-1DWQ6FL)RXQGDWLRQ6UL/DQND DOI: http://dx.doi.org/10.4038/jnsfsr.v45i3.8190
RESEARCH ARTICLE
Morphological, SSR and ISSR marker based genetic diversity assessment of mountain papaya germplasm in comparison with Carica papaya
W.A.D.L.R. Warnakula 1, N.S. Kottearachchi 1* and K. Yakandawala 2 1 Department of Biotechnology, Faculty of Agriculture and Plantation Management, Wayamba University of Sri Lanka, Makandura, Gonawila (NWP). 2 Department of Horticulture and Landscape Gardening, Faculty of Agriculture and Plantation Management, Wayamba University of Sri Lanka, Makandura, Gonawila (NWP).
Revised: 02 October 2016; Accepted: 16 February 2017
Abstract: The genetic diversity in papaya cultivars is 3DSD\DLVDJRRGVRXUFHRIYLWDPLQVDQGLWLVZLGHO\JURZQ essentially important as it provides the basis for varietal for consumption as a fresh fruit and for use in drinks, LPSURYHPHQW,QWKLVUHJDUGDVWXG\ZDVLQLWLDWHGWRDVVHVVWKH jams, candies, etc. Papaya also has pharmaceutical and genetic diversity of the commercial Carica papaya cultivars industrial values due to its proteins and alkaloids. Of in Sri Lanka and to introduce mountain papaya ( Vasconcellea these, papain is the most important industrially valuable cundinamarcensis ), ZKLFKLVUHSRUWHGWRFRQWDLQFROGUHVLVWDQFH DQG SDSD\D ULQJ VSRW YLUXV 35693 UHVLVWDQW WUDLW 7ZHQW\ SURWHRO\WLFHQ]\PHWKDWLVSURGXFHGLQWKHPLON\ODWH[RI one accessions of Carica papaya DQGPRXQWDLQSDSD\DZHUH green, unripe papaya fruits (Moussaoui et al. , 2001). In assessed by morphological, simple sequence repeat (SSR) and Sri Lanka there are several C. papaya cultivars including inter simple sequence repeat (ISSR) markers. A total of 33 introduced varieties from other countries, hybrid varieties DOOHOHVZHUHJHQHUDWHGZLWKDQDYHUDJHIUHTXHQF\RIDOOHOHV and local-traditional cultivars. Currently 7,108 ha are per marker from 11 SSR and 2 ISSR markers. Morphological under cultivation and the annual production is 84,606 mt and molecular marker based cluster analyses revealed that (DOA, 2014). WKHUHZDVQRFOHDUGLVWLQFWLRQDPRQJWKH C. papaya cultivars JURZQ LQ GLIIHUHQW JHRJUDSKLFDO DUHDV LQ 6UL /DQND ZKLOH The family Caricaceae FRPSULVHV ¿YH JHQHUD DQG C. papaya PRXQWDLQSDSD\DZDVKLJKO\GLVWLQFWIURPWKHRWKHU DERXWíVSHFLHV &DUYDOKR et al., 2015). Previously, accessions. Except three SSR markers, all the other markers the genus Carica ZHUH SRO\PRUSKLF EHWZHHQ PRXQWDLQ SDSD\D DQG C. papaya FRQWDLQHG VSHFLHV DQG IROORZLQJ DFFHVVLRQV2IWKHDOOHOHVSURGXFHGDOOHOHVZHUHFRPPRQ the recommendation of Badillo (2000) Carica ZDVVSOLW for both C. papaya and mountain papaya indicating the LQWR WZR JHQHUD FUHDWLQJ DQ DGGLWLRQDO JHQXV FDOOHG potential relatedness to C. papaya. This investigation revealed Vasconcellea FRPSULVLQJZLOGVSHFLHV3UHVHQWO\WKH both the genetic diversity and the relatedness of mountain genus Carica contains only one species, Carica papaya , SDSD\DZLWK C. papaya so as to use it as a potential source for ZKLFK LV WKH PRVW HFRQRPLFDOO\ YDOXDEOH Caricaceae the improvement of C. papaya by hybridisation. VSHFLHVZRUOGZLGH
Keywords: Carica papaya , ISSR markers, morphological The mountain papaya plant has been studied by characterisation, mountain papaya, SSR markers. PDQ\ VFLHQWLVWV LQ WKH ZRUOG GXH WR LWV HFRQRPLFDOO\ important traits such as the potential for cold tolerance INTRODUCTION (Muthulakshmi et al. , 2007), papaya ring spot virus (PRSV-P) resistance (Dillon et al ., 2006), higher sugar Most of the Caricaceae species are considered as FRQWHQW RI WKH IUXLW 'UHZ et al. ,1998), and genetic unexploited species except for Carica papaya /ZKLFK relatedness to C. papaya 2¶%ULHQ 'UHZ $V LV FRPPHUFLDOO\ JURZQ LQ PDQ\ SDUWV RI WKH ZRUOG the closest relative of C. papaya species, mountain
