have become the markers of choice for studying genetic variation at molecular level. DNA markers are helpful both in basic and applied research (Somasundaram & Kalaiselvam, 2010).

Numerous molecular markers have been used for genetic variation, DNA fingerprinting and plant systematic etc. Among these, RAPD are the most widely used markers which can amplify many loci in a single PCR reaction. RAPD markers are inherited in a simple Mendlian fashion. They are less expensive, faster and require only a small amount of DNA. The results are reproducible, the technique require less skill for operation and does not require any radioisotopes for detection. Therefore, RAPD have proven to be used in land race identification and gene mapping (Demeke & Adams, 1994). They are convenient in performance and do not require sequencing of the amplified band (Weder, 2002). On the basis of these characteristics, RAPD technology has proven to be a powerful tool used for evaluation of genetic diversity, differentiation and taxonomic study of any species including pears.

In the present work, 28 RAPD markers were found to amplify 304 reproducible bands from 304 loci. The band sizes of the amplicons ranged from 150 bp-2600 bp. A total of 1983 polymorphic bands were generated from these 304 loci across 36 Pyrus land races. All the bands were polymorphic in nature and could be used for differentiation of the local land races of pears. Monte-Corvo et al., (2000) applied 25 RAPD markers on different cultivars of Pyrus, generating a total of 324 bands among which 271 were polymorphic. The bands represented 84% polymorphism and were found to be highly reproducible. Lee et al., (2004) used RAPD markers for identification and classification of P. pyrifolia and P. communis cultivars. Beside RAPD markers, other molecular markers, SSR were applied on different wild and cultivated individuals of Pyrus for

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clustering into different groups belonging to different geographical regions (Volk, et al., 2006). For example, 56 land races of Pyrus were evaluated through 12 SSR markers, a total of 106 polymorphic alleles and 11.8 alleles per locus were identified. The band sizes were ranged from 83 to 328 bp and polymorphism ranged from 0.78% to 0.84% (Ahmed et al., 2010). Similarly, 18 SSR markers were applied on 228 pear accessions belonging to different eco-geographical regions which amplified 308 alleles. Analysis revealed that the Anatolian pears accessions were intermixed among different eco- geographical regions due to gene flow and shifting of germplasm (Akcay et al., 2014). A sum of 60 RAPD primers were used for differentiation of 36 pear land races in which 28 markers generated 304 reproducable bands with band size ranging from 150-2600bp (Islam et al., 2014).

In the present work, a total of 1983 reproducaible bands were generated by 304 loci. Among these, the maximum number of bands (111) were produced by land races 9- Mamosranga and 21-Zira Mamothy (Zm) while the minimum number of band (01) was produced by land race 29-Shaker Batang (Sb-B). All the bands were polymorphic, therefore showing 100% polymorphism. Oliveira et al., (1999) used 60 RAPD primers to obtain polymorphic patterns of Occidental and Oriental pear land races. Out of the 60 primers tested, 22 primers generated 358 prominent and reproducible bands. Among these 358 bands, 327 (91%) were polymorphic. In another study, a total of 25 Pyrus communis cultivars including eight traditional Portuguese pears and 17 Pyrus pyrifolia were evaluated through 25 RAPD markers. The markers genehrated 324 bands, among which 271 (84%) were polymorphic (Monte-Corvo et al., 2000). Similarly, a total of 28 RAPD primers were applied on 36 Pyrus land races, which amplified from 304 loci, a total of 1983 bands produced and showed 100%polymorphism (Islam et al., 2014). Similarly, RAPD technique was also used for the investigation of genetic diversity of leguminous plants (Gomez et al., 2011). In the present investigation, out of 28 RAPD primers, 14 primers showed specificity to some land race by producing 35 different band sizes, ranging from 150 to 2100bp (Islam et al., 2014). Teng et al., (2001) and Teng & Tanabe (2002) evaluated and analysed 118 species and cultivars for genetic similarities and variations through RAPD markers and revealed that some markers showed specificity to species and 176

cultivars. Similarly, RAPD technology has been used for markers specificity, Oliveira et al., (1999) reported that some RAPD primers were cultivars and species specific and can be used for identification in pears. RAPD, SCAR and 18S rDNA were used for identification of pear cultivars and their comparison of specificity with respect to pear cultivars (Lee et al., 2004). Kesseli et al. (1994) carried out a study for the targeted and randomly produced primers such RFLP, RAPD, SCAR and AFLP etc for crop breeding programs and their suitability in forest genetic research. Some RAPD markers have shown specific linkage to major genes that controllskin colour in Japanese pear, which is important with respect to market value and external pressures. The RAPD marker

(OPH-19425) was found specific to a particular trait such as green fruit color, useful in breeding program (Inoue et al., 2006). Muhammad et al., (2013) used RAPD primers for the investigation of sugarcane cultivars and recorded 11 cultivar specific loci. In the present finding, fourteen (14) land race specific RAPD primers produced 35 different size bands/loci in the range of 150-2100bp specific (Table 3.5) to twenty (20) different Pyrus land races. These findings were also supported by (Islam et al., 2014). Similar work was also conducted in the sugarcane cultivars (Muhammad et al., 2013).

For molecular and phenotypic characterization of different cultivars and landraces of P. communis, P. pyrifolia and other wild species of the genus Pyrus were evaluated through RAPD primers (Oliveira et al., 1999). The generation of SCAR primers, derived from RAPD primers were used for identification of Pyrus pyrifolia (ChungSun et al., 2000; Lee et al., 2004). Intraspecific and interspecific genetic distances and similarities have been studied through various DNA markers in East Asian pears. Similarly, RAPD primers were used for discrimination of mutant Rhododendron land races (Atak et al., 2011).

A homology tree constructed on the basis of similarity and discriminated 36 Pyrus land races into six major groups, shown by Roman letters i.e I, II, III, IV, V and VI. Oliveira et al. (1999) reported 4 major groups of 13 Pyrus land races on the bases of 22 RAPD markers and supported the Occidential and Oriental geographical pattern of Pyrus classification. Monte-Corvo et al., (2000) and Botta et al., (1997) used RAPD, AFLP for genetic similarity of Pyrus cultivars and varieties of Pyrus. Similarly

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different cultivars of P. pyrifolia and P. communis were identified by RAPD and SCAR primers (Lee et al., 2004). Group-I was further subdivided into 3 sub- group/clusters i.e IA, IB and IC. Kim, (2004) conducted a similar study by using RAPD primers on 33 Asian pear cultivars and identified 4 groups. Group-II consisted of nine (9) sub-groups, Group-III consisted of three sub-group i.e IIIA, IIIB and IIIC Group-IV consisted of 3 sub-groups. ChungSun et al., (2000) successfully differentiated 19 Japanese pear landraces through 82 RAPD primers. In another study, three main clusters were reported in Pyrus germplam which showed various affinity levels in the dendrogram (Ahmed et al., 2010).

Group-V consists of two sub-groups that is VA and VB .Similarly Group-VI comprises of land races (OTUs) 21- Zira Mamothy (Zm) and 22- Spina Mamothy (Sm). Therefore, all 36 land races of Pyrus were distributed into 19 sub-groups. Monte- Corvo et al., (2000) carried out a comparative study of RAPD and AFLP primers to evaluate the relationships among pear cultivars. Pyrus pyrifolia cultivars possessed a closer genetic similarities and formed a cluster located far away from the cluster of P. communis cultivars. Most of the Portuguese land races clustered together and showed an independent genetic pool which can be helpful in pear breeding programs. Six RAPD markers and fourteen (SCAR) specific primers with two negative markers were used for identification of pear cultivars of P. pyrifolia (DongHyeon & KiHyun, 2000; Kim et al., 2005). Similarly in the last few decades various types of DNA markers such as RAPD, SSR, AFLP etc have been used for evaluation of genetic diversity and discrimination of Pyrus land races (Yamamoto & Chevreau, 2009). In the present study, a phylogenetic tree has been developed on the basis of RAPD data and discriminated 36 Pyrus land races into 6 major groups and match the homology tree by 95%.

In addition to above discussion, RAPD technique has also been applied to various types of plants land races ranging from herbaceous crop to horticultural trees and herbs to wild trees. Different species and cultivars of Allium and Brassica have been evaluated through RAPD echnique (Wilkie et al., 1993). Similarly, different medicinal plants have

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been evaluated for authentication, identification and taxonomic study (Arif et al., 2010; Khan et al., 2010).

5.4 Molecular Characterizations and Discrimination through 18S ribosomal rRNA

The genus Pyrus belongs to family Rosaceae and sub-family Maloideae (Challice & Westwood, 1973). On the basis of lmorphological and geographical features, the genus Pyrus has been divided into two groups i.e. the Oriential and Occidential pears (Zhukovsky & Zeelinski, 1965). On the basis of morphological and chemical characteristics, it has been divided into four groups, the East Asian pea pears, the larger fruited East Asian pears, the North African pears and the European & West Asian pears (Challice & Westwood, 2008).

The genus Pyrus contains 22 primary species, more than 3,000 cultivars, 10 naturally occurring interspecific and two intergeneric hybrids, distributed in North Africa, Europe and Temperate Asia (Bell & Itai, 2011). In Asia there are 25 species while 14 species have been reported from China which include 8 endemic species (Cuizhi & Spongberg, 2003). Rescently, a total of 14 species of Pyrus were morphologically identified from northern Pakistan (Islam & Ahmad, 2014). Similarly, fourteen (14) land races belonging to the genus Pyrus have been reported from northern Pakistan (Islam & Ahmad, 2012).

Morphological characterization is the first step in the description, characterization and classification of crop germplasm (Singh & Tripathi, 1985; Smith & Smith, 1989). Identification of Pyrus is mainly carried out through morphological and geographical distribution but identification to species level is difficult because of (i) poor wild population (ii) poor morphological characteristics and diversity and (iii) wide spread crossability and interspecific hybridization and introgression among different species. On the basis of morphological parameters species identification are very difficult because morphological markers are influenced by environmental factors, plant biology, developmental stages and anthropogenic activities (Paul et al., 2010; Ali et al., 2011). In near past, many DNA markers have been used for Taxonomical relationships and evolution in Pyrus (Yamamoto & Chevreau, 2009).

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Therefore, DNA barcoding, molecular taxonomy in combination with morphological data generates an integrated taxonomy. DNA barcoding generate a universal molecular identification key that is assembled in logical sequences in reference library. The DNA barcoding extensively strengthen the field of molecular identification (Teletchea, 2010). Phylogeny and several other molecular techniques were used for species identification. Among these techniques, a group of techniques, broadly termed ―DNA barcoding‖ (Hebert et al., 2003), In which different genes such as 18S rRNA or rDAN, rbc L and matK have been used for plants identification (Kress et al., 2005; Little, 2007) and COI for animals identification. In these techniques, the species identity of a query sequence is assigned on the basis of its similarity to a set of reference sequences. From the above mention genes, 18S rRNA technique can be used for phylogenetic and taxonomic study (Nickrent & Franchina, 1990). Similarly 18S ribosomal DNA (rDNA) sequences were used for parsimony analyses of 223 species of angiosperms (Soltis et al., 1997).

