Genetic Diversity of Superior Walnut Genotypes Revealed by ISSR Markers in Shahrood, Iran

Document Type : Research Article

Authors

• Fatemeh Davoodi ¹
• Mehdi Rezaei ²
• Parviz Hidari ²
• Hossein Hokmabadi ³

¹ Shahrood unversity of technology

² Shahrood University of Technology

³ Agriculture and Natural Resources Research

https://doi.org/10.22067/jhorts4.v32i4.71228

Abstract

Introduction: Iran is known as the origin of Persian walnut in the world, and so the study of genetic diversity, identifying and introducing superior walnut genotypes from the main walnut production areas in Iran is very important. From the beginning of the year 2001, seeds of selected walnut genotypes from important walnut production area of Iran were collected and cultivated in a walnut collection in Semnan Research Center (Shahrood), Iran. From this collection, promising walnut genotypes were selected based on climate adaptability to Shahrood area and pomological characteristics.  In this research, in addition to introducing fruit characters of these walnut selected genotypes, genetic diversity and their genotypes relationship in the genome level have been also investigated with the ISSR markers.
Materials and Methods: 40 selected genotypes of important walnut regions of Iran (Tuyserkan, Orumieh, Karaj and Khorasan province) were cultivated in a walnut collection at the Shahrood Research Center in 2001. From these seedlings, 21 genotypes were selected on the base of pomological characteristics and they were grafted onto Persian walnut seedlings. Pomological traits of the fruits of these selected genotypes including the average of nut weight, kernel weight, kernel percentage, shell attachment to the kernel, kernel color, shell thickness and nut size and shape were measured. DNA was extracted from young leaves of walnut genotype by CTAB method and its quality and quantity evaluated on the agarose gel as well as the Nano drop, and then genomic DNA was amplified with the 10 ISSR primers in PCR. The amplified bands were separated by Metaphor agarose gel and stained with Ethidium bromide. For each primer, the amplified band range, the total number of bands, the number of polymorphic bands, the polymorphic percentage, Average informative band (AvIb) and resolving power (Rp) was determined. UPGMA dendrogram based on Jaccard similarity matrix was the performance by Ntsys 2.0 software. Principle Coordinate analysis was performed based on the genetic distance matrix with GenAlex 6.2 software.
 Results and Discussion: The mean of fruit and kernel weight in selected walnut genotypes were 14.34 and 7.33 g, respectively. The highest nut and kernel weight was found to be 17.5 and 10.3 g, respectively in OR23. The Kernel weight was more than kernel weight reported by Shamlu et al. (2015), Yarilgak et al. (2001) and Tasmuris et al. (2002) (9.40, 8.88 and 6.32 g). The average of kernel percentage in selected walnuts was 51.5%. The highest percentage of the kernel (62.7%) was observed in T12 genotype. from the ten ISSR primers in 21 walnut genotypes, 112 DNA fragments were amplified and 102 DNA bonds of them were polymorphic and they were used for genetic variation analysis. The highest number of amplified DNA bands was observed in UBC826 and UBC888 primers with 14 bands. The highest average band informative (AvIb), 0.62, was observed in UBC887 and UBC886 primers. The highest resolving power (Rp) was found to be 7.71 in UBC826 primer and then in UBC.887 primer. The Jaccard' similarity coefficient of genotypes varies from 0.51 to 0.88. The results showed that the genetic distance of selected genotypes of walnut is medium. The dendrogram analysis of 21 genotypes of walnuts was divided genotypes into two main groups and three subgroups in the first group, which greatly matched the results of the PCoA. The genotypes were collected from Shahrood were inserted on the first subgroup of GI in dendrogram analysis, except of KH4 and OR37 genotypes, and the highest genetic similarity was observed between some of these genotypes (R1G2 with R2G1 and R1G7). The second group includes genotypes KH34, OR26, and SH1. Subgroup 3 of the first group contains R2G8, K26, OR23, T12, and K28. The second group has high genetic distances including R2G4, KH31, T1, OR4, and T9.
Conclusion: The ISSR marker technology is an inexpensive, easy and satisfactory way to evaluate genetic relationships and genetic variation among walnut cultivars. The results of this research showed that these markers (ISSR) are suitable for study of variation in walnut genotypes. The genotypes collected from Shahrood had a lower genetic diversity, and the observed diversity is probably related to human interactions. The Tuyserkan and Urmia genotypes showed more genetic variation. The results of clustering based on molecular markers were largely matched with the grouping based on qualitative traits. The results showed that there is a moderate genetic distance between selected walnuts genotypes. The genotypes collected from Urmia and Tuyserkan regions have a higher genetic distance than other genotypes and which according to their superior characteristics, they can be used as parent along with selected walnut genotypes from the Shahrood area in a breeding program.
 