* Corresponding author ( [email protected] ) W.A.D.L.R. Warnakula et al. papaya has the likelihood to hybridise and exchange In Sri Lanka, there are four main types of commercial JHQHVZKLFKDUHDVVRFLDWHGZLWKHFRQRPLFDOO\LPSRUWDQW papaya: Rathne, Sinta, Red Lady and local/ traditional traits (Badillo, 2000). Both Carica and Vasconcellea are varieties, and their genetic relatedness has not been genetically diploids and share the same chromosome studied at molecular level using simple sequence repeat number, 2n = 18 (Storey, 1976). Several research studies (SSR) and inter simple sequence repeat (ISSR) markers. have indicated that pollination of C. papayaZLWKSROOHQ Allelic variation of the economically important traits IURPZLOGSDSD\DV DQG vice versa ) successfully facilitate needs to be utilised in breeding programmes and they can WKH IHUWLOLVDWLRQ 0DQVKDUGW :HQVODII 'UHZ be revealed by SSR and ISSR marker based assessments. et al ., 1998). Although mountain papaya naturally possesses a number of desirable traits, its proper characterisation has not 0RXQWDLQ SDSD\D H[LVWHG LQ WKH PRQWDQH ]RQH RI EHHQ FRQGXFWHG DQG QHJOHFWHG DV LW JURZV LQ WKH ZLOG Sri Lanka has been recognised as Carica pubescence 7KHUHIRUHWKLVUHVHDUFKVWXG\ZDVDLPHGDWDVVHVVLQJWKH in the Revised Handbook to the Flora of Ceylon genetic diversity of the commercial papaya accession in (Dasanayake, 1995) and in the Check List of the 6UL/DQNDDORQJZLWKPRXQWDLQSDSD\DDQGWRH[SORUHLWV )ORZHULQJ3ODQWVRI6UL/DQND 6HQDUDWQD ,WKDV potential traits to improve C. papaya accessions in the EHHQ UHSRUWHG LQ WKH +DOJROOD (VWDWH ZKLFK LV ORFDWHG country. LQ WKH VRXWKHDVWHUQ HGJH RI WKH .HJDOOH 'LVWULFW DQG FORVH WR WKH ZHVWHUQ ERXQGDU\ RI WKH 1XZDUD (OL\D METHODOLOGY District (Weerakoon et al ., 2009) as an uncultivated papaya species. Mountain papaya is a sparsely branched Plant materials herbaceous tree, habituated in the highest elevation OHYHO LQ 6KDQWKLSXUD 1XZDUD (OL\D7D[RQRP\ RI WKH /HDI VDPSOHV ZHUH FROOHFWHG IURP JHQRW\SHV RI mountain papaya accession in Sri Lanka has not been C. papaya distributed in the Central, North Western explained clearly and naturally hybridised genotypes and Western provinces of Sri Lanka and three mountain have not been reported in Sri Lanka. SDSD\D VDPSOHV ZHUH FROOHFWHG IURP 6KDQWKLSXUD
Table 1: Details of the papaya germplasm and their locations
6DPSOHQR *HUPSODVP &RGH 6RXUFH 'LVWULFW
5HG/DG\ 5'/ .DWXJDVWKRWD .DQG\ /RFDO /&/ .DWXJDVWKRWD .DQG\ 3 Sinta SIN01 PVIC, Homagama Colombo 4 Breeders line BRL01 PVIC, Homagama Colombo 5 Breeders line BRL02 PVIC, Homagama Colombo 6 Local LCL02 PVIC, Homagama Colombo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
39,&3ODQW9LUXV,QGH[LQJ&HQWUH 6&66HHG&HUWL¿FDWLRQ6WDWLRQ +5)+RUWLFXOWXUDO5HVHDUFK)DUP RARDC - Regional Agricultural Research and Development Centre
September 2017 Journal of the National Science Foundation of Sri Lanka 45(3) Genetic diversity assessment of mountain papaya 257
1XZDUD(OL\D6UL/DQNDIRU'1$H[WUDFWLRQ7KHORFDO descriptor list published by the International Board for or common names ascribed to the individual accessions 3ODQW*HQHWLF5HVRXUFHV ,%3*5 $VWKHVDPSOHV ZHUH UHFRUGHG DV D UHIHUHQFHWR WKH PDWHULDOFROOHFWHG ZHUHFROOHFWHGIURPGLIIHUHQWJHRJUDSKLFDODUHDVLQWKH DQGHDFKDFFHVVLRQZDVDVVLJQHGDWD[RQFRGHEDVHGRQ FRXQWU\RQO\WKHTXDOLWDWLYHWUDLWVZHUHDFFRXQWHGLQWKLV their local name (Table 1). VWXG\ IROORZLQJ WKH VWXG\ FRQGXFWHG E\ 0DGDUERNXV and Ranghoo-Sanmukhiya (2012). The sex of each Morphological analysis YDULHW\ ZDV GHWHUPLQHG E\ WKH ÀRUDO PRUSKRORJ\ -LPpQH] et al. , 2012). Visual morphological characters A total of 14 vegetative and reproductive characters RIWKHSODQWVSHFLPHQVRIPRXQWDLQSDSD\DZHUHIXUWKHU ZHUH REVHUYHG in situ from the selected papaya FODUL¿HG E\ H[DPLQLQJ GLJLWDO LPDJHV RI KHUEDULXP DFFHVVLRQV 7DEOH $VFRUHZDVDVVLJQHG 7DEOH VSHFLPHQVLQWKH.HZ+HUEDULXPGDWDEDVH KWWS ZZZ WRHDFKPRUSKRORJLFDOFKDUDFWHUIROORZLQJWKHSDSD\D NHZRUJVFLHQFH ).