Therefore, for better evaluation and understanding of the present problem, 18S rRNA technology was used to discriminate different land races of the genus Pyrus selected from northern Pakistan. A total of 24 land races were sequenced and subjected for molecular analysis. Among these, ―Kacha Tora Tangai‖showed 99% maximum identity and 99% query cover with P. pyrifolia cultivars Sunwhang, Miwhang and Imamuraaki. For phylogenetic relationship of the land race ―Kacha Tora Tangai (Ktt)‖, Maximum Likelihood showed a total of 1222 characters in which conserved, variable, parsimony informative and singleton characters were 259, 232, 209 and 23, respectively. Douglas et al., (1997) applied 18S rDNA and rbcL gene for parsimony analyses and phylogeny of angiosperm. The local land race ―Kacha Tora Tangai‖ has made close cluster with P. pyrifolia cultivar Shinil (Fig.1.19). Similarly, the nucleotide sequence of 18S rRNA of Ghata Tora Tangai (Gtt) showed 100% maximum identity and query cover with P.pyrifolia cv.Imamuraaki and Ghata Zara Tangai (Gzt) showed maximum identity and query cover with P. communis cv. Conference (Islam & Ahmad, 2014). Similarly, Nickrent & Franchina (1990) have been reported 18S rRNA sequences from 3 representativ genera of three families i.e Olacaceae, Santalaceae and Viscaceae within the Santalales and 6 dicotyledons outgroup families and observed that Santalales was holophyletic taxon, most closely related to Euronymus, Celastraceae. Similarly, the 180

whole genome of P.bretschneideri was sequenced and several vital genes were reported which controlling the key horticulture traits. These traits play an important role in the development of commercially important varieties (Wu et al., 2013).

In the present investigation, on the basis of 18S rRNA analysis, local land race ‖Zira Mamoothay‖ (Zm) showed 99% maximum identity and 98% query cover with Pyrus communis cultivars Pachkan's Triumph and Conference. The maximum likelihood tree (Fig.3.20) of Zira Mamoothay (Zm) showed a total of 1213 characters in which 258 were conserved, 234 variable, 209 parsimony-informative, 25 were singleton sites and the highest log likelihood was -885.7129. Wu et al., (2013) had sequened the whole genome of P. ussuriensis to determine different putative genes. In the present study, the phylogenetic tree showed that the local land race (Zm) fall in sub clade I and has made cluster to P. communis cultivar Clapp's Favorite (Fig. 3.20). Similarly 18S ribosomal DNA (rDNA) sequences were used for parsimony analyses of 223 species of angiosperms; their topologies are highly supporting the chloroplast gene rbcL. The general similarity of 18S rDNA and rbcL topologies further clarifies the broad picture of angiosperm phylogeny. In all these analyses, the first-branching angiosperms were Amborellaceae, Austrobaileyaceae, Illiciaceae, and Schisandraceae and the taxa were always followed by family Nymphaeaceae (Soltis et al., 1997).

The molecular characterization of ―Khan Tango (Kt)‖ showed 97% maximum identity and 100% query cover with P. communis cultivars Pachkan's Triumph, Beurre and Cascade while for Maximum Likelihood a total of 41 sequences were considered and a total of 1170 characters were used for analysis, in which 258 were conserved, 254 were variable, 227 were parsimony-informative and 27 were singleton sites. Zimmer et al., (1989) have been evaluated 60 species of vascular plants to establish the phylogenetic relationship on the basis of 18S rRNA. In the present work, the phylogenetic tree (Fig. 3. 21), with the highest log likelihood, consists of two major and five sub clades. The local land race ―Khan Tango (Kt)‖ fall in the sub_clade_I (Pyrus clade) but does not make a cluster with any of the available database species or cultivars in the phylogenetic tree (Fig. 3.21) and fall independently within the sub_clade_I. This shows that it may be a separate species of the genus Pyrus and need further study for it identification.

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Similarly, Soltis et al., (1997) have been carried out a study to demonstrate 18S rDNA sequences, having a lot of information for phylogenetic study at higher taxonomic levels in the seed plants. Recently, whole genome of pear (Pyrus bretscheideri) was recently sequenced (Wu et al., 2013), making it possible to develop genetic markers of pear (Fan et al., 2013). With the development of next generation sequencing technologies, a set of 1096 SNPs identified from three Europea n pear cultivars and 7692 apple SNPs were combined together to develop useful markers for genotyping in pears.

The molecular characterizations of ―Ghata Zira Tangai‖showed 97% maximum identity and 99% query cover with P. communis cultivars Pachkan's Triumph and Beurre. For phylogenetic relationship, a total of 41 sequences were considered and the final data sheet had 1170 characters, in which 33 were singleton sites, 235 were conserved, 258 were variable and 225 were parsimony-informative. Similarly maximum likelihood and maximum parsimony methods were used for evaluation of nucleotide sequences of seven different genes of 192 land plants (Qiu et al., 2007). Similarly, Ghata Zara Tangai (Gzt) showed 97% maximum identity and query cover with P. communis cv. Conference and P. pyrifolia cv. Okusankichi (Islam & Ahmad, 2014). Further, the Phylogenetic tree (Fig.3.22) of the current work showed that the local land race ―Ghata Zira Tangai‖ fell at end of sub clade I or Pyrus clade and does not make a close cluster to any one of the Pyrus species/ cultivars. The branch length of ―Ghata Zira Tangai‖does not match with other Pyrus species or cultivars. Therefore, it may be a new species or cultivar and need further work for its taxonomic retification. Several varieties of P. pashia have been taxonomically identified including var. pashia, kumaoni, obtusata and grandiflora (Yu et al. 1986). Katayama et. al. (2012) found a close phylogenetic relationship of P. pashia to Oriental pears. In addition to 18S rRNA gene analysis, pear cultivars were also evaluated through SSR technique for genetic variability and relationships (Song et al., 2013).

Several chloroplast gene regions are typically used as plant barcodes such as maturase K (matK) and ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) as barcodes (Hollingsworth et al., 2009). In the present work, a 500 bp fragment of 18S

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rRNA was amplified, sequenced and BLAST evaluated. The BLAST result of ―Nashpati‖ revealed 99% identity and 100% query cover with Pyrus pyrifolia cultivars Sunwhang, Miwhang and Imamuraaki while the phylogenetic relationship based on a total of 1223 characters.The final data sheet showed that the conserved, variable and parsimony informative characters were 245, 231 and 208, respectively. Similarly, 18S ribosomal RNA and DNA sequences were also used for phylogenetic relationship of higher taxonomic levels in animals (Chapman, 1991; Bakker, 1994). For phylogenetic tree (Fig.3.23), a total of 41 sequences of different genera were evaluated and the phylogenetic tree distributed all the 41 reference sequences into two major clades and five sub clades. The local land race ―Nashpati‖ falls in sub clade I independently and did not make cluster to the available database species and cultivars. However, molecular data confirm the position of ―Nashpati‖ to the genus level. The 18S rRNA technique does not support the morphological data. Therefore, further study is required for its identification to species and cultivar levels. Hamby & Zimmer (1992) have concluded that the classification of plants into classes, orders, families, genera and species has been based on shared morphological, chemotaxonomical and ecological parameters. Both molecular and morphological data generate an integrated taxonomy while a set of molecular techniques establish a universal molecular identification key on the basis of molecular information that is assembled in logical sequence in reference libraries. The DNA barcoding extensively strengthen the field of molecular identification (Teletchea, 2010), DNA sequencing is used to enhance understanding of species limitation in angiospems, via contributing towards the clarification of ambiguous species (Lahaye et al., 2008). Therefore, this technique should be used for species and cultivars level of identification.

The molecular characterizations reveal that the local landrace ―Parawoo Tango (Pt)‖ possess 99% maximum identity and 99% query cover with P.communis cultivars Pachkan's Triumph, Beurre, Cascade and Clapp's Favorite. The sequence of the candidate species with other 41 sequences of reference genera, species and cultivars were compared and aligned. This procedure was also supported by (Nickrent, 1995; Hamby & Zimmer, 1992). The claldogram (Fig.3.24) thus constructed, comprised of 183

two majors and five sub-clades. The local land race ―Parawoo Tango‖ falls in the sub_clade_I, Pyrus clade and does not develop a cluster with the available Pyrus species or cultivars but falls independently and shows a separate identity.The work has been supported by Islam & Ahmad (2014), as taxonomically it is identified as Pyrus sinkiangensis. Doyle et al., (1994) conducted a study in which 18S and 26S rRNAs were used for finding the phylogenic relationship of various groups of angiosperm and revealed that the bootstrap support were strong for genetelies and angiosperms. Through genome sequencing, a study was conducted to understand the molecular mechanism of persistancy and nonpersistancy of calyx with the fruits of P.sinkiangensis to facilitate the selection of new chemical agents and accelerate genetic strategies for the development of more effective pear calyx abscission for commercial purposes (Qi et al., 3023).

Our local land race, ―Pekhawry Tango, Pkt ‖showed 97% maximum identity and 94% query covers with Pyrus communis cv. Pachkans Triumph and Beurre. The Phylogenetic tree (Fig. 3.25) consisted of two major and three sub clades and the local land race fell independently near the Pyrus clade and did not construct a cluster with any of the available Pyrus species or cultivars. On the basis of morphological markers it is identified as P. hopeiensis, the result was supported by Islam et al. (2014). DNA sequences of nine genes (plastid: atpB, matK, and rbcL; mitochondrial: atp1, matR, mtSSU, and mtLSU; nuclear: 18S and 26S rDNAs) from 100 species of basal angiosperms and gymnosperms were analyzed using parsimony, bayesian, and maximum likelihood methods. The result supported the consensus of relationships among basal angiosperms (Qiu et al., 2005). Here, 18S rRNA was used for evaluation of the local landrace, ―Nak Tango (Nk)‖. The result showed 97% maximum identity with P. communis cultivar Pachkan's Triumph, Conference and Beurre. Islam et al., (2014) obtained similar results for the landrace, Nak Tango (Nk) through 18S rRNA analysis. Similarly for determination of phylogenetic relationship, Kranz et al. (1995) used sequences of 18S rRNA of diverse group of plants and concluded that Charophyceae was continuously placed on the branch of land plants. In the present investigation, the phylogenetic tree has placed the ―Nak Tango (Nk)‖ (Fig. 3.26) in between sub_clade_II (Prunus spp) and sub_clade_IV (Cydonia spp). It did not make a close cluster with any of 184

the available species. Taxonomically it has been identified as Pyrus ovoideae and the results were supported by (Islam & Ahmad, 2014).