Keywords

• Walnut genotypes
• ISSR markers
• Genetic diversity
• Clustering

20.1001.1.20084730.1397.32.4.9.4

References

1- Aradhya M., Woeste K., and Velasco D. 2010. Genetic diversity, structure and differentiation in cultivated walnut (Juglans regia L.). Acta Horticulturae, 861: 127-132.

2- Arzani K., Mansouri Ardakan H., and Vezvaei A. 2008. Morphological variation among Persian walnut (Juglans regia L.) genotype from central Iran. New Zealand Journal of Crop and Horticulteral Science, 36: 159-168.

3- Attar S.H., Davarinezhad G.R., Samiee L., and Moghaddam M. 2017. Study of genetic diversity of some Persian walnut genotypes in Mashhad commercial orchards by using ISSR marker. Iranian Journal of Horticultural Science, 31(3): 611-620. (In Persian with English abstract)

4- Bayazit S., Kaza K., Golbitti S., Cevik V., Ayanogla H., and Ergul A. 2007. AFLP analysis of genetic diversity in low chill requiring walnut (Juglans regia L.) genotyping from Hatay, Turkey. Scientia Horticulturae, 111: 394-398.

5- Browicz K. 1976. Juglandaceae. In: Rechinger, K.H. (Ed.), Flora Irinica. Akademische Druck-u Verlagsanstalt, 121: 1–5

6- Christopoulos M.V., Rouskas D., Tsantili E., and Bebeli P.J. 2010. Germplasm diversity and genetic relationships among walnut (Juglans regia L.) cultivars and Greek local selections revealed by Inter-Simple Sequence Repeat (ISSR) markers. Scientia Horticulturae, 125(4): 584-592.

7- Dogan Y., Kafkas S., Sütyemez M., Akca Y., and Türemis N. 2014. Assessment and characterization of genetic relationships of walnut (Juglans regia L.) genotypes by three types of molecular markers. Scientia Horticulturae, 168: 81–87.

8- Ebrahimi A., Fatahi R., and Zamani Z. 2011. Analysis of genetic diversity among some Persian walnut genotypes (Juglans regia L.) using morphological traits and SSRs markers. Scientia Horticulturae, 130: 146-151.

9- FAO. 2016. FAO statistical yearbook. Agricultural production, Food and Agriculture Organization of the United Nations (http: faostat.fao.org.site.291.default.aspx).

10- Fornari B., Canata F., Spada M., and Malvolti M.E. 1999. Alozyme analysis of genetic diversity differentiation in European and Asiatic walnut (Juglans regia L.) populations. Forest Genetics, 6(9): 115-127.

11- Gerald D.S., Woeste K., Aradhya M.K., Koehmstedt A., Simon C., Potter D., Leslie C.A., and McGranahan G.H. 2005. Characterization of 14 microsatellite markers for genetic analysis and cultivar identification of walnut. Journal of the American Society for Horticultural Science, 130: 348–354.

12- Jaccard P. 1908. Nouvelle reserches sur la distribution florale. Bulletin de la societe des sciences naturelles, 44: 223–227.

13- Kafkas S., Ozkan H., and Sutyemez M. 2005. DNA polymorphism and assessment of genetic relationships in walnut genotypes based on AFLP and SAMPL markers.Journal of the American Society for Horticultural Science, 130: 585–590.

14- Li H., and Chen G. 2008. Genetic relationship among species in the genous Sonneratia in China as revealed by inter-simple sequence repeat (ISSR) markers. Journal Biochemical Systematic and Ecology, 36: 392–398.

15- Liu W., Liu D., Zhang A., Feng C., Yang J., Yoon J., and Li S. 2007. Genetic diversity and phylogenetic relationships among plum germplasm resources in China assessed with Inter-simple Sequence Repeat markers. Journal of the American Society for Horticultural Science, 132: 619–628.