Table 2: 4XDOLWDWLYHWUDLWVDQGWKHLUUHVSHFWLYHVFRUH
1R 4XDOLWDWLYHWUDLW 6FRUHV
1 Stem type 1 - Single, 2 - Branched 6WHPFRORXU /LJKWJUH\*UH\LVKEURZQ 3 Stem pigmentation 1 - Mostly upper, 2 - Indiscriminate &RORXURIPDWXUHOHDISHWLROH 3DOHJUHHQ'DUNJUHHQ5HGSXUSOH*UHHQSXUSOH 5 Waxiness on leaf surface 1 - Present, 2 - Absent 6 Leaf segments (lobes) 1 - Nine segments, 2 - Seven segments 7\SHRIÀRZHULQJ 6ROLWDU\,QÀRUHVFHQFH &RORXURILQÀRUHVFHQFHVWDON &UHDP Genomic DNA extraction IRU PLQ 7KH SUHFLSLWDWHG '1$ ZDV ZDVKHG ZLWK 70 % ice-cold ethanol by centrifugation at 15,000 rpm $ KHDOWK\ WHQGHU OHDI ZDV XVHG IRU H[WUDFWLQJ '1$ IRUDQRWKHUPLQ7KH'1$SHOOHWVZHUHWKHQGULHGDQG )UHVKOHDIWLVVXHV PJ ZHUHJURXQGLQȝ/RI UHVXVSHQGHGLQȝ/RI7(EXIIHUDQGVWRUHGDW& cetyl trimethyl ammonium bromide (CTAB) extraction before using for SSR and ISSR analyses. buffer [2 % CTAB, 5 M NaCl, 0.5 M ethylene diamine WHWUDDFHWLFDFLG ('7$ DWS+7ULV+&/ WUL]PDEDVH PCR assay hydrochloric acid) at pH 8 and 2 % ß-mercaptoethanol] XVLQJDPRUWDUDQGSHVWOH 'R\OH 'R\OH 7KH 3&5DPSOL¿FDWLRQRIWKHH[WUDFWHG'1$ZDVFRQGXFWHG H[WUDFW ZDV WUDQVIHUUHG WR D FOHDQ HSSHQGRUI WXEH DQG ZLWK665SULPHUV 5DPRV et al ., 2011), and 2 ISSR (da WKHQLQFXEDWHGLQDZDWHUEDWKDW&IRUPLQZLWK Costa et al. , 2011) primers in a thermal cycler (Mycycler, RFFDVLRQDO VZLUOLQJ$IWHU LQFXEDWLQJ WKH PL[WXUH ZDV BioRAD, CA, USA). The annealing temperatures centrifuged at 12,000 rpm for 5 min in a microcentrifuge (T A ZHUH RSWLPLVHG IRU HDFK SULPHU 7DEOH 3&5 3ULVP&/DEQHW1-86$ DQGWKHVXSHUQDWDQWZDV DPSOL¿FDWLRQZDVFDUULHGRXWLQȝ/YROXPHVRI3&5 WUDQVIHUUHGWRDFOHDQHSSHQGRUIWXEH7ZRWKLUGVYROXPH mixture. Each reaction mixture contained 100 ng of RIFKORURIRUP LVRDP\ODOFRKRO YY ZDVDGGHGWR JHQRPLF'1$ȝ0RIHDFKSULPHU[3&5EXIIHU WKHWXEHDQGWKHWXEHVZHUHFHQWULIXJHGDWUSPIRU ȝ0RIG1738 Taq DNA polymerase (Dream PLQ7KHDTXHRXVSKDVHZDVWUDQVIHUUHGWRDQHZWXEH 7DT)HUPHQWDV DQG3&5JUDGHZDWHU7KHUHDFWLRQPL[ DQG'1$ZDVSUHFLSLWDWHGE\WKHDGGLWLRQRIWZRWKLUGV ZDVSUHKHDWHGDW &IRUPLQIROORZHGE\F\FOHVRI YROXPHRILFHFROGLVRSURSDQRO7KHWXEHZDVLQFXEDWHG PLQGHQDWXUDWLRQDW &VDQQHDOLQJDWRSWLPLVHG DW & IRU PLQ DQG FHQWULIXJHG DW USP WHPSHUDWXUHDQGH[WHQVLRQDW&IRUPLQZLWKWKH Journal of the National Science Foundation of Sri Lanka 45(3) September 2017 258 W.A.D.L.R. Warnakula et al. Table 3: Variation in morphological characters among papaya germplasm 4XDOLWDWLYHFKDUDFWHU colour Code Stem type Fruit shape )ORZHUW\SH Stem colour Flesh colour Seed colour Flesh aroma )ORZHUFRORXU Leaf segments Ripen fruit skin /HDIZD[LQHVV Sample number Stem pigmentation Stem Leaf petioleLeaf colour )ORZHUVWDONFRORXU 1 RDL01 1 2 1 1 1 1 1 1 1 7 1 3 1 2 2 LCL01 1 2 2 2 1 1 2 1 1 4 2 1 2 1 3 SIN01 1 1 2 1 1 1 1 1 1 1 2 1 1 2 4 BRL01 1 1 2 1 1 1 2 1 1 4 2 1 1 2 5 BRL02 1 1 2 1 1 1 2 1 1 4 2 1 1 2 6 LCL02 1 2 2 1 1 1 2 1 1 5 2 1 1 1 7 LCL03 1 2 2 1 1 1 2 1 1 2 3 2 1 2 8 SIN02 1 2 2 1 1 1 2 1 1 3 2 2 1 2 9 RDL02 1 1 2 1 1 1 1 1 1 4 2 3 1 2 10 RTN01 1 1 2 1 1 1 2 1 1 3 2 2 1 2 11 LCL04 1 2 1 3 1 1 1 2 1 5 2 4 2 1 12 RDL03 1 1 2 1 1 1 1 1 1 1 2 3 1 2 13 SIN03 1 2 2 1 1 1 2 1 1 3 2 1 1 2 14 LCL05 1 1 2 1 2 1 1 1 1 2 2 1 1 1 15 MTP01 2 2 2 2 2 2 2 3 2 6 2 4 3 3 16 MTP02 2 2 2 2 2 2 2 3 2 6 2 4 3 3 17 MTP03 2 2 2 2 2 2 2 3 2 6 2 4 3 3 18 LCL06 1 2 1 1 1 1 1 1 1 1 2 1 1 1 19 LCL07 1 1 2 1 1 1 1 1 1 7 2 1 1 1 20 LCL08 1 1 2 1 1 1 1 1 1 3 3 3 1 1 21 LCL09 1 1 2 1 1 1 1 1 1 3 3 2 1 1 ¿QDOH[WHQVLRQRIPLQDW&$IWHUFRPSOHWLRQRIWKH RESULTS F\FOLQJSURJUDPPHUHDFWLRQVZHUHKHOGDW&7KH DPSOL¿HG 3&5 SURGXFWV ZHUH HOHFWURSKRUHVHG E\ agarose gel containing 0.5 µg/mL ethidium bromide. Morphological