The BLAST result of 500 bp sequence of our local landrace, ―Spina Malmothy, Sm‖ showed 98% maximum identity and 100% query cover with P. communis cultivars Pachkan's Triumph, Conference and cultivar Beurre. Ross et al., (2008) studied evolutionary relationship on simulation, based on sequence comparison, BLAST search and genetic distances and shape of the tree. The query specific sequences were included in the reference alignment. It is further reported that the high rate of identification was related to the degree of differentiation among species/land races nucleotide sequences. In the present work, the phylogram (Fig. 3.27) can be divided into Pyrus clade, Prunus clade, Cydonia clade, Rubus calde and Frageria calde. The local landrace, ―Spina Mamothy‖ fall in the Pyrus_clade which form a close cluster with P. communis cultivar Clapp's Favorite. On the basis of 18S rRNA gene analysis the local land race, ―Asmasy Tango, At‖ showed 99% maximum identity and 100% query cover with Pyrus communis cultivars Pachkans Triumph, Beurre and Cascade. Gottschalk & Blanz (1984) were used 5.8S rRNA for phylogenetic evaluation of different species of fungi. Similarly, 18S rDNA has also been used for molecular analysis of fern rusts and their allies (Sjamsuridzal et al., 1999). In the present study, the phylogenetic tree (Fig.3.28) segregated the candidate landrace and reference sequences into three major clades, i.e clade I (Pyrus+Prunus) and clade II (Cydonia) and clade III (Rusbus+Frageria). Clade I was divided into two sub clades, sub clade Pyrus and sub clade Prunus. The local land race ―Asmasy Tango‖ falls in the Sub_clade_Pyrus and clustered with similar species/cultivars i.e P. communis cultivar Beurre.

On the basis of 18S rRNA sequence analysis, our local landrace, ―Mamosay Batal-8‖ showed 97% maximum identity and 100% query cover with Pyrus communis cultivar Pachkans Triumph. The phylogeney relationship was established and the phylogenetic tree (Fig. 3.29) is divided into two major clades, clade I (Pyrus+Prunus clade) and clade II (Cydonia+Rubus+Frageria Clade).

Clade I was further divided into two sub clades, sub-clade I and sub clade II. Our local landrace, ―Mamosay Batal-8‖ falls in Sub_clade_1 and clustred with similar 185

species/cultivar, P. pyrifolia cultivar Shinsui. The ―Mamosay Batal-8‖ is being described and reported for the first time from Pakistan. Phylogenetic analyses using direct sequencing of rRNA from 60 species of vascular plants, of which 29 were dicots and 17 monocots were carried out (Zimmer et al., 1989; Hamby & Zimmer, 1992). Maier et al., (2003) evaluated 52 rust fungi through rDNA. The phylogenetic tree segregated the data into nine families and three outgroup species. Similarly, the local landrace,―Mamosay Bat_12‖ revealed 97% maximum identity and 100% query cover with Pyrus pyrifolia cultivars Imamuraaki, Soowhang and Wonwhang on the basis of 18S RNA gene. A total of 1173 characters were subjected to Maximum Likelihood to determine the phylogenetic relationship of ―Mamosay Bat-12‖. Similarly, Hamby & Zimmer, (1992) reported that many new tools have been developed for the establishment of phylogenetic relationships in which the nucleotide sequences of different populations or species are aligned and compared with the known or reference sequences. In the present work, the query sequence was evaluated and the phylogenetic tree (Fig.3.30) placed the local land race/species, ―Mamosay Bat_12‖ in sub_clade_II with P. pyrifolia cultivar Miwhang and P. communis cultivar Conference. Taxonomically this taxon is still undocumented and need further study for its identification. Similarly, for other genera, Ashfaq et al., (2013) have studied three plastid regions i.e matK, rbcL, and ITS2 as standard markers for plant DNA barcoding in the genus Gossypium.

Wu, et al., (2013) reported a high-quality draft genome sequence of P. bretschneideri cv. Dangshansuli having an abundant of repetitive DNA sequences. In the present study, the 500bp sequence of ―Mamosay Batal-14‖ were used for BLAST search and result showed 97% maximum identity and 100% query cover with Pyrus communis cultivars Pachkan's Triumph and Beurre. A total of 1175 characters were evaluated and phylogenetic relationship was established. Similarly, Hao et al., (2012) collected more than 9100 plant sequences. The inter- and intra-specific variations of the individual genus were calculated and investigated through barcoding. In the present work, phylogenetic relationship was established on the basis of barcoding and the cladogram (Fig. 3.31), was divided into two major clades, clade_I and clade_II. Clade_I was further divided into two sub_clades, sub-clade_I and sub_clade_II. The local land race fall in 186

sub_clade_1 independently and did not make cluster to any of the available species/cultivars. Similarly, taxonomically it is unidentified due to lack of previous published work. Therefore, for further investigation both morphological markers and DNA barcoding is needed for investigating its identity. DNA barcoding has multiple applications and has been used for ecological survey (Dick & Kress, 2009), cryptic taxon identification (Lahaye et al., 2008), and confirmation of medicinal plant specimens (Xue & Li, 2011).

For molecular characterizations, ―Mamosoay B15‖ BLAST result showed 97% maximum identity and 88% query cover with Pyrus communis cultivars Pachkan's Triumph. A total of 1573 characters in the final data sheet were subjected to Maximum Likelihood (ML) to determine phylogenetic relationships. Kelch, (1998) conducted maximum parsimony analyses of the genera of Podocarpaceae by using 18S ribosomal DNA sequence data. Similarly, phylogenetic relationships of the Uredinales was reported by Maier et al., (2003) where sequence data of LS rDNA of 52 rust fungi were used for phylogenetic relationship. The Phylogenetic tree (Fig.3.32), was divided into two major clades, clade_I and clade_II. Clade_I was further divided into two sub_clades, sub-clade_I and sub_clade_II. The local land race ―Mamosoay B15‖ falls in the Pyrus sub_ clade_1 in between its similar species/cultivars of P. communis cultivar Clapp's Favorite and P. pyrifolia cultivar Niitaka. It does not cluster with any of the available Pyrus species/cultivars. Taxonomically, it is not reported from Pakistan and needs further work for its evaluation. Angiosperms possess relatively few morphological characters for comparison at higher levels (Donoghue & Doyle, 1989) and all seed plants (Doyle et al., 1994). As reported by Doyle et al., (1994), that careful analysis of both morphological and molecular data was required to understand angiosperm phylogeny.

The initial BLAST result of our local land race ―Gultar Tango‖ showed 100% maximum identity and 100% query cover with Pyrus communis cultivars Pachkan's Triumph and Conference. For phylogenetic relationships Maximum Likelihood (ML) was used. The final data sheet had a total of 1138 characters. Out of these characters, 229 were conserved, 251 were variable, 226 were parsimony informative and 25 were singleton

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sites. Similarly, Ro et al., (1997) evaluated 35 species belonging to the family Ranunculaceae (31) and Berberidaceae (4), respectively to demonstrate phylogentic relationships through 26S rDNA sequences. The results were compared with that of the previously published DNA sequences of rbcL, atpB, 18S rDNA. The Phylogenetic tree (Fig.3.33) of the present work was divided into two major clades, clade_I and clade_II. Clade_I was further divided into two sub_clades, sub-clade_I and sub_clade_II. The local land race ―Gultar Tango‖ falls in sub-clade_I and clustered with similar species/cultivars of P. communis cultivar Clapp's Favorite, represented by sub_clade_IA. Similarly, Hebert et al., (2003) obtained sequences of the barcoding region from different individuals and phylogeny was established. In the phylogenetic tree, similar, putatively related individuals were clustered together and each cluster was assumed to present a separate species. Both, molecular and taxonomic work is needed for further elaboration.

The molecular characterizations revealed that ‖Hary Tango-Batal‖ possess 95% maximum identity and 100% query cover with Pyrus communis cultivars Pachkan's Triumph and Beurre. Similarly, BLAST and Maximum Likelihood (ML) were used for phylogenetic relationships. A total of 1173 characters in the final data sheet were subjected for maximum likelihood. Out of these characters, 280 were conserved, 249 were variable, 223 were parsimony informative and 26 were singleton sites. Similarly, Velasco et al., (2010) studied the comparision of apple and pear genes and concluded that the genes of both, apple and pear were sharing high similarity in their nucleotide sequences. The classification of plants into classes and species level has been based on shared morphological, chemotaxonomical and ecological parameters. However, with the advancement in molecular biology, many new tools for the investigation of phylogenetic relationships have been developed and with the help of these tools we can identify species/cultivars (Hamby & Zimmer, 1992). In the present study, phylogenetic tree (Fig.3.34), divided into two major clades, clade_I and clade_II. Clade_I was further divided into two sub_clades. The land race ‖Hary Tango-Batal‖ falls in sub-clade_I and cluster with P. communis cultivar Clapp's, represented by sub clade IA. Taxonomically, the local land race ‖Hary Tango-Batal‖ is not reported from Pakistan. A description and evaluation being reported for the first time from Pakistan. 188

The initial BLAST result of ―Kado batang (Kb)‖ showed 99% maximum identity and 100 query cover with Pyrus communis cultivars Beurre and Cascade. The maximum Likelihood was used for phylogenetic relationship. The candidate land race and 41 refrence species/ cultivars were aligned. Similarly, Hillis & Dixon (1991) had aligned and compared a number of ribosomal DNA (rDNA) sequences which provide a lot of information about phylogenetic relationships. In the present work, a total of 1221 characters were used. Among these, 244 were conserved, 230 were variable, 209 were parsimony-informative and 21 were singleton sites. Phylogenetic tree (Fig.3.35) was divided into two major clades, clade_I and clade_II. The local land race ―Kado batang (Kb)‖ falls in sub-clade_I, Pyrus clade, and does not cluster with any species/cultivars of Pyrus but falls idependently. Sequences of the barcoding region were obtained from different individuals and the resulting data were used for construction of phylogenetic tree (Hebert et al., 2003). In the phylogenetic tree, similar, putatively related individuals were clustered together and each cluster was assumed to present a separate species. In the present work, it shows an idependent identity. Taxonomically, this local landrace is unidentified because of unavailability of previous reports. On the basis of current molecular work, it is being reported for the first time from Pakistan. The evolutionary histories and relationships among the eukaryotes have been revolutionized by molecular systematics. Five prominent eukaryotic groups have been defined on the basis of phylogenetic comparison of 18S rRNA sequences. A worthy model was presented on the basis of rRNA and protein based phylogenies to understand the evolutionary history of eukaryotes (Sogin, 1991).

The molecular analysis of local land race, ―Malyzay Tango‖ showed 99% maximum identity and 98% query cover with Pyrus communis cultivar Pachkan's Triumph, Beurre and Cascade. In the final data sheet a total of 1278 characters were subjected to Maximum Likelihood (ML) method for determination of phylogenetic relationships. Out of these characters, 243 were conserved, 196 were variable, 170 were parsimony- informative and 26 were singleton sites. Hillis & Dixon, (1991) had aligned and compared a number rDNA sequences which provide a lot of information about phylogenetic relationships. Phylogenetic tree (Fig. 3.36) was divided into two major clades, clade_I, and clade_II. Clade_I is further divided into two sub_clades, sub- 189

clade_I, clade_II and sub_clade_III. Nickrent & Franchina (1990) had reported 18S rRNA sequences from 3 representative genera of three families i.e Olacaceae, Santalaceae and Viscaceae within the Santalales and 6 dicot outgroup families. In the present investigation, the local land race ―Malyzay Tango‖ falls in sub-clade_II and cluster with P.communis cv. Clapp's Favorite. The molecular data showed that candidate land race is clearly related to P. communis cultivar Clapp's Favorite. Previously, the local landrace ―Malyzay Tango‖ is not reported from Pakistan. On the basis of molecular data, it is being reported for the first time from Pakistan. Schor & Showalter (2011) had also conducted a study to discriminate and differentiate medicinal plants through DNA barcoding. Similarly, Bassil et al., (2009) had determined synonymy, homology, and phylogeny of apples, pears and examined potential relationships of the Island pears with standard apples and pears of Portuguese and American ancestry.