16- McGranahanp G.P., and Leslie C. 1990. Walnuts (Juglans). Acta Horticulturae, 290: 907-951.

17- Niceses F.P., Hormaza J.I., and McGranahan G.H. 1998. Molecular characterization and genetic relatedness among walnut (Juglans regia L.) genotypes based on RAPD markers. Euphytica, 101: 199-206.

18- Oliveira C.M., Mota M., Monte-Corvo L., Goulao L., and Silva D.M. 1999. Molecular typing of Pyrus based on RAPD markers. Scientia Horticulturae, 79: 163–174.

19- Peakall R., and Smouse P.E. 2012. GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research-an update. Bioinformatics. 28: 2537-2539.

20- Pollegioni P., Bartoli S., Cannata F., and Malvoti ME. 2003. Genetic differentiation of four Italian walnut varieties by inter simple sequence repeat. Journal of Genetics and Breeding, 57(3): 231–240.

21- Pollegioni P., Major A., Bartoli S., Ducci F., Proietti R., and Malvolti M.E. 2005. Application of microsattelite and dominant molecular markers for the discrimination of species and interspecific hybrids in genus Juglans. Acta Horticulturae, 705: 191-197.

22- Potter D., Gao F., Aiello G., Leslie C., and McGranahan G.H. 2002. Intersimple sequence repeat markers for fingerprinting and determining genetic relationships of walnut (Juglans regia L.) cultivars. Journal of the American Society for Horticultural Science, 127: 75-81.

23- Prevost A., and Wilkinson M.J. 1999. A new system of comparing PCR primers applied to ISSR fingerprinting. Theoretical Applied Genetics, 98: 107–112.

24- Rahimipanah M., Hamedi M., and Mirzapour M. 2010. Antioxidant activity phenolic contents of Persian walnut (Juglans regia L.) green husk extract. American Journal of Food Science and Technology, 1: 105-111.

25- Reddy M.P., Sarla N., and Siddiq E.A. 2002. Inter simple sequence repeat (ISSR) polymorphism and its application in plant breeding. Euphytica, 128: 9–17.

26- Rohlf F.J. 1998. NTSyS-p.c. Numerical Taxonomy and Multivariate Analysis System (Version 2.0). Exeter Software Publishers Ltd., Setauket.

27- Sahli-Hannachi A., Chatti K., Mars M., Marrakchi M., and Trifi M. 2005. Comparative analysis of diversity in two Tunisian collections of fig cultivars based on random amplified polymorphic DNA and inter simple sequence repeats fingerprints. Genetic Resources and Crop Evolution, 52: 563–573.

28- Shamlu F., Rezaei M., Biabani A., and Khanahmadi A. 2015. Morphological Diversity among Walnut Genotypes of in Azadshar, Iran. Journal of Horticulture Science, 30(3): 469-479. (In Persian)

29- Terzopoulos P.J., Kolano B., Bebeli P.J., Kaltsikes P.J., and Metzidakis I. 2005. Identification of Olea europaea L. cultivars using inter-simple sequence repeat markers. Scientia Horticulturae, 105: 45–51.

30- Terzopoulos P.J., and Bebeli P.J. 2008. Genetic diversity analysis of Mediterranean faba bean (Vicia faba L.) using ISSR markers. Field Crops Research, 108: 39–44.

31- Tian L., xiang A., Li-SI Z., Hai Rong W., and Qing L. 2011. ISSR Analysis of Genetic Diversity among Seedling Walnut (Juglans spp.)Populations. Journal of Plant Genetic Resources, 12(4): 640-645.

32- Tsmouris G., Hatziantoniou S., and Demetzos C. 2002. Lipid analysis of Greek walnut oil (Juglans regia L.). Zeitschrift für Naturforschung C, 57: 51- 56.

33- Vijayan K., Awasthi K., Sprivasava P., and Sarachandra B. 2006. Genetic Analysis of Indian Mulberry Varieties Through Molecular Markers. Hereditas, 141:8-14.

34- Woeste K., Burns R., Rhodes O., and Michler C. 2002. Thirty polymorphic nuclear microsatellite loci from black walnut. Journal of Heredity, 93: 58–60.

35- Yarilgac T., Koyuncu F., Koyuncu M.A., Kazankaya A., and Sen S.M. 2001. Some promising walnut selections (Juglans regia L.) Acta Horticulturae, 544: 93-96.