analysis Data analysis Phenotypic trait assessment of all examined C. papaya DQGPRXQWDLQSDSD\DDFFHVVLRQVVKRZHGDYDULDWLRQLQ all qualitative traits assessed (Table 4). Out of all the 7KH DOOHOHV DPSOL¿HG E\ 665 DQG ,665 SULPHUV ZHUH scored for each primer across all genotypes. The number JHQRW\SHV WKHUH ZHUH KHUPDSKURGLWH SODQWV DQG of alleles per locus, gene diversity, and polymorphism female plants. All observed mountain papaya plants LQIRUPDWLRQ FRQWHQW 3,& ZHUH FDOFXODWHG XVLQJ ZHUH KHUPDSKURGLWH LQ WKH 6KDQWKLSXUD 1XZDUD (OL\D 323*(1( YHUVLRQ September 2017 Journal of the National Science Foundation of Sri Lanka 45(3) Genetic diversity assessment of mountain papaya 259 EURZQ WR OLJKW JUH\ 3LJPHQWDWLRQ RI WKH VWHP ZDV YHLQV ZKLOH PRXQWDLQ SDSD\D DQG RQH ORFDO SDSD\D indiscriminate for almost all the samples excluding a DFFHVVLRQ /&/ IURP 'LNNHOH .XUXQHJDOD 'LVWULFW IHZ ORFDO SDSD\D JHQRW\SHV /&/ DQG /&/ DQG VKRZHG OHDYHV WKDW ZHUH GHHSO\ GLYLGHG LQWR PDLQ RQH 5HG /DG\ JHQRW\SH 5'/ IRXQG LQ WKH .DQG\ VHJPHQWV:D[LQHVVRQWKHOHDIVXUIDFHZDVSUHVHQWRQO\ 'LVWULFW7KH\VKRZHGDGLIIHUHQWSLJPHQWDWLRQSDWWHUQ in mountain papaya plants. ZKLFK LV PRVWO\ FRQ¿QHG WR WKH XSSHUPRVW SDUW RI WKH stem. 7KH ÀRZHU VL]H FRORXU DQG WKH W\SH RI ÀRZHULQJ VKRZHGVLPLODULWLHVDPRQJ C. papaya accessions. Their All C. papaya cultivars including commercially ÀRZHUV ZHUH ERUQ RQ LQÀRUHVFHQFHV ZKLFK DSSHDU JURZQ6LQWD5HG/DG\DQG5DWKQHVKRZHGVLPLODUOHDI in the axils of the leaves. All C. papaya cultivars bear FKDUDFWHUV 7KH\ SURGXFHG ODUJH SDOPDWH OHDYHV ZLWK FUHDPFRORXUÀRZHUV&RPSDUDWLYHO\PRXQWDLQSDSD\D actinodromous venation, arranged in a spiral pattern GLVSOD\HG GLVWLQFW ÀRZHU FKDUDFWHUV WKH\ KDG VPDOO and clustered in the upper section of the plant. Except JUHHQLVKFRORXUÀRZHUVERUQHRQLQÀRUHVFHQFHVH[KLELWHG WZR C. papaya genotypes, all the others possessed pale DOODORQJWKHWUXQNDQGVKRZHGGHQVHLQÀRUHVFHQFHV green leaf petioles. One local papaya genotype (LCL04) IRXQGLQWKH.XUXQHJDOD'LVWULFWVKRZHGGDUNSXUSOHOHDI 7KHIUXLWVKDSHVKRZHGDZLGHGLVVLPLODULW\DPRQJ SHWLROHV ZKLOH DQRWKHU ORFDO JHQRW\SH /&/ LQ WKH all papaya cultivars. Of all the cultivars, Red Lady and .DQG\'LVWULFWKDGGDUNJUHHQOHDISHWLROHVZKLFKZDV 6LQWDFXOWLYDUVVKRZHGFRPSDUDWLYHO\ODUJHUIUXLWVZKLOH similar to the colour of mountain papaya leaf petioles. PRXQWDLQSDSD\DKDGWKHVPDOOHVWVL]HIUXLWV0RXQWDLQ The blade of C. papaya OHDYHVZDVGLYLGHGLQWR PDLQ SDSD\DIUXLWVVKRZHG¿YHSURPLQHQWEURDGORQJLWXGLQDO VHJPHQWV DQG KDG SURPLQHQW \HOORZLVK PLG ULEV DQG ribs from the base to apex of the fruit (Figure 1). The Table 4: 665DQG,665SULPHUVXVHGIRU3&5DPSOL¿FDWLRQ a 3ULPHU 0RWLI 6HTXHQFH ĺ 6L]H ES 7A & P&S&,5 &7 *$ ) $7&*7&7&&77777&7**77 5 7&7*&&7&&&$$7$&$&7$$7 P&S&,5 7& ) $*&&$&$$&&7$&***$$$7 5 $*7$$&**$**$$$$7*$*7 P&S&,5 7& ) $7&*7&7&&77777&7**77 5 77&7*&&7&&&$$7$&$&7$ P&S&,5 &7 $& ) $&&&$&&$*&$$7&7&&$7 5 $*&$$$&&$&7&$&7&7&$7$ P&S&,5 &7 ) 7*$&*$7$$$$&&&7$$&*$ 5 7$$*$$$&$*&*$$$&&&7$ P&S&,5 &7 ) 7$&$&7*&&7$$&$&&&$77 5 $$&&$$&&$7$$&7*&&777 P&S&,5 *$ ) $&$$$&$$*7&&&&$$$7&7 5 7$&$&7*&&7$$&$&&&$77 P&S&,5 7& 7& ) 7&**77&7&$**777&77&7$$ 5 $&$$7&$&$**&$&$&$7 P&S&,5 *$ ) $$$$**$&*$$$$**$*$&7 5 777*$$&7$&&7$&$&*$$&7 6 *$7 ) $$7*7*7*$*$$7$**77 5 $$7&7$7&&7&&7&$7*7$ 6 $& ) $77&77$*&&$*$7*$7*7 5 $77*&$7*7$&$&$7$&&*7 ,665 &$ *$ &$ *$ ,665 ***7 ****7 * ***7 ****7 * a Annealing temperature Journal of the National Science Foundation of Sri Lanka 45(3) September 2017 W.A.D.L.R. Warnakula et al. peel colour of ripe C. papaya fruits ranged from light A B \HOORZ WR UHGGLVK RUDQJH ZKHUHDV PRXQWDLQ SDSD\D IUXLWVZHUHOLJKWRUSDOH\HOORZLQFRORXU7KHÀHVKFRORXU ZDVGLVWLQFWDPRQJDOOFXOWLYDUVEXWDFOHDUVHSDUDWLRQ based on it could not be seen in C. papaya cultivars. +RZHYHUDOOWKUHHPRXQWDLQSDSD\DJHQRW\SHVVKRZHGD GLVWLQFWÀHVKFRORXUZKLFKZDVQRWVLPLODUWRDQ\RIWKH C. papaya cultivars and they released a strong pleasant DURPDZKHQULSHQLQJZKLFKZDVDQRWKHUKLJKO\GLVWLQFW trait. 