Traditional methods of assessing genetic identity and relatedness depend on the observation of morphological and phonological traits. Molecular marker analysis offers an independent estimation of genetic identity, diversity and ancestry through direct determination of genetic differences (Bassal et al., 2009). Here, in the current work, a sequence of 18S rRNA were used and the BLAST result of local landrace ‖Mamosranga‖ showed 99% maximum identity and 82% query cover with Pyrus communis cultivars Pachkan's Triumph, Beurre and Cascade. In the final data sheet a total of 1208 characters were subjected to Maximum Likelihood (ML) method. Out of these characters, 253 were conserved, 142 were variable, 210 were parsimony-informative and 32 were singleton sites. Phylogenetic tree (Fig.3.37) was divided into two major clades, clade_I and clade_II. Clade_I was further divided into three sub_clades. The local land race‖Mamosranga‖ falls independently on outside of sub-group_II and is represented by sub-clade_III. It does not cluster with any of the available Pyrus or Prunus species or cultivars. Previously, it is not reported from Pakistan, both taxonomic and molecular studies are needed for its further identity. Nickrent & Franchina (1990) had revealed that in flowering plants, determination of relationship in different species is difficult task because of poor, less prominent morphological characteristics and a

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small number of molecular markers to be useful for phylogenetic relationship in angiosperms.

The molecular characterizations of ―Guraky Tango‖ showed 99% maximum identity and 88% query cover with P. communis cultivars Pachkan's Triumph, Conference and Beurre. For assessment of phylogenetic relationships Maximum Likelihood (ML) was used. Gottschalk & Blanz (1984) had used 5.8S rRNA for molecular characterization and establishment of phylogenetic relationships of different species of fungi. Similarly, Sjamsuridzal, et al., (1999) had also been used 18S rDNA for molecular analysis of fern rusts and their allies. In the present investigation, a total of 1217 characters were used and the Phylogenetic tree (Fig.3.38) was divided into two major clades, clade_I and clade_II. Clade_I was further divided into three sub_clades. The local land race ―Guraky Tango‖ clustred with Pyrus pyrifolia cultivar Nijisseiki and presented in the sub_clade_III. Similarly, Chaw et al., (1993) had conducted a sudy on the basis of 18S rRNA data to clearify the position of Taxus sp with respect to Pinus species, Ginko species and Podocarus species. The results showed that Taxus species are more closely related to Pinus species than Podocarpus species.

Schnabel et al., (1999) had amplified 18S rDNA, ITS1 and ITS2 regions, and the 5.8S rDNA gene from populations of Venturia inaequalis and evaluated for genetic characterization. The sequences of 6 different species of Venturia and Cladosporium caryigenum were compared through phylogenetic analysis. The results had shown that Venturia species were placed in three distinct monophyletic groups in the phylogenetic tree. In the present study, sequence of 18S rRNA of local landrace ―Ghata Tora Tangai (Gtt)‖ were BLAST and the BLAST result showed 100% maximum identity and 100% query cover with P. pyrifolia cultivar Imamuraaki 99% maximum identity and 100% query cover with Pyrus communis cultivar Pachkans Triumph. In the final data sheet a total of 1175 characters were subjected to Maximum Likelihood (ML) for determination of phylogenetic relationships. Out of these characters, 291 were conserved, 194 were variable, 07 were parsimony informative and 187 were singleton sites. Phylogenetic tree (Fig. 3.39) was divided into two major clades, clade_I and clade_II. Clade_I was further divided into two sub_clades. Barker et al., (2001) had reported an integrated study

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included representative set of 62 grasses and four out group taxa by using six molecular techniques and morphological data too. Parsimony analysis, using 2143 characters resulted a single parsimonious tree of 8752 steps. The bootstrap support was > 90% for more than half of the internal nodes. In the present study, the local land race ―Ghata Tora Tangai (Gtt)‖ falls independently in between Pyrus pyrifolia cultivar Nijisseiki and Pyrus pyrifolia cultivar Okusankichi and presented in the sub_clade_I. The local land race does not cluster with any of the available refrence species and cultivars. Therefore, further work needs to be carried out for its confirmation. A morphological and molecular work was carried out to study the seed plant phylogeny (Doyle, et al., 1994). In flowering plants, determination of relationship in different species is difficult task because of poor, less prominent morphological characteristics and a small number of molecular markers to be useful for phylogenetic relationship in angiosperms. Therefore, the small subunit 18S rRNA sequences technique can be used to solve such sorts of problems (Nickrent & Franchina, 1990).

A total of 13 SSR markers were applied on total population of 145 P.communis individuals for establishment of phylogenetic relationship. The results showed that the Bayasian cluster method had segregated these individuals into 12 groups (Volk et al., 2006). In the present work, 18S rRNA was used for evaluation of local land race ‖Shaker Tango‖, the results showed 97% maximum identity and 93% query cover with Pyrus communis cultivar Pachkans Triumph, Conference and Beurre. In the final data sheet, a total of 1169 characters were used for establishment of phylogenetic relationships. Nickrent et al., (1995) had evaluated 64 taxons having 1853 bp of sequences used for establishment of phylogenic relationship. In the present investigation, phylogenetic tree (Fig. 3.42), divided into two major clades, clade_I and clade_II. Clade_I was further divided into three sub_clades, sub-clade_I and sub_clade_II. The local land race ‖Shaker Tango‖ falls independently between Pyrus pyrifolia cultivar Okusankichi and Pyrus pyrifolia cultivar Nijisseiki and does not cluster with any of available refrence species and cultivars. Teletchea (2010) reported that using DNA barcoding in combination with morphological data, a taxon can easily be identified. Similarly, Schori & Showalter (2011) reported that DNA barcoding is being used to identify several medicinal plants, Acacia nilotica, Calotropis procera, Justicia adhatoda, Nigella sativa and 192

Rosa damascene etc. They were able to distinguish these taxa from suspected species and provide quality control and standardization of the plant material supplied to the pharmaceutical industry. Sasaki et al., (2002) differentiated the Chinese and Japanese Curcuma drugs on the basis of comparision of 18S rRNA gene and trnK gene sequences with those of six Curcuma species reported previously.

In the current investigation, the BLAST results of our local landrace ―Pak_24‖ showed 100% maximum identity with Pyrus pyrifolia cultivar Wonwhang and Kimitsukawase. For phylogenetic relationship of ―Pak 24‖, a total of 1168 characters were subjected to (ML) Maximum Likelihood method. Out of 1168 characters, 242 were conserved, 251 were variable, 226 were parsimony-informative and 25 were singleton sites. Phylogenetic tree (Fig.3.41), was divided into two major clades, clade_I and clade_II. Clade_I was further divided into three sub_clades, sub-clade_I and sub_clade_II. Our local landrace, ―PAK_24 falls independently between Pyrus pyrifolia cultivar Okusankichi and Pyrus pyrifolia cultivar Nijisseiki. The results showed that ―Pak 24‖, may be a hybrid of these two cultivars or an independent species. Further study is needed for confirmation of its identity. Smith (1989) had used 18S rRNA sequences for assessment of phylogenetic relationships through parsimony analysis. Chagne et al., (2014) had reported a total of 2,279 SNP markers, absession initio gene prediction and other 43,419 putative genes. Out of these, 1219 are unique to European pear. Further study revealed that the genome assembly of P.communis is a useful tool for identifying the genetic control of horticultural traits in pear and will enable the wide application of marker-assisted and genomic selection that will enhance the speed and efficiency of pear cultivar development.

The 18S rDNA fragment possess sufficient information for phylogenetic analysis at higher taxonomic levels in the angiosperms. However, phylogenetic analyses of angiosperms had evaluated through 18S rDNA which unproved taxon samples of Magnoliidae and Dilteniidae (Soltis et al., 1997). In the present study, phylogenetic analysis was carried out on the basis of 18S rRNA gene sequences for the evaluation of local landrace ―Shaker Tango‖, the results showed 97% maximum identity and 93%

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query cover with Pyrus communis cultivars Pachkans Triumph, Conference and Beurre. For phylogenetic relationships, both, qurrey and reference sequences were compared, aligned and chopped. A total of 1210 characters were considered for establishment of phylogenetic tree. The phylogenetic tree showed that the candidate land race ―Shaker Tango‖ falls adjuscent to the P. communis cultivar Pachkan's Triumph in the sub_clade_1 of Pyrus_clade but does not cluster with any of the available reference cultivars, species and showing an independent identity. Further work is required for its identification through taxonomic and molecular markers. Smith & Smith (1989) had reported that morphological characterization is the first step in evaluation and classification of plants. However, both DNA barcoding and morphological data produce an integrated taxonomy (Teletchea, 2010). In angiosperms, establishment of relationships of different species is difficult because of diverse morphological traits and environmental factors. Till now, a limited number of molecular markers were used for phylogenetic relationships in angiosperms (Nickrent & Franchina, 1990). DNA restriction mapping provides useful information and suitable for studies of molecular evolution and systematics of plants. Although DNA sequence data are often used for systematic studies, restriction mapping analysis has an advantage when a relatively large number of samples were analyzed (Burns, et al., 1991). The angiosperms possess relatively few morphological characters for comparison at higher levels (Donoghue & Doyle, 1989; Doyle et al., 1994). As reported by Doyle et al., (1994), that careful analysis of both morphological and molecular data is necessary for better understanding of angiosperm phylogeny.

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APPENDICES

Appendix 1 1 From the Excel table, copy only the contents without sample names and paste into a new Elxcel sheet. 2 Under the ―Edit‖ pull down menu, choose . 3 In the pop-up find window, and C. 4 In the pop-up find window, 1 and A. 5 Now you have a non-labeled table with ―A‖ or ―C‖ values. Select and copy the contents into the original Excel table. 6 Select the contents including the sample names and hit or keys. 7 Open a Microsoft Word window, under the ―Edit‖ pull down menu, choose , and select . 8 Now you have a data sheet with sample names followed by ―A or C with a tab space‖. 9 Under the ―Edit‖ pull down menu, choose . (Go to the word file, highlight and select A(tab) and paste into the pop-up find window in the next). 10 In the pop-up find window, A (tab) and A. 11 Again in the pop-up find window, C(tab) and C. 12 Now you have a DNA sequence of A or C following sample names. 13 Cut and paste the sequence into DNAMAN window file and save it as a DNAMAN file. 14 Cut and paste the sample name into pop-up save as window. 15 Continue with the remaining samples.

