Morphological, Chemical Characters and Genetic Analysis Discrimination of Five Naturalized Populus Species Inhabiting Four Gover
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Arab Univ. J. Agric. Sci., Ain Shams Univ., Cairo, Egypt 27(4), 2273-2290, 2019 Website: http://ajs.journals.ekb.eg 7722 MORPHOLOGICAL, CHEMICAL CHARACTERS AND GENETIC ANALYSIS DISCRIMINATION OF FIVE NATURALIZED POPULUS SPECIES INHABITING FOUR GOVERNORATE TERRITORIES OF EGYPT [181] Ahmed1 M.F., Hosni2 A.M., Hewidy2* M., Abd El razik1 A.B. and Bahnasy3 M.I. 1- Genetic. Dept., Fac. of Agric., Ain Shams Univ., P.O. Box 68, Hadayek Shobra 11241, Cairo, Egypt 2- Hortic. Dept., Fac. of Agric., Ain Shams Univ., P.O. Box 68, Hadayek Shobra 11241, Cairo, Egypt 3- Forestry and Timber Tree Research Dept., Hortic. Res. Inst., Agric. Res. Center, Giza, Egypt *Corresponding author: [email protected] Received 7 October, 2019 Accepted 12 November, 2019 ABSTRACT euramericana female and male from Giza showed close relation at morphological level. From this Populus is a fast growing tree that attract the study it can be concluded that: firstly; leaf parame- attention of people. This genus provides environ- ters i.e. leaf area width and length were suitable for ment protection due to their ability in carbon se- identifying the differences among species, and questration, phytoremediation and wildlife inhabit- secondly; genetic analysis using molecular mark- ant. Populus species are widely used as a source ers was enough to discriminate between species. of wood, veneer, paper and bioenergy. This survey Our results finally suggest that Populus species study aimed to identify the current status of Popu- possess genetic variation and can adapt to new lus species inhabiting four different governorates of environmental conditions, which in its self-valuable the Egyptian territories. In this survey, four different information would potentially lead to promising locations were surveyed, i.e. Cairo, Giza, Qaliobia applications. and Gharbia governorates. According to the site survey, five species of Populus were found. Re- Keywords: Egypt, Survey study, Woody trees, search findings indicate that the length and diame- Poplar, Populus, Morphology, Genetic analysis, ter of the trees varied according to various species ISSR in various localities. Selected trees of various spe- INTRODUCTION cies were subjected to different morphological, chemical parameters and genetic assessments. P. Poplar (Populus spp.) is regarded as a group of deltoides from Giza in addition to P. deltoides and species growing all over the world. Due to its fast- P. nigra from Cairo gave somewhat close relation growing feature and acceptable quality of timber; it in their vegetative parameters. Furthermore, both plays significant roles in silviculture. Therefore, P. nigra and P. alba from Gharbia location gave paying attention to its characteristics along with high similarity due to overall vegetative parameters gathering more information on it can enable us to and also both P. euramericana female from the apply and treat correctly. To our knowledge no Gharbia and P. nigra from Qaliobia. Genetic diver- survey was conducted up till now on the im- sity was analyzed using molecular markers. Three portance of popular trees diversity and various different Inter-simple sequence repeat (ISSR) pri- morphological and biometric analyses in Egypt. mers were used for the reaction (17899A, 17899B Plant surveys are designed to search in geo- and HB13). ISSR primers markers yielded 67.5% graphic areas to determine the presence of exotic polymorphic loci among the surveyed species and species, like Populus amongst other species and cluster analysis enabled separation of these popu- evaluate its their habitat suitability (Haber and lations on the basis of their genetic distances. P. Network, 1997). 2274 Ahmed; Hosni; Hewidy; Abd El-razik and Bahnasy Poplar include 33 species belonging to the fam- ISSR represent rather new insight for curious dif- ily Salicaceae. The poplar species are native to ferentiation among species (Bandyopadhyay et al North America. The wood of poplars is relatively 2013). Inter simple sequence repeat (ISSR) re- soft and hence is mostly used to make cardboard quires very small amount of template and is con- boxes, crates, paper, and veneer (Isebrands & venient in result recording and highly reproducible Richardson, 2000) (Zietkiewicz et al 1994). Some Populations of Populus face severe Accordingly, the aim of this study was to con- threats. Three main factors have been recognized. duct basic research to identify and find out similari- The first one is the alteration of riparian ecosys- ties and differences amongst Populus species sur- tems throughout the species’ distribution area. veyed at the morphological, chemical and genetic Second, faster growing hybrid poplars have been levels. planted to replace black poplar or they have just been removed due to over-exploitation. Finally, MATERIAL AND METHODS there is the potential threat of introgression from cultivated poplars such as the male clone ‘Italica’ (Cagelli and Lefèvre 1995, Lefèvre et al 1998). Location and duration Leaf morphology may offer precise differentia- tion