7KH 83*0$ RI PRUSKRORJLFDO WUDLWV JURXSHG WKH C. papaya DQG PRXQWDLQ SDSD\D JHQRW\SHV LQWR WZR main clusters (Figure 2). One main cluster (cluster A) Figure 1: Ripen fruit of mountain papaya. A: External appearance of comprised all the mountain papaya genotypes collected WKHIUXLWZLWKSURPLQHQWORQJLWXGLQDOULEV % ORQJLWXGLQDO IURPWKH1XZDUD(OL\D'LVWULFW 073í WRJHWKHU section of the fruit ZLWKRQH5HG/DG\JHQRW\SH 5'/ FROOHFWHGIURP Figure 2: Dendrogram showing the relationships among Carica papaya and mountain Figure 2: 'HQGURJUDP VKRZLQJ WKH UHODWLRQVKLSV DPRQJ C. papaya and mountain papaya JHUPSODVPDVREWDLQHGIURPPRUSKRORJLFDOWUDLWDQDO\VLVXVLQJ(XFOHGLDQGLVWDQFHZLWK 83*0$DOJRULWKP WKH .DQG\ 'LVWULFW DQG WZR ORFDO JHQRW\SHV /&/ SSR/ ISSR analysis DQG FROOHFWHGIURPWKH.XUXQHJDOD'LVWULFW$OOWKUHH mountain papaya accessions grouped into a single sub 7KLUWHHQSULPHUSDLUVLGHQWL¿HGDOOHOHVZLWKDQDYHUDJH cluster in the main cluster A. LCL 04 shared similar frequency of 2.5 alleles per primer. Eight SSR primer FKDUDFWHUV ZLWK PRXQWDLQ SDSD\D ZKLOH 5'/ DQG SDLUVDQGWZR,665SULPHUSDLUV )LJXUH SURGXFHGD /&/ VKDUHG PRUH VLPLODU FKDUDFWHUV ZLWK HDFK WRWDO RI SRO\PRUSKLF DOOHOHV DPRQJ WKH WZHQW\ RQH other. The other main cluster (cluster B) encompassed papaya genotypes assessed. SSR primers mCpCIR02, DOO WKH RWKHU JHQRW\SHV WKDW VKRZHG VLPLODULW\ 7KH P&S&,5 DQG 6 VKRZHG D PRQRPRUSKLF DOOHOH morphological traits-based clustering pattern did not SDWWHUQ ZKLOH 665 SULPHUV P&S&,5 P&S&,5 VXSSRUWDQ\UHJLRQDOZLVHVHSDUDWLRQ mCpCIR08, mCpCIR09, mCpCIR16, mCpCIR17, September 2017 Journal of the National Science Foundation of Sri Lanka 45(3) *HQHWLFGLYHUVLW\DVVHVVPHQWRIPRXQWDLQSDSD\D FigureFigure 3: 3&5DPSOL¿FDWLRQRISDSD\DVDPSOHVZLWK665DQG,665PDUNHUV3: A: Primer mCpCIR05 ; B: primer ISSR01; lane L: 100 bp ladder; lane 1: RDL01; lane 2: LCL01; lane 3: SIN01; lane 4: BRL01; lane 5: BRL02; lane 6: LCL02; lane 7: LCL03; lane 8: SIN02; lane 9: RDL02; lane 10: RTN01; lane 11: LCL04; lane 12: RDL03; lane 13: SIN03; lane 14: LCL05; lane 15-17: MTP01-03; lane 18-21: LCL 06-09 Figure 4: 83*0$GHQGURJUDPVKRZLQJWKHUHODWLRQVKLSVDPRQJ C. papaya and mountain papaya JHUPSODVPDVREWDLQHGIURP665DQG,665PDUNHUDQDO\VLVXVLQJ-DFFDUG¶VFRHI¿FLHQW P&S&,5 DQG 6 VKRZHG SRO\PRUSKLVP DPRQJ IUHTXHQF\ZKLOHWKHRWKHUSULPHUVKDGDOOHOHIUHTXHQFLHV mountain papaya and C. papaya accessions. ranging from 0.1 to 0.9. The highest major allele IUHTXHQF\ ZDV REVHUYHG ZLWK SULPHUV P&S&,5 7ZHQW\RQHDFFHVVLRQVVKRZHGDPHDQJHQHGLYHUVLW\ P&S&,5DQG6ZKHUHDVWKHORZHVWDOOHOHIUHTXHQF\ RIZLWKDOOWKHSULPHUVHYDOXDWHGDQGWKHSULPHU ZDVZLWKWKHSULPHU,665 P&S&,5VKRZHGWKHKLJKHVWJHQHGLYHUVLW\RI SSR primers resulting in monomorphic allele pattern had The polymorphism information content (PIC) value WKHORZHVWJHQHGLYHUVLW\ SURYLGHV DQ HVWLPDWHG ¿JXUH RI JHQHWLF GLYHUVLW\ DQG the average PIC of the 11 SSR and 2 ISSR loci in the Major allele frequencies of each SSR and ISSR DFFHVVLRQVZDV0DUNHUP&S&,5KDGWKH locus for the 21 accessions are presented in Table 5. KLJKHVW3,&YDOXHRIZKHUHDVPDUNHUVP&S&,5 Monomorphic primers had the highest major allele and ISSR1 KDGWKHORZHVW3,&RI Journal of the National Science Foundation of Sri Lanka 45(3) September 2017 W.A.D.L.R. Warnakula