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6.1 A N A L Y S I S O F V A R I A N C E T A B L E FOR PETIOLE LENGTH

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 35.728 17.864 0.4922 2 Factor A 13 1919.403 147.646 4.0685 0.0011 -3 Error 26 943.546 36.290 ------Total 41 2898.676 ------Coefficient of Variation: 14.62%

6.2 A N A L Y S I S O F V A R I A N C E T A B L E L FOR LEAF AREA

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 781331.193 390665.596 0.7914 2 Factor A 13 10582799.267 814061.482 1.6491 0.1345 -3 Error 26 12834691.360 493641.975 ------Total 41 24198821.820 ------Coefficient of Variation: 98.85%

6.3 A N A L Y S I S O F V A R I A N C E T A B L E FOR PEDICEL LENGTH

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 0.733 0.366 0.0093 2 Factor A 13 8852.655 680.973 17.2392 0.0000 -3 Error 26 1027.041 39.502 ------Total 41 9880.428 ------Coefficient of Variation: 21.03%

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6.4 A N A L Y S I S O F V A R I A N C E FOR FRUIT LENGTH

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 20.110 10.055 1.1893 0.3205 2 Factor A 13 20621.807 1586.293 187.6217 0.0000 -3 Error 26 219.823 8.455 ------Total 41 20861.740 ------Coefficient of Variation: 6.06%

6.5 A N A L Y S I S O F V A R I A N C E T A B L E FOR FRUIT WIDTH

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 31.459 15.729 2.6584 0.0890 2 Factor A 13 6133.518 471.809 79.7416 0.0000 -3 Error 26 153.835 5.917 ------Total 41 6318.811 ------Coefficient of Variation: 5.92%

6. 6 A N A L Y S I S O F V A R I A N C E T A B L E FOR FRUIT WEIGHT

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 480.491 240.245 1.4899 0.2440 2 Factor A 13 114743.600 8826.431 54.7393 0.0000 -3 Error 26 4192.366 161.245 ------Total 41 119416.457 ------Coefficient of Variation: 19.35%

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6.7 COLLECTION OF VOUCHAR SPECIMENS OF PYRUS GENOTYPES FROM DIFFERENT LOCALITIES OF NORTHERN PAKISTAN

S. No Land races Accession No Locality GPS references Altitude N E (ft) 1 Nashpati Nashpati-1 Chail, Swat 35 08.19 72 38. 96 5149 Nashpati-2 Shawar, Swat 34 59.02 72 18. 68 4818 Nashpati-3 Shawar, Swat 34 59.057 72 18. 642 4816 Nashpati-4 Shawar, Swat 34 59.063 72 18. 642 4785 Nashpati-5 Shawar, Swat 34 59.060 72 18. 609 4785 2. Ghata Zyara Gzt-1 Chail, Swat 35 08. 486 72 33. 151 4650 Tangy Gzt-2 Shawar, Swat 34 59.064 72 18. 626 4797 Gzt-3 Shawar, Swat 34 59.064 72 18. 626 4797 Gzt-4 Chail, Swat 35 08. 367 72 33. 576 4615 Gzt-5 Chail, Swat 35 11.436 72 34. 675 4670 3 Parawo Pt-1 Chail, Swat 35 08. 364 72 33. 086 4825 Tangoo Pt-2 Chail, Swat 35 08. 344 72 33. 086 4840 Pt-3 Shawar, Swat 34 99.077 72 18. 664 4850 Pt-4 Shawar, Swat 34 59.064 72 18. 626 4788 Pt-5 Shawar, Swat 34 59.082 72 18. 654 4790 4 Khan Tango Kt-1 Shawar, Swat 34 49.044 72 18. 661 4830 (Kt) Kt-2 Shawar, Swat 34 48.193 72 18. 551 4810 5 Nak Tango Nt-1 Chail, Swat 35 08. 364 72 32. 956 5140 Nt-2 Shawar, Swat 34 25.093 72 14.086 3537 Nt-3 Shawar, Swat 34 25.093 72 14.086 4811 Nt-4 Shawar, Swat 34 59.044 72 18.662 4820 Nt-5 Chail, Swat 35 08. 191 72 32. 356 5160 6 Shaker St_1 Shawar, Swat 34 59.02 72 18. 68 4818 Tango

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7 Soor Tango St-1 Chail, Swat 35008. 151 72 32.967 5175 St-2 Chail, Swat 35 08. 151 72 32 967 5200 St-3 Chail, Swat 35 08. 151 72 32.967 5150 St-4 Chail, Swat 35 08. 261 72 32. 988 5175 St-5 Chail, Swat 35 08. 356 72 32.987 5250 8 Mamosrang Mamosrang-1 Chail, Swat 35 08. 151 72 32.987 5210 Mamosrang-2 Chail, Swat 35 08. 362 72 33.085 4790 Mamosrang-3 Chail, Swat 35 08. 151 72 32.987 5190 Mamosrang-4 Chail, Swat 35 08. 051 72 32.987 5190 Mamosrang-5 Chail, Swat 35 09. 463 72 33.185 4795 9 Mamosay Mamosay-1 Chail, Swat 35 08.188 72 33.161 4801 Mamosay-2 Chail, Swat 35 08. 186 72 33.642 4801 Mamosay-3 Chail, Swat 35 08. 156 72 33.625 4501 Mamosay-4 Chail, Swat 35 09. 186 72 33.621 4811 Mamosay-5 Chail, Swat 35 08. 186 72 33.641 4801 10 Shankwlay Shankwlay-1 Chail, Swat 35 08. 151 72 32.967 5200 11 Kacha Tora tangai Ktt Chail, Swat 35 08. 421 72 33.008 4720 12 Ziara Mamothai Zm-1 Shawar, Swat 34 40.014 72 19.621 4850 13 Spina Mamothay Sm-1 Shawar, Swat 34 40.014 72 19.621 4850

14 Khapa Tango Kt-1 Shawar, Swat 34 49.033 72 18.650 4812 15 Gultar Tango Gt-1 Shawar, Swat 34 50.045 72 19.561 4855 16 Malyzay Tango Mt-1 17 Pikhawary Tango Pkt-1 Shawar, Swat 34 50.045 72 19.561 (maluch) 18 Gurakay Tango Gkt-1 Shawar, Swat (aray) 19 Haray Tango Ht-1 Shawar, Swat (malak abad) 20 Asmasae At Shawar, Swat 35 08. 364 72 33. 957 4860 Tango 21 Mamosranga Mamosranga Shawar, Swat 35 08. 365 72 32. 957 4855 22 Batangi Batangi-1 Batal, Mansehra 34 32.878 73 10.271 4692 Sp -1 Batangi-2 Batal, Mansehra 34 33.145 73 11.125 4712

23 Niki Kali Nkb Batal, Mansehra 34 32.971 73 10.381 5180 Batangae 24 Kado Batang Kb_1 Batal, Mansehra 34 32.946 73 08.750 4541

Kb_2 Batal, Mansehra 34 32.812 73 08.410 4520

25 China Nespati Cn-1 Batal, Mansehra 34 32.924 73 08.713 4560

Cn-2 Batal, Mansehra 34 32.924 73 08.713 4540

Cn-3 Batal, Mansehra 34 32.924 73 08.713 4570

26 Kado Batng Kb-1 Batal, Mansehra 34 32.344 73 08.628 4280 Mark- 5 Kb-2 Batal, Mansehra 34 32.350 73 08.640 4281

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Kb-3 Batal, Mansehra 34 32.340 73 08.620 4278

Kb-4 Batal, Mansehra 34 32.360 73 08.655 4285

27 Batangi Batangi-1 Batal, Mansehra 34 32.141 73 08.413 4450 Mark- 6 Batangi-2 Batal, Mansehra 34 32.150 73 08.419 4460

Batangi-3 Batal, Mansehra 34 32.145 73 08.415 4455

Batangi-4 Batal, Mansehra 34 32.130 73 08.410 4470

Batal, Mansehra 34 33.250 73 09.3400 5070

28 Mamosay Bat-8 Mamosay-b8 Batal, Mansehra 34 35.289 73 09.290 5040

29 Batangi Bat-9 Batangi-B9 Batal, Mansehra 34 35.290 73 09.291 5010

Batangi-B9 Batal, Mansehra 34 35.300 73 09.298 500

Batangi-B9 Batal, Mansehra 34 35.270 73 09.189 5010

30 Hary Tango Htb-b1 Batal, Mansehra 34 35.301 73 09.201 4490 Batal 31 Shaker Bataal-10 Shaker- B10 Batal, Mansehra 34 35.291 73 09.411 5079

Sbp_2 Shawar Swat 34 35.298 73 09.412 5090

32 Shaker Tango St_1 Shawar, Swat 34 59.02 72 18. 68 5200

Mamosay-Bat-12 Mamosay-B12 Batal, Mansehra 34 35.277 73 09.587 5200

33 Kado Batang Kb-1 Batal, Mansehra 34 35.272 73 09.587 5191 Mark-13 Kb-2 Batal, Mansehra 34 35.490 5189 73 09.580 Kb-3 Batal, Mansehra 34 35.270 73 09.588 5190

34 Mamosay Bat-14 Mamosay-B14 Batal, Mansehra 34 35.277 73 09.587 5160 35 Mamosay Bat-15 Mamosay-B15 Batal, Mansehra 34 35.262 73 09.600 5180

36 Kado Batang Kb-1 Batal, Mansehra 34 35.069 73 08.869 4870 Mark-16 Kb-2 Batal, Mansehra 34 35.070 73 08.875 Kb-2 Batal, Mansehra 34 35.075 73 08.878

37 Mamosay Bat-18 Mamosay-B18 Batal, Mansehra 34 35.069 73 08.869 4870

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Mamosay-18 Batal, Mansehra 34 35.080 73 08.878 4890

38 Mamosay Bat-19 Mamosay-B19 Batal, Mansehra 34 35.275 73 08.587 5166

39 Kushbago Kbb-1 Sangar-Balakot 34 34.658 73 22.345 5085 Batang 40 Kado Batang Kb balakot-1 Sangar-Balakot 34 34.708 73 22.386 5157 Balakot 41 Batangi Batangi-1 Sangar-Balakot 34 34.658 73 22.345 5085

42 Tanchi Batang Kb balakot-4 Sangar-Balakot 34 34.687 73 22.579 5306

43 Nak Btang Nb balakot-1 Sangar-Balakot 34 34.674 73 22.579 4980

Nb balakot-2 Sangar-Balakot 34 34.701 73 22.279 5140

Nb balakot-3 Sangar-Balakot 34 34.671 73 22.671 5140

Nb balakot-4 Sangar-Balakot 34 34.701 73 22.380 5140

44 Gahata Zira Gzt-1 Sangar-Balakot 34 34.992 73 22.313 4881 Tangi (golden) Gzt-1 Sangar-Balakot 34 34.986 73 22.201 4401

45 Franci Batang Fb-1 Sangar-Balakot 34 34.700 73 22.396 5146

Fb-1 Sangar-Balakot 34 34.689 73 22.390 5142

Fb-1 Sangar-Balakot 34 34.641 73 22.793 5143

46 Kala Batang Kb-1 Sangar-Balakot 34 34.701 73 22.423 5166

47 Shardi Tanchi St-1 Sangar-Balakot 34 34.721 73 22.291 5344 (Srt) 48 Ati Batng Ab-1 Sangar-Balakot 34 34.621 73 22.812 5505

Ab-2 Sangar-Balakot 34 34.986 73 22.620 5505

49 Kashmiry Kshb-1 Sangar-Balakot 34 34.986 73 22.620 5320 Batangi 50 Kashmiry Kshbf-1 Sangar-Balakot 34 34.986 73 22.620 5320 Batangi Flexable 51 China Batang Cb-1 Sangar-Balakot 34 34.670 73 22.515 5351