because it varies remarkably among species A rapid reconnaissance survey was carried out and within species with respect to structure, di- at four research stations belonging to the Agricul- mensions, types of margins, form, size of petiole, ture Research Center at four different gover- venation pattern, dry weight per unit area, moisture norates of Egypt, viz. Cairo, Giza, Qaliobia and content, canopy, stomata density, presence of tri- Gharbia. Survey result data in Table (1) demon- chomes and cuticular composition (Rodriguez et strate the presence of five species of Populus at al 2016). the Horticulture Research Stations, located in four Interesting enough, genetic discrimination governorates from which plant material were col- analyses using various techniques, i.e. AFLP or lected in June and August of 2016. Table 1. Naturalized Populus species at research stations in four governorates of Egypt Governorate Research Station Populus species Populus euramericana ♀ Giza Horticulture research institute P. euramericana ♂ P. deltoides Cairo Zohria garden P. nigra Horticulture research station in Qaliobia P. nigra Qanater P. nigra Agriculture research station in P. deltoides Gharbia Gemiza P. alba P. euramericana ♀ Data collected - Number of branches. - Leaf area, width and length of leaf, they were Morphological characteristics estimated selecting completely grown leaves - Tree height was estimated with the use of a from the middle of branches. Leaves were pho- clinometer. tographed using a digital camera as presented - Tree diameter at Breast Height (DBH), i.e. the in Plate (1) then resulted photos were elabo- stem diameter measured in cm at 1.5 m above rated using Image J program (Schneider et al., ground level. Number of branches along the 2012) which calculated individual leaf area, main trunk were counted. width and length. AUJASCI, Arab Univ. J. Agric. Sci., 27(4), 2019 Morphological, Chemical Characters and Genetic Analysis Discrimination of Five 2275 Naturalized Populus Species Inhabiting Four Governorate Territories of Egypt P. euramericana P. euramericana P. deltoides – P. nigra - P. nigra – female - Giza male - Giza Giza Cairo Gharbia P. deltoides - P. alba – P. euramericana P. nigra – Gharbia Gharbia female - Gharbia Qaliobia Plate 1. Photographed mature leaves of surveyed Populus species from four governorates in Egypt, viz. Giza, Cairo, Qaliobia and Gharbia in June-August 2016 - Leaf fresh and dry weight, 20 leaves were Molecular Marker Bioassay Analysis (ISSR) collected then weight to obtain fresh weight then oven-dried at 70 °C to estimate dry bio- The ISSR molecular marker was applied to cal- mass. culate the variation in Populus plant (Meyer et al - Leaf greenness was estimated using SPAD. 1993). Three different ISSR primers were used for the reaction (17899A, 17899B and HB13) as de- Internal chemical analysis scribed in Table 2. The content of N, P, K, Ca and Mg elements Statistical Analysis were determined in oven dried samples of leaves by digestion using concentrated H2SO4. N per- Survey data were statistically analyzed accord- centage was determined in the digested solution ing to the separation among means using Duncan by the modified microkjeldahl method as described multiple range test (Duncan, 1995). To identify the by Plummer (1971). P percentage was determined relation between each individual characteristic of calorimetrically according to the method of Jack- various Populus species, the correlation was ana- son (1958). K percentage was determined against lyzed using the CoStat. a standard using a flame photometer (Piper, Image results from electrophoresis were ana- 1950). Ca and Mg percentage was determined by lyzed by the presence of a band which scored as an atomic absorption spectrophotometer, accord- 1, whereas the absence of the band was coded as ing to David (1959). 0. A pair wise similarity matrix was generated using Jaccard’s similarity coefficient and using the un- DNA Extraction weighted pair group method with the arithmetic averaging algorithm (UPGMA), cluster analysis DNA extraction was applied for the nine differ- was performed to develop a dendrogram. These ent treatments of Populus plant. DNA extraction calculations were carried out using Bio-Rad Quan- followed CTAB method according to Doyle and tity one (4.6.2) as prescribed by Li et al (2008). Doyle (1990) and modified by Edwards et al (1991). AUJASCI, Arab Univ. J. Agric. Sci., 27(4), 2019 2276 Ahmed; Hosni; Hewidy; Abd El-razik and Bahnasy Table 2. List of primer names (ID) utilized, their nucleotide sequences in the ISSR analysis Temperature No Primer name Primers nucleotide sequence GC% (°C) 1 17899A 5’-CACACACACACAAG-3’ 50 42 2 17899B 5’-CACACACACACAGG-3’ 57.1 50 3 HB13 5’-GAGGAGGAGGC-3’ 72.7 38 RESULTS and 1.2 g/leaf). Whereas, the lowest fresh weight was found in P. alba from Gharbia (1 g/leaf), in Morphological parameters respect order and lowest dry weight was found in P. nigra from Cairo (0.4 g/leaf). Morphological parameters were demonstrated in Fig. 1. Diameter of trees at breast height (DBH) Greenness of leaves showed significant variation among different spe- cies. P. deltoides and P. euramericana female Gi- SPAD readings in Fig.