et al. Table 5: 'HWDLOVRIWKHDOOHOHSRO\PRUSKLVPSURGXFHGZLWK665DQG LCL 07, and one Red Lady cultivar (RDL 01) shared ISSR markers VLPLODU PRUSKRORJLFDO FKDUDFWHUV ZLWK PRXQWDLQ papaya accessions. Morphological markers alone are Primer NSB a NPB b MAF c *' d PIC e QRW UHOLDEOH WR DQDO\VH WKH LQWUDVSHFL¿F UHODWLRQVKLS EHWZHHQ C. papaya and mountain papaya to examine mCpCIR01 3 2 0.5682 0.2489 0.4908 the likelihood of intra-generic hybridisation. Therefore, mCpCIR02 1 0 1.0000 0.0000 0.0000 '1$ EDVHG PROHFXODU DQDO\VLV ZDV FRQGXFWHG DV LW LV mCpCIR05 2 2 0.5000 0.6931 0.5000 the preferred method for diversity analysis among plant mCpCIR08 3 3 0.5909 0.9369 0.5580 breeders. mCpCIR09 2 2 0.5909 0.6765 0.4836 mCpCIR16 2 2 0.6818 0.6255 0.4340 0ROHFXODU DQDO\VLV VKRZHG FOHDU LQIRUPDWLRQ RQ mCpCIR17 2 2 0.7273 0.5860 0.3967 LQWHUDQGLQWUDVSHFL¿FUHODWLRQVKLSVJLYLQJHVWLPDWHVRI mCpCIR40 2 2 0.8636 0.3983 0.2356 WKHJHQHWLFUHODWLRQVKLSEHWZHHQ C. papaya and mountain mCpCIR45 1 0 1.0000 0.0000 0.0000 papaya, and among C. papaya cultivars. Thirty three S285 2 2 0.5455 0.6890 0.4959 DOOHOHVZHUHLGHQWL¿HGE\665DQG,665PDUNHUV S414 1 0 1.0000 0.0000 0.0000 proving their ability to be used as polymorphic markers ISSR1 8 3 0.1363 0.2489 0.2356 in both C. papaya and mountain papaya accessions. ISSR2 4 2 0.8636 0.8923 0.4616 Although RAPD markers have been extensively used to Average 2.53 1.6923 0.6975 0.4611 0.3127 assess some traits in papaya (Sondur et al ., 1996; Jobin- a Number of scored band; b number of polymorphic band; c major Decor et al WKHUHDUHRQO\DIHZUHSRUWVLQSDSD\D allele frequency; d gene diversity (Shannon’s information index); analysed by SSR and ISSR (Carrasco et al ., 2009; de e polymorphic information content Oliveira et al ZKLFKDUHPRUHUHOLDEOHDQGGHOLYHU UHSURGXFLEOHUHVXOWV,QWKLVVWXG\P&S&,5VKRZHG the highest PIC value thereby indicating its usefulness 7KH 83*0$ EDVHG RQ 665 DQG ,665 JURXSHG WKH in the screening of C. papaya accessions. DNA based C. papayaDQGPRXQWDLQSDSD\DDFFHVVLRQVLQWRWZRPDLQ cluster analysis also revealed that C. papaya cultivars clusters (Figure 4). This result is described by the distinct DUHQRWPXFKGLYHUJHQWIURPHDFKRWKHUZKLFKPD\EH SRO\PRUSKLF DOOHOH SDWWHUQ EHWZHHQ C. papaya and due to sharing the same alleles in most of the SSR and mountain papaya accessions obtained from mCpCIR01, ISSR markers. Results of the molecular analysis revealed mCpCIR40, ISSR1 and ISSR2 primers. Cluster A that mountain papaya and C. papaya are genetically comprised all the C. papaya JHQRW\SHVZKLOHFOXVWHU% GLVWDQW UHODWLYHV +RZHYHU RXW RI WKH DOOHOHV comprised 3 mountain papaya genotypes collected from SURGXFHGDOOHOHVZHUHFRPPRQIRUERWK C. papaya WKH1XZDUD(OL\D'LVWULFW(LJKWHHQ C. papaya genotypes and mountain papaya and hence, it can be assumed that LQFOXVWHU$ZHUHIXUWKHUGLYLGHGLQWRVHYHUDOVXEFOXVWHUV mountain papaya could be more closely aligned to C. Local cultivars (LCL 07, 08 and 09) collected from three papaya'UHZ et al . (1998) have reported the possibility GLIIHUHQWORFDWLRQVLQWKH.XUXQDJDOD'LVWULFWZHUHVXE RILQWHUJHQHULFK\EULGLVDWLRQEHWZHHQVRPHVSHFLHVRI clustered separately in the main cluster A although such the genus, Vasconcelleae and C. papaya, proving their VHSDUDWLRQZDVQRWREVHUYHGIURPPRUSKRORJLFDOEDVHG close relationship. clusters. 