Cb-2 Sangar-Balakot 34 34.615 73 22.256 5509

52 Glass Batang Gb-1 Sangar-Balakot 34 34.992 73 22.202 4773

Gb-1 Sangar-Balakot 34 34.986 73 22.200 4400

53 Glass Batang Gb-Hs-1 Sangar-Balakot 34 34.698 73 22.692 5417 ( Hard Skin) 54 Pyrus Pyrus Dhear Koat, 34 07.467 73 54.325 6000 218

A.K. 55 Pyrus sp Pyrus Dhear Koat, 34 00.007 73 54.374 6000 A.K. 56 Pyrus sp Pyrus Dhear Koat, 34 00.007 73 54.374 5090 A.K. 57 Pyrus sp Pyrus Dhear Koat, 34 01.653 73 35.345 5090 A.K. 56 Pyrus sp Pyrus Dhear Koat, 34 01.653 73 35.345 A.K. 57 Pyrus sp Pyrus Narwal, A.K. 34 00.740 73 36.106 4015 58 Pyrus sp Pyrus Narwal, A.K. 33 59.801 73 37.452 4014 59 Pyrus sp Pyrus Ghazi Abad, A. 33 59.821 73 37.375 4014 K 60 Pyrus sp Pyrus Ghazi Abad, A. 33 59.821 73 37.375 4014 K 61 Nakh Batang Nb-AK Ghazi Abad, A. 33 59.821 73 37.375 4075 K 62 Raigh Batangi Rb Chuck saver 29 42.775 98 20.807 4075 Mutawali, A.K 63 Bahu Gosha Bg Chuck saver 29 42.775 98 20.807 7298 Mutawali 64 Ghata Zara Gzt Suddhen Gali 34 04.624 73 44.498 4000 tangae Top, A.K 65 Gal koti Batang Gkb Oppi Bala- 34 06.327 73 54.007 4000 Chakoti, A.K 66 Raj Batangi Rjb Oppi Bala- 34 06.327 73 54.007 4000 Chakoti, A.K 67 Gosho Bago Gbb Oppi Bala- 34 06.327 73 54.007 4000 batang Chakoti, A.K 68 Batangae Batangae Nelum Valley 34 24.675 73 36.939 3209

69 Kado Batang Kb-AK Panch Ghar, 34 26.675 73 36.939 3209 Nelum, A.K 70 Ghata Zara Gzb-P Panch Ghar, 34 26.675 73 36.939 3209 Batangae Nelum, A.K

219

6.8 Consensus Sequences of 18S rRNA of Pyrus Land races

>Malyzay Tango (Mt)

GACTSGTAGAGCCCGGTATTGTTATTTRTWGTCACTACCTCCCCGTGTCAGGATT GGRTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAGGCTC CCTCTCCGGAATCGAACCCTAATTCTCCGTCACCCGTCACCACCATGGTAGGCC ACTATCCTACCATCGAAAGTTGATAGGGCAGATATTTGAATGATGCGTCGCCAG CACGATGGCTGTGCGATCCGTCGAGTTATCATGAATCATCAGAGCAACGGGCA GAGCCCGCGTCGACCTTTTATCTAATAAATGCGTCCCTTCCAGAAGTCGGGGTT TGTTGCACGTATTAGCTCTAGAATTACTACGGTTATCCGAGTAGCAGATACCAT CAAACAAACTATAACTGATTTAATGAGCCATTCGCAAGTTTTTCAAAA

> Nashpati

CCTCCTATGGATCCTCGTTARGGGATTTAGATTGTACTCMTTCCAATTACCAGAC TCGTAGAGCCCGGTATTGTTATTTATTGTCACTACCTCCCCGTGTCAGGATTGGR TAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAGGCTCCCT CTCCGGAATCGAACCCTAATTCTCCGTCACCCGTCACCACCATGGTAGGCCACT ATCCTACCATCGAAAGTTGATAGGGCAGATATTTGAATGATGCGTCGCCAGCA CGATGGCTGTGCGATCCGTCGAGTTATCATGAATCATCAGAGCAACGGGCAGA GCCCGCGTCGACCTTTTATCTAATAAATGCGTCCCTTCCAGAAGTCGGGGTTTGT TGCACGTATTAGCTCTAGAATTACTACGGTTATCCGAGTAGCAGGTACCATCAA ACAAACTATAACTGATTTAATGAGCCATT

> Nak Tango (Nt)

CCGTACTGCGACAACTTAWGTATACGCTATTGGRGATTTARAGWGCCAYTCSTM MKTTWMCAGACTGGTARAGTYMGGTATTGTYWTTTGTWGWCMCTACCTCCCC GTGTCAGGATTGGRTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGT TTCTCAGGCTCCCTCTCCGGAATCGAACCCTAATTCTCCGTCACCCGTCACCACC ATGGTAGGCCACTATCCTACCATCGAAAGTTGATAGGGCAGATATTTGAATGAT GCGTCGCCAGCACGATGGCTGTGCGATCCGTCGAGTTATCATGAATCATCAGAG CAACGGGCAGAGCCCGCGTCGACCTTTTATCTAATAAATGCGTCCCTTCCAGAA GTCGGGGTTTGTTGCACGTATTAGCTCTAGAATTACTACGGTTATCCGAGTAGC AGATACCATCAAACAAACTATAACTGATTTAATGAGCCATTCGA

> Mamosranga

ARKTGKCACAGTACTGCGACAACTTAWGKATACTATTGGRGATTTAGAGAGCA CWYTCKTCRWTTMCAGACTGGWAAASYCMGGTATTGTTATTTRTTGTCACTACC 220

TCCCCGTGTCAGGATTGGRTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTA GCCGTTTCTCAGGCTCCCTCTCCGGAATCGAACCCTAATTCTCCGTCACCCGTCA CCACCATGGTAGGCCACTATCCTACCATCGAAAGTTGATAGGGCAGATATTTGA ATGATGCGTCGCCAGCACGATGGCTGTGCGATCCGTCGAGTTATCATGAATCAT CAGAGCAACGGGCAGAGCCCGCGTCGACCTTTTATCTAATAAATGCGTCCCTTC CAGAAGTCGGGGTTTGTTGCACGTATTAGCTCTAGAATTACTACGGTTATCCGA GTAGCAGRTACCATCAAACAAACTATAACTGATTTAATGAGCCATTC

> Guraky Tango (Gt)

CCGTACTGCGACAACTTATGTATACkCTATmGGGGATTTAGAGTGCACTyCTACT TTACCAGACTGGTAGAGyCCGGTATTGTyATTTGTTGTCACTACCTCCCCGTGTC AGGATTGGrTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTC AGGCTCCCTCTCCGGAATCGAACCCTAATTCTCCGTCACCCGTCACCACCATGG TAGGCCACTATCCTACCATCGAAAGTTGATAGGGCAGATATTTGAATGATGCGT CGCCAGCACGATGGCTGTGCGATCCGTCGAGTTATCATGAATCATCAGAGCAA CGGGCAGAGCCCGCGTCGACCTTTTATCTAATAAATGCGTCCCTTCCAGAAGTC GGGGTTTGTTGCACGTATTAGCTCTAGAATTACTACGGTTATCCGAGTAGCAGA TACCATCAAACAAACTATAACTGATTTAATGAGCC

> Pekhawary Tango (Pt)

CTGCTKKCACCRKWCTGGGACMTCYTAWGGATCCKCKATAGGGGATTTARAKT GYACTTCCATTTMCCAGACTGGKAGAGCCCGGTATTGTTATTTATTGTCACTACC TCCCCGTGTCAGGATTGGGTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTA GCCGTTTCTCAGGCTCCCTCTCCGGAATCGAACCCTAATTCTCCGTCACCCGTCA CCACCATGGTAGGCCACTATCCTACCATCGAAAGTTGATAGGGCAGATATTTGA ATGATGCGTCGCCAGCACGATGGCTGTGCGATCCGTCGAGTTATCATGAATCAT CAGAGCAACGGGCAGAGCCCGCGTCGACCTTTTATCTAATAAATGCGTCCCTTC CAGAAGTCGGGGTTTGTTGCACGTATTAGCTCTAGAATTACTACGGTTATCCGA GTAGCAGATACCATCAAACAAACTATAACTGATTTAATGAGCCATT

> Ghata Tora Tangai (Gtt)

AACTTGCCCTCCAATGGATCCTCGTTAAGGGATTTAGATTGTACTCATTCCAATT ACCAGACTCGTAGAGCCCGGTATTGTTATTTATTGTCACTACCTCCCCGTGTCAG GATTGGGTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAG GCTCCCTCTCCGGAATCGAACCCTAATTCTCCGTCACCCGTCACCACCATGGTA GGCCACTATCCTACCATCGAAAGTTGATAGGGCAGATATTTGAATGATGCGTCG CCAGCACGATGGCTGTGCGATCCGTCGAGTTATCATGAATCATCAGAGCAACG GGCAGAGCCCGCGTCGACCTTTTATCTAATAAATGCGTCCCTTCCAGAAGTCGG

221

GGTTTGTTGCACGTATTAGCTCTAGAATTACTACGGTTATCCGAGTAGCAGGTA CCATCAAACAAACTATAACTGATTTAATGAGCCATTCGCAAGTTTC

>Kado Batang (Kb)

CTTGCCTCCATGGATCCTCGTTAAGGGATTTAGATTGTACTCATTCCAATTACCA GACTCGTAGAGCCCGGTATTGTTATTTATTGTCACTACCTCCCCGTGTCAGGATT GGGTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAGGCTC CCTCTCCGGAATCGAACCCTAATTCTCCGTCACCCGTCACCACCATGGTAGGCC ACTATCCTACCATCGAAAGTTGATAGGGCAGATATTTGAATGATGCGTCGCCAG CACGATGGCTGTGCGATCCGTCGAGTTATCATGAATCATCAGAGCAACGGGCA GAGCCCGCGTCGACCTTTTATCTAATAAATGCGTCCCTTCCAGAAGTCGGGGTT TGTTGCACGTATTAGCTCTAGAATTACTACGGTTATCCGAGTAGCAGATACCAT CAAACAAACTATAACTGATTTAATG

>Zira Mamoothay (Zm)

GCTGGCACAGKACTKGSACCTCYWATGGATCCTCGTTAGGGGATTTAGATTGTA CTCMTTCCAATTACCAGACTCGTAGAGCCCGGTATTGTTATTTATTGTCACTACC TCCCCGTGTCAGGATTGGGTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTA GCCGTTTCTCAGGCTCCCTCTCCGGAATCGAACCCTAATTCTCCGTCACCCGTCA CCACCATGGTAGGCCACTATCCTACCATCGAAAGTTGATAGGGCAGATATTTGA ATGATGCGTCGCCAGCACGATGGCTGTGCGATCCGTCGAGTTATCATGAATCAT CAGAGCAACGGGCAGAGCCCGCGTCGACCTTTTATCTAATAAATGCGTCCCTTC CAGAAGTCGGGGTTTGTTGCACGTATTAGCTCTAGAATTACTACGGTTATCCGA GTAGCAGRTACCATCAAACAAACTATAACTGATTTAATGAGCCATT

> Shaker Tango (St)

TACTGCAACAACTTATGrAwACGCTATwrGGGGATTTArAGwGCACTCCTTAAwT TcCCAGACTGGTArAGyCCGGTATTGTTATTTGTTGTCACTACCTCCCCGTGTCAG GATTGGGTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAG GCTCCCTCTCCGGAATCGAACCCTAATTCTCCGTCACCCGTCACCACCATGGTA GGCCACTATCCTACCATCGAAAGTTGATAGGGCAGATATTTGAATGATGCGTCG CCAGCACGATGGCTGTGCGATCCGTCGAGTTATCATGAATCATCAGAGCAACG GGCAGAGCCCGCGTCGACCTTTTATCTAATAAATGCGTCCCTTCCAGAAGTCGG GGTTTGTTGCACGTATTAGCTCTAGAATTACTACGGTTATCCGAGTAGCAGATA CCATCAAACAAACTATAACTGATTTAATGAGCCAT

> Parawoo Tango (Pt)

ACGNTGCAACAAYTAAATATACGCTATTGGAGCTGGAATTACCGCGGCTGCTG GCACCAGACTTGCCCTCCAATGGATCCTCGTTAAGGGATTTAGATTGTACTCAT TCCAATTACCAGACTCGTAGAGCCCGGTATTGTTATTTATTGTCACTACCTCCCC 222

GTGTCAGGATTGGGTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGT TTCTCAGGCTCCCTCTCCGGAATCGAACCCTAATTCTCCGTCACCCGTCACCACC ATGGTAGGCCACTATCCTACCATCGAAAGTTGATAGGGCAGATATTTGAATGAT GCGTCGCCAGCACGATGGCTGTGCGATCCGTCGAGTTATCATGAATCATCAGAG CAACGGGCAGAGCCCGCGTCGACCTTTTATCTAATAAATGCGTCCCTTCCAGAA GTCGGGGTTTGTTGCACGTATTAGCTCTAGAATTACTACGGTTATCCGAGTAGC AGATACCATCAAACAAACTATAACTGATTTAATGAGCCATTC

L2

> Khan Tango (Kt)

ACTGCGAATGGCTCATTAAATCAGTTATAGTTTGTTTGATGGTAYCTGCTACTCG GATAACCGTAGTAATCTCTAGAGCTAATACGTGCAACAAACCCCGACTTCTGG AAGGGACGCATTTATTAGATAAAAGGTCGACGCGGGCTCTGCCCGTTGCTCTGA TGATTCATGATAACTCGACGGATCGCACAGCCATCGTGCTGGCGACGCATCATT CAAATATCTGCCCTATCAACTTTCGATGGTAGGATAGTGGCCTACCATGGTGGT GACGGGTGACGGAGAATTAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACG GCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCTGACACG GGGAGGTAGTGACAATAAATAACAATACCGGGCTCTACGAGTCTGGTAATTGG AATGAGTACAATCTAAATCCCTTAACGAGGATCCATTGGAGGGCAAGTCTGGT GCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTTAAGTT

> Gultar Tango (Gt)

CAACAACTTAAATATACGCTATTGGAGCTGGAATTACCGCGGCTGCTGGCACC AGACTTGCCCTCCAATGGATCCTCGTTAAGGGATTTAGATTGTACTCATTCCAAT TACCAGACTCGTAGAGCCCGGTATTGTTATTTATTGTCACTACCTCCCCGTGTCA GGATTGGGTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCA GGCTCCCTCTCCGGAATCGAACCCTAATTCTCCGTCACCCGTCACCACCATGGT AGGCCACTATCCTACCATCGAAAGTTGATAGGGCAGATATTTGAATGATGCGTC GCCAGCACGATGGCTGTGCGATCCGTCGAGTTATCATGAATCATCAGAGCAAC GGGCAGAGCCCGCGTCGACCTTTTATCTAATAAATGCGTCCCTTCCAGAAGTCG GGGTTTGTTGCACGTATTAGCTCTAGAATTACTAC

>Shaker Batang (Sb)

CAACTTAAATATACGCTATTGGAGCTGGAATTACCGCGGCTGCTGGCACCAGA CTTGCCCTCCAATGGATCCTCGTTAAGGGATTTAGATTGTACTCATTCCAATTAC CAGACTCGTAGAGCCCGGTATTGTTATTTATTGTCACTACCTCCCCGTGTCAGGA TTGGGTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAGGC TCCCTCTCCGGAATCGAACCCTAATTCTCCGTCACCCGTCACCACCATGGTAGG 223

CCACTATCCTACCATCGAAAGTTGATAGGGCAGATATTTGAATGATGCGTCGCC AGCACGATGGCTGTGCGATCCGTCGAGTTATCATGAATCATCAGAGCAACGGG CAGAGCCCGCGTCGACCTTTTATCTAATAAATGCGTCCCTTCCAGAAGTCGGGG TTTGTTGCACGTATTAGCTCTAGAATTACTACGGTTATCCGAGTAGCAGRTACCA TCAAACAAACTATAACTGATTTAATGAGCCATTCGCAG

> Ghata Zira Tangai (Gzt)

CTGCTACTCGGATAACCGTAGTAATTCTAGAGCTAATACGTGCAACAAACCCC GACTTCTGGAAGGGACGCATTTATTAGATAAAAGGTCGACGCGGGCTCTGCCC GTTGCTCTGATGATTCATGATAACTCGACGGATCGCACAGCCATCGTGCTGGCG ACGCATCATTCAAATATCTGCCCTATCAACTTTCGATGGTAGGATAGTGGCCTA CCATGGTGGTGACGGGTGACGGAGAATTAGGGTTCGATTCCGGAGAGGGAGCC TGAGAAACGGCTACCACATCCAAGGAAGGCcGCAGGCGCGCAcATTACCCAAT CCTGACACGGGGAGGTAGTGACAATAAATAACAATACCGGcCTCTACCACTCT GGTAATTGGAATGAGTATAATCTAAATCCCTTAACTAGGATCCATTGGAGGGCA AGTCTGGAGCCATCACCCgCGGTATTTCCAAgTCCAATANCNNATATTTAANT

>Asmasay Tango

ATATACGCTATTGGAGCTGGAATTACCGCGGCTGCTGGCACCAGACTGTGCCCT CCWATGGATCCTCGTTAAGGGATTTAGATTGTACTCATTCCAATTACCAGACTC GTAGAGCCCGGTATTGTTATTTATTGTCACTACCTCCCCGTGTCAGGATTGGGTA ATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAGGCTCCCTCT CCGGAATCGAACCCTAATTCTCCGTCACCCGTCACCACCATGGTAGGCCACTAT CCTACCATCGAAAGTTGATAGGGCAGATATTTGAATGATGCGTCGCCAGCACG ATGGCTGTGCGATCCGTCGAGTTATCATGAATCATCAGAGCAACGGGCAGAGC CCGCGTCGACCTTTTATCTAATAAATGCGTCCCTTCCAGAAGTCGGGGTTTGTTG CACGTATTAGCTCTAGAATTACTACGGTTATCCGAGTAGCAGRTACCATCAAAC AAACTATAACTGATTTAATGAGCCATT

> Kacha Tora Tangai (Ktt)

AATATACGCTATTGGAGCTGGAATTACCGCGGCTGCTGGCACCAGACTTGCCCT CCAATGGATCCTCGTTAAGGGATTTAGATTGTACTCATTCCAATTACCAGACTC GTAGAGCCCGGTATTGTTATTTATTGTCACTACCTCCCCGTGTCAGGATTGGGTA ATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAGGCTCCCTCT CCGGAATCGAACCCTAATTCTCCGTCACCCGTCACCACCATGGTAGGCCACTAT CCTACCATCGAAAGTTGATAGGGCAGATATTTGAATGATGCGTCGCCAGCACG ATGGCTGTGCGATCCGTCGAGTTATCATGAATCATCAGAGCAACGGGCAGAGC CCGCGTCGACCTTTTATCTAATAAATGCGTCCCTTCCAGAAGTCGGGGTTTGTTG

224

CACGTATTAGCTCTAGAATTACTACGGTTATCCGAGTAGCAGGTACCATCAAAC AAACTATAACTGATTTAATGAGCCATTCG

> Mamosy Batal_14

ATATACGCTATTGGAGCTGGAATTACCGCGGCTGCTGGCACCAGACTTGCCCW CYWATGGATMCTCGWTWRGGGATTTARAKTGTACTCATTYCAATTACCAGACT CGTAGAGCCCGGTATTGTTATTTATTGTCACTACCTCCCCGTGTCAGGATTGGGT AATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAGGCTCCCTC TCCGGAATCGAACCCTAATTCTCCGTCACCCGTCACCACCATGGTAGGCCACTA TCCTACCATCGAAAGTTGATAGGGCAGATATTTGAATGATGCGTCGCCAGCACG ATGGCTGTGCGATCCGTCGAGTTATCATGAATCATCAGAGCAACGGGCAGAGC CCGCGTCGACCTTTTATCTAATAAATGCGTCCCTTCCAGAAGTCGGGGTTTGTTG CACGTATTAGCTCTAGAATYACTACCGGKTATCMSAGTAKCAGAYAMCATCAA ACAAACTATAACTGATTTAATGAGCCATTC

> Mamosay Batal-8

TAAATATACGCTATTGGAGCTGGAATTACCGCGGCTGCTGGCACCAGACTTGCC CTCYWATGKATCCTCKWTWRGGGATTTAGAKTGTAYTCATTCCAATTACCAGA CTCGTAGAGCCCGGTATTGTTATTTATTGTCACTACCTCCCCGTGTCAGGATTGG GTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAGGCTCCC TCTCCGGAATCGAACCCTAATTCTCCGTCACCCGTCACCACCATGGTAGGCCAC TATCCTACCATCGAAAGTTGATAGGGCAGATATTTGAATGATGCGTCGCCAGCA CGATGGCTGTGCGATCCGTCGAAGTTATCTGAATCATCAGAGCAACGGGCAGA GCCCGCGTCGACCTTTTATCTAATAAATGCGTCCCTTCCAGAAGTCGGGGTTTGT TGCACGTATTAGCTCTAGAATTACTACGGTWWTCMSAGTAGCAGATACCATCA AACAAACTATAACTGATTTAATGAGCCATT

> Hary Tango Batal

TAAATATACGCTATTGGAGCTGGAATTACCGCGGCWGCTKKCACMRKAYKYGM CCTCYWAWGGATCCTCGWTWRGGGATTTARAKTGTACTCATTCCAATTACCAG ACTCGTAGAGCCCGGTATTGTTATTTATTGTCACTACCTCCCCGTGTCAGGATTG GGTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAGGCTCC CTCTCCGGAATCGAACCCTAATTCTCCGTCACCCGTCACCACCATGGTAGGCCA CTATCCTACCATCGAAAGTTGATAGGGCAGATATTTGAATGATGCGTCGCCAGC ACGATGGCTGTGCGATCCGTCGAGTTATCATGAATCATCAGAGCAACGGGCAG AGCCCGCGTCGACCTTTTATCTAATAAATGCGTCCCTTCCAGAAGTCGGGGTTT GTTGCACGTATTAGCTCTAGAATTACTACSGTTATCMSAGTAKSMRAYAMCATC AAACAAACTATAACTGATTTAATGAGCCATT