9DVFRQFHOOHD FXQGLQDPDUFHQVLV 9 FDXOLÀRUD DISCUSSION V. quercifolia and V. stipulata are considered as the major VRXUFHVIRUH[WUHPHUHVLVWDQFHWR35693 0DQVKDUGW The genetic improvement of any crop is dependent on :HQVODII 'UHZ et al. , 1998). Resistance to PRSV-P WKHXWLOLVDWLRQRIZLOGUHODWLYHVWUDGLWLRQDOYDULHWLHVDQG in C. papaya KDV QRW EHHQ LGHQWL¿HG WR GDWH ,QWHU modern breeding techniques. The assessment of genetic JHQHULF K\EULGLVDWLRQ FRQGXFWHG EHWZHHQ Vasconcellea diversity at a given level is a requirement to select species and C. papaya has yielded resistant hybrids resistant and high yielding varieties (Mondini et al ., indicating the possibility of donating PRSV-P resistant 2009). In this study, the cluster analysis of morphological JHQHV 0DQVKDUGW :HQVODII 'UHZ et al ., 1998). traits revealed that there is no distinct separation among +RZHYHUWKHVHK\EULGVZHUHQRWFRPPHUFLDOO\YDOXDEOH the C. papayaJHQRW\SHVJURZQLQGLIIHUHQWJHRJUDSKLF due to the presence of unfavourable traits. Therefore, it is DUHDV LQ 6UL /DQND &XOWLYDUZLVH VHSDUDWLRQ FRXOG QRW QHFHVVDU\WRFRQWLQXHEDFNFURVVEUHHGLQJZLWK C. papaya EHREVHUYHGLQPRVWRIWKHFKDUDFWHUVVWXGLHG+RZHYHU as the recurrent parent to maximise the C. papaya genetic LWZDVUHYHDOHGWKDWPRXQWDLQSDSD\DLVKLJKO\GLVWLQFW EDFNJURXQGZKLOHPLQLPLVLQJWKHUHJLRQRIGRQRU'1$ from the C. papaya accessions although LCL 04 and to the target loci of PRSV-P resistance inherited from September 2017 Journal of the National Science Foundation of Sri Lanka 45(3) *HQHWLFGLYHUVLW\DVVHVVPHQWRIPRXQWDLQSDSD\D Vasconcellea species. This backcross breeding is only 4. GD &RVWD )5 3HUHLUD 71 *DEULHO $3& 3HUHLUD SRVVLEOHZLWKJHQHWLFPDUNHUVWKDWDUHSUHVHQWWKURXJKRXW 0* ,665 PDUNHUV IRU JHQHWLF UHODWLRQVKLSV WKHJHQRPH7KHUHIRUH¿QGLQJRXWSRO\PRUSKLFPDUNHUV in Caricaceae and sex differentiation in papaya. Crop EHWZHHQ PRXQWDLQ SDSD\D DQG C. papaya is important Breeding and Applied Biotechnology 11 DOI: https://doi.org/10.1590/S1984-70332011000400009 WRSURGXFHFXOWLYDUVZLWK35693YLUXVUHVLVWDQFHFROG 5. 'DVVDQD\DNH0')RVEHUJ)5 &OD\WRQ:' resistance etc., via marker assisted backcrossing utilising Revised Handbook to the Flora of Ceylon , volumes VIII- mountain papaya genetic resource. ,;$PHULQG3XEOLVKLQJ&R3YW/WG1HZ'HOKL,QGLD 6. de Oliveira E.J., Silva A.D.S., de Carvalho F.M., dos Santos CONCLUSION /) &RVWD -/ $PRULXP 9%'2 'DQWDV -// (2010). Polymorphic microsatellite marker set for Carica papaya L. and its use in molecular-assisted selection. Morphological and molecular marker-based cluster Euphytica 173: 279 – 287. DQDO\VLV UHYHDOHG WKDW WKHUH ZDV QR FOHDU JHQHWLF DOI: https://doi.org/10.1007/s10681-010-0150-y separation among the C. papaya FXOWLYDUV JURZQ 7. 'LOORQ65DPDJH&$VKPRUH6 'UHZ5$ in different geographic areas in Sri Lanka. SSR Development of a codominant CAPS marker linked to PDUNHUV P&S&,5 P&S&,5 DQG 6 VKRZHG PRSV-P resistance in highland papaya. Theoretical and D PRQRPRUSKLF DOOHOH SDWWHUQ LQ DJDURVH ZKLOH Applied Genetics 113 SSR markers: mCpCIR01, mCpCIR05, mCpCIR08, DOI: https://doi.org/10.1007/s00122-006-0375-2 8. Department of Agriculture (DOA) (2014). Agstat. Pocket mCpCIR09, mCpCIR16, mCpCIR17, mCpCIR40, Book of Agricultural Statistics . Department of Agriculture, and S285 and ISSR markers: ISSR01 and ISSR02 Peradeniya. ZHUH SRO\PRUSKLF EHWZHHQ PRXQWDLQ SDSD\D DQG 9. 'R\OH -- 'R\OH -/ $ UDSLG '1$ LVRODWLRQ C. papaya accessions. Out of the 13 markers tested, procedure for small quantities of fresh leaf tissue. 665 PDUNHU P&S&,5 ZRXOG EH XVHIXO LQ VFUHHQLQJ Phytochemistry Bulletin 19 C. papaya accessions due to the highest PIC value. 10. 