> Mamosay Batal_12

225

TAAATATACGCTATTGGAGCTGGAATTACCGCGGCTGCTGGCACCAKACTGYG MCMTCYWATGGATCCTCGWTWRGGGATTTAGATTGTACTCATTCCAATTACCA GACTCGTAGAGCCCGGTATTGTTATTTATTGTCACTACCTCCCCGTGTCAGGATT GGGTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAGGCTC CCTCTCCGGAATCGAACCCTAATTCTCCGTCACCCGTCACCACCATGGTAGGCC ACTATCCTACCATCGAAAGTTGATAGGGCAGATATTTGAATGATGCGTCGCCAG CACGATGGCTGTGCGATCCGTCGAGTTATCATGAATCATCAGAGCAACGGGCA GAGCCCGCGTCGACCTTTTATCTAATAAATGCGTCCCTTCCAGAAGTCGGGGTT TGTTGCACGTATTAGCTCTAGAAYTACTACGGKTATCMSAGTAKCAGAYACCAT CAAACAAACTATAACTGATTTAATGAGCCAT

> Mamosy Batal_15

TAAATATACGCTATTRKARCTGSARYYAYYGCSGCWGCTKKCACMRKAYKYGMC MTCYWATGGATCCTCGWTWRGGGATTTAGAKTGTACTCATTCCAATTACCAGA CTCGTAGAGCCCGGTATTGTTATTTATTGTCACTACCTCCCCGTGTCAGGATTGG GTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAGGCTCCC TCTCCGGAATCGAACCCTAATTCTCCGTCACCCGTCACCACCATGGTAGGCCAC TATCCTACCATCGAAAGTTGATAGGGCAGATATTTGAATGATGCGTCGCCAGCA CGATGGCTGTGCGATCCGTCGAGTTATCATGAATCATCAGAGCAACGGGCAGA GCCCGCGTCGACCTTTTATCTAATAAATGCGTCCCTTCCAGAAGTCGGGGTTTGT TGCACGTATTAGCTCTAGAAYTACTACSGKTATCMSAGTAGCAGATACCATCAA ACAAACTATAACTGATTTAATGAGCCATTCCACGTTTTCACAAAA

> Spina Mamothy

GCACAGGACKTGCCCTCCTATGKRTCCTCGTTAGGGGATTTAGATTGTACTCATT CCAATTACCAGACTCGTAGAGCCCGGTATTGTTATTTATTGTCACTACCTCCCCG TGTCAGGATTGGGTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTT TCTCAGGCTCCCTCTCCGGAATCGAACCCTAATTCTCCGTCACCCGTCACCACC ATGGTAGGCCACTATCCTACCATCGAAAGTTGATAGGGCAGATATTTGAATGAT GCGTCGCCAGCACGATGGCTGTGCGATCCGTCGAGTTATCATGAATCATCAGAG CAACGGGCAGAGCCCGCGTCGACCTTTTATCTAATAAATGCGTCCCTTCCAGAA GTCGGGGTTTGTTGCACGTATTAGCTCTAGAATTACTACGGTTATCCGAGTAGC AGRTACCATCAAACAAACTATAACTGATTTAATGAGCCATTCCC

>Pak_24

TGCTACTCGGATAACCGTAGTAATTCTAGAGCTAATACGTGCAACAAACCCCG ACTTCTGGAAGGGACGCATTTATTAGATAAAAGGTCGACGCGGGCTCTGCCCGT TGCTCTGATGATTCATGATAACTCGACGGATCGCACAGCCATCGTGCTGGCGAC GCATCATTCAAATATCTGCCCTATCAACTTTCGATGGTAGGATAGTGGCCTACC ATGGTGGTGACGGGTGACGGAGAATTAGGGTTCGATTCCGGAGAGGGAGCCTG

226

AGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATC CTGACACGGGGAGGTAGTGACAATAAATAACAATACCGGGCTCTACGAGTCTG GTAATTGGAATGAGTACAATCTAAATCCCTTAACGAGGATCCATTGGAGGGCA AGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTTAAGTT

227

PAPERS PUBLISHED

Int. J. Biosci. 2012

International Journal of Biosciences (IJB) ISSN: 2220-6655 (Print) 2222-5234 (Online) Vol. 2, No. 12, p. 1-6, 2012 http://www.innspub.net

R ESEARCH PAPER OPEN ACCESS

Morphological evaluation of Pyrus genotypes of Kaghan Valley, Pakistan through quantitative parameters Mohammad Islam*, Habib Ahmad

Department of Genetics, Hazara University, Mansehra, Pakistan

Received: 05 November 2012 Revised: 28 November 2012 Accepted: 28 November 2012 Key words: Pyrus, morphology, quantitative parameters, Kaghan, Pakistan.

Abstract

In the present study, 14 genotypes of Pyrus were evaluated and compared for quantitative parameters viz. Petiole length, Leaf Area, Pedicle length, Fruit length, Fruit width and Fruit weight with the help of local names belonging to Kaghan Valley, Pakistan. The study revealed that the genotypes are highly significant with respect to above parameters except leaf area. Mean value shows that genotypes Kushbago, Atti Bating and Shardi Tanchi have maximum value while genotypes Black batangi and Glass batangi have minimum value while all others have intermediate values with respect to petiole length. For Pedicle length, genotypes Black batangi, Brown batangi, Nak Hard Skin, Shardi tanchi have minimum value (ranges 15-20 mm), genotype kushbago has maximum (65.5 mm), Tanchi and Glass batang have similar (55.8mm) values and all others ranges from 21.5-30 mm. Similarly for fruit length, genotypes Glass Batang has highest value fallowed by genotypes Kado and China batang while genotypes Black Batangi, Golden Batangi and Shardi have minimum value. For fruit weidth, genotype Chiana batang has highest value, genotypes black batangi, golden batangi and Shardi tanchi have the lowest value while genotypes Kado and Glass batang have similar values and all others genotypes have values ranges from 35.17- 46.1 mm. For fruit weight, genotypes China and Glass batang have maximum value, followed by genotypes Kado, Kushbago and Nak Hard Skin batang while the minimum values showed by genotype black batangi, Golden batangi and Shardi Tanchi. From above discussion it is concluded that these parameters play an important role in the identification of these genotypes belonging to the genus Pyrus. *Corresponding Author: Mohammad Islam  [email protected]

228

1 Islam and Ahmad

Int. J. Biosci. 2013

International Journal of Biosciences | IJB | ISSN: 2220-6655 (Print) 2222-5234 (Online) http://www.innspub.net Vol. 3, No. 4, p. 122-127, 2013

RESEARCH PAPER OPEN ACCESS

An efficient protocol for DNA isolation from the genus Pyrus

Mohammad Islam*, Habib Ahmad, Imtiaz Ahmad Khan

Department of Genetics, Hazara University, Mansehra, KP, Pakistan

Key words: DNA Isolation, CTAB, economical protocol, Pyrus species. doi: http://dx.doi.org/10.12692/ijb/3.4.122-127 Article published on April 22, 2013

Abstract

This paper communicates an economical, handy, quicker and reliable protocol of DNA isolation from different parts e.g. herbarium specimens, bark, wood, leaves etc. of pears. The protocol is recommended under the normal lab conditions without using any sophisticated equipment and costly kits/chemicals. Best quality of DNA was extracted from any part of the plant. The PCR product was amplified for 18S rRNA genes of various Pyrus genotypes. The genes were cleaned with gel elution Kit (K0513, Fermentas) and sequenced accordingly. The sequence data obtained was BLAST against the entire available nucleotide database using online tool at NCBI access and the land races were successfully discriminated. The protocol presented here is recommended for reliable extraction of best quality and high yield of DNA from woody plants. * Corresponding Author: Mohammad Islam  [email protected]

229

122 Islam et al.

6.9 ABSTRACTS PUBLISHED

TheThird International Symposium on theBiology of RareandEndemicPlantSpecies (BIORARE-2014) April 19-23 2014Antalya,Turkey

OP48: DISTRIBUTION OF Pyrus SPECIES ACROSS THE HINDU KUSH- HIMALAYN REGION OF NORTHERN PAKISTAN

Mohammad Islam*, Habıb Ahmad

Department of Genetics, Hazara University, Mansehra, Pakistan

Hindu Kush Himalayas form the northern temperate mountainous terrain of Pakistan. The area has witnessed variety of civilization entering and disappearing here, wherein their remains are not only available as archeological records but has also left behind diverse biological resources including food crops among which the temperate fruits like pears are most important. Extensive collection of specimen pear trees available in nature or traditional farms in Northern Pakistan and Azad Kashmir were made during 2008-2011. The collected specimens were processed and deposited in Herbarium Hazara University, Mansehra. Valid names and synonyms of all the recognized taxa were typified. Key to the species along with detailed morphological description were developed. Sum 14 species viz., P. Pashia, Pyrus calleryana, Pyrus bretschneideri, Pyrus pyrifolia, Pyrus pseudopashia, Pyrus communis, Pyrus sinkiangensis, Pyrus hopeienses, Pyrus serrulata, Pyrus ovoidea, P. turcomanica, Pyrusussuriensis, Pyrus xerophila and Pyrus armenicefolia. Among these, only two species i.e. P.pashia and P. communis were previously reported from here.

Keywords:Hindu Kush, Himalaya, Pakistan, pear, Pyrus. *Presenting author; email:[email protected]

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TheThird International Symposium on theBiology of RareandEndemicPlantSpecies (BIORARE-2014) April 19-23 2014Antalya,Turkey

PP56: SPECIES DIVERSITY OF PEARS IN PAKISTAN: 1. PRELIMINARY REPORT OF THE 18S RRNA ANALYSES

Muhammad Islam*, HabibAhmad

Department of Genetics, Hazara University, Mansehra, Pakistan

Pear allies belonging to the genus Pyrus are the traditional selections widely available as landraces in the traditional field boundaries, conventional forms, kitchen garden and as feral in the wild, mostly in temperate areas of Pakistan. These land races possesses overlapping morphological traits and lacks sharply differentiating taxonomic characters, thus not established taxonomically up-till now. Thus 18S rRNA nucleotide sequence analyses were started to differentiate three landraces at molecular level. We communicate the preliminary results obtained for three land races viz. Kacha Tora Tangai (Ktt), Ghata Tora Tangai (Gtt) and Ghata Zira Tangai (Gzt). The BLAST nucleotide sequence of Ktt revealed 99% maximum identity and quarry cover with the reference sequences of P.pyrifolia cvs. Sunwhang and Miwhang, available at NCBI. The land race Gtt proved 100% maximum identity and quarry cover with P. pyrifolia cv. Imamuraaki. Similarly Gzt provided 97% maximum identity and quarry cover with P. communis cvs. Pachkan's Triumph, Beurre, and 97% maximum identity and quarry cover with P. communis cv. Conference and P. pyrifolia cv. Okusankichi. The study concluded that both the land races Ktt and Gtt are P. pyrifolia in origin, whereas Gzt might be a hybrid of P. communis and P. pyrifolia.

Keywords:Pakistan, pyrus, pear, species diversity, 18S rRNA *Presenting author; email:[email protected]

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