'UHZ 5$ 2¶%ULHQ &0 0DJGDOLWD 30 Both morphological and molecular analysis proved that 'HYHORSPHQW RI LQWHUVSHFL¿F Carica hybrids. Acta mountain papaya and C. papaya are genetically distant Horticulturae 461 DOI: https://doi.org/10.17660/ActaHortic.1998.461.31 UHODWLYHV +RZHYHU WKH SUHVHQFH RI FRPPRQ JHQHWLF 11. ,QWHUQDWLRQDO%RDUGIRU3ODQW*HQHWLF5HVRXUFHV ,%3*5 DQG PRUSKRORJLFDO IHDWXUHV EHWZHHQ C. papaya and (1988). Descriptors for Papaya . International Board for PRXQWDLQSDSD\DVXJJHVWWKHSRVVLELOLW\RILQWHUVSHFL¿F 3ODQW*HQHWLF5HVRXUFHV5RPH,WDO\ hybridisation. 12. -LPpQH]900RUD( *XWLpUUH]90 Biology of the Papaya Plant . Available at KWWS ZZZFXOWLYRSDSD\D Acknowledgement RUJZSFRQWHQWXSORDGV FSGI , Accessed 15 May 2014. 13. -RELQ'pFRU 03 *UDKDP *& +HQU\ 5- 'UHZ The authors express their gratitude to the Wayamba 5$ 5$3' DQG LVR]\PH DQDO\VLV RI JHQHWLF 8QLYHUVLW\RI6UL/DQND 5HVHDUFK*UDQW1R 65+'& UHODWLRQVKLSV EHWZHHQ Carica papaya DQG ZLOG UHODWLYHV 53 IRU JLYLQJ ¿QDQFLDO VXSSRUW IRU WKLV Genetic Resources and Crop Evolution 44 research. 14. 0DGDUERNXV 6 5DQJKRR6DQPXNKL\D 90 ,GHQWL¿FDWLRQRIJHQHWLFGLYHUVLW\DPRQJSDSD\DYDULHWLHV in Mauritius using morphological and molecular markers. REFERENCES International Journal of Life Sciences Biotechnology and Pharma Research 1 1. Badillo V.M. (2000). Carica L. vs . Vasconcella St.-Hil. 15. 0DQVKDUGW 50 :HQVODII 7 ,QWHUVSHFL¿F (Caricaceae) con la rehabilitacionde este ultimo. Ernstia K\EULGL]DWLRQ RI SDSD\D ZLWK RWKHU Carica species. 10 Journal of the American Society for Horticultural Science 2. &DUUDVFR%$YLOD33HUH]'LD]-0XQ×R]3*DUFÕD5 114 /DYDQGHUR%=XULWD6LOYD$5HWDPDOHV-% &DOLJDUL 16. 0RQGLQL/1RRUDQL$ 3DJQRWWD0$ $VVHVVLQJ 3'6 *HQHWLF VWUXFWXUH RI KLJKODQG SDSD\DV plant genetic diversity by molecular tools. Diversity (Vasconcellea pubescens ) cultivated along a geographic 1 gradient in Chile as revealed by inter simple sequence DOI: https://doi.org/10.3390/d1010019 repeats (ISSR). Genetic Resources and Crop Evolution 56 : 17. Moussaoui A., Nijs M., Paul C., Wintjens R., Vincentelli 331 – 337. -$]DUNDQ0 /RR]H< 5HYLVLWLQJWKHHQ]\PHV DOI: https://doi.org/10.1007/s10722-008-9367-1 stored in the laticifers of Carica papaya in the context of 3. &DUYDOKR)$)LOHU' 5HQQHU66 7D[RQRP\LQ their possible participation in the plant defense mechanism. the electronic age and an e-monograph of the papaya family Cell and Molecular Life Sciences 58 (Caricaceae) as an example. Cladistics 31 DOI: https://doi.org/10.1007/PL00000881 Journal of the National Science Foundation of Sri Lanka 45(3) September 2017 W.A.D.L.R. Warnakula et al. 18. Muthulakshmi S., Balamohan T.N., Amutha R., Baby Foundation, Maitland Place, Colombo 07, Sri Lanka. 5DQL : 0DUHHVZDUL 3 ,QGLUD . %UHHGLQJ 22. 6RQGXU610DQVKDUGW50 6WLOHV-, $JHQHWLF for cold tolerance in papaya ( Carica papaya L.) through OLQNDJH PDS RI SDSD\D EDVHG RQ UDQGRPO\ DPSOL¿HG K\EULGL]DWLRQ Research Journal of Agriculture and polymorphic DNA markers. Theoretical and Applied Biological Sciences 3 Genetics 93 19. 2¶%ULHQ &0 'UHZ 5$ 0DUNHUDVVLVWHG DOI: https://doi.org/10.1007/BF00417946 K\EULGL]DWLRQ DQG EDFNFURVVLQJ EHWZHHQ Vasconcellea 23. Storey W.A. (1976). Evolution of Crop Plants . Longman, species and Carica papaya for PRSV-P resistance. Acta /RQGRQ8. Horticulturae 859 24. :HHUDNRRQ'*RRQDWLODNH6$:HHUDWXQJH93HUHUD1 DOI: https://doi.org/10.17660/ActaHortic.2010.859.43 &KDQGUDQLPDO' Halgolla Estate, Kelani Valley 20. 5DPRV+&&3HUHLUD0*6LOYD))*RQFDOYHV/6$ Plantation Management Plan for the Local Nature Reserve . 3LQWR)2GH6RX]D)LOKR*$ 3HUHLUD761 International Union for Conservation of Nature, Sri Lanka. *HQHWLFFKDUDFWHUL]DWLRQRISDSD\DSODQWV Carica papaya 25. September 2017 Journal of the National Science Foundation of Sri Lanka 45(3)