Study of Genetic Diversity of Some Persian walnut Genotypes in Mashhad Commercial Orchards by using ISSR Marker

علوم باغبانی

Article 3, Volume 31, Issue 3, September 2017, Pages 611-620   

Document Type: Research Article

DOI: 10.22067/jhorts4.v31i3.60521

Authors

Shadi Attar^{* 1}; Gholamhossein Davarynejad¹; Leila Samiee²; Mohammad Moghadam¹

¹Ferdowsi University of Mashhad

²Research Center for Plant Sciences, Ferdowsi University of Mashhad

Abstract

Introduction: Persian walnut (Juglans regia L.), belonging to the Juglandaceae family, has its natural origin in the mountainous regions of central Asia and especially northern forests of Iran. Most walnut genotypes are seedling and sexually reproduced. Conducting studies on the genetic structure of these genotypes to identify, select and maintain their genetic resources is important. Identifying and collecting local varieties of fruit trees is considered as the first step on the path of breeding programs and lack of information regarding plants genetic characteristics causes the breeding work to be done slowly. Various methods have been used for studying genetic diversity and determining the genetic relationship between European and Asian varieties of walnut and identifying commercial walnut varieties, among which we can mention: Morphologic indices, Alozyme, Isozym, RFLP, RAPD, AFLP and ISSR markers. ISSR molecular marker was used in order to investigate genetic diversity of some genotypes of Persian walnut (Juglans regia L.) in Mashhad orchards. .
Materials and methods: To begin with, about 56 walnut trees from 4 orchards in Mashhad (Esteghlal (1), Golestan (2), Alandasht (3) and Emam Reza (4)) were selected and tagged from 2014 to 2016. In the spring of 2014 with the beginning of trees growth and opening of leaves, a number of leaves from each genotype were collected. After DNA extraction, the quality of samples by agarose gel (1 percentage) and electrophoresis method and quantity of them via spectrophotometer device at 260 and 280 nm wavelengths were determined. First, 24 primers of ISSR marker were prepared and after initial evaluation on 5 random genotypes, 9 primers with high polymorphism and repeatability were selected for further investigation. For PCR reaction, Amplicon kit (code 180 301, made in Denmark) was used. Gel electrophoresis images of primers that produced polymorphic bands with suitable resolution were analyzed manually. After scoring the bands, in which 0 used for showing the absence of a band and 1 showing the presence of a band in each sample, 1 and 0 numbers were changed to matrix by using NTedit software. Genetic similarities of samples were calculated by using NTSYSpc software, SIMQUAL method and DICE similarity coefficient. Dendrogram by cluster analysis was drawn by using UPGMA method. Principle coordinate analysis (PCO) was performed using the NTSYS software and grouping samples were evaluated in a two-dimensional plot.
Results and discussion: Results showed that from 9 primers in total, 118 bands amplified were in 300 to 3000 base pairs, while 29 bands were polymorphic. The number of amplified fragment for each primer was different so that UBC 844 (14 bands) had the highest and UBC 890 (8 bands) had the lowest amplified bands. The average amplified fragments for each primer was 9.83. The percentage of polymorphic in various primers was different. Maximum polymorphism (80%) of the primers was observed in UBC830. In general, according to the percentage of polymorphic bands, low adjustment to any changes in the environment was indicated. This can be used as an indicator to determine the value of diversity and genetic erosion. In genotypes cluster analysis, clustering was performed based on Dice similarity coefficient and UPGMA method, and 10 clusters were formed. ISSR molecular marker somewhat revealed genetic diversity among walnuts genotypes, whereas the genetic diversity was lower than expected. In general, by reviewing the findings in other parts of the world about walnut genetic diversity and comparing them with the results of this study, despite existing high genetic diversity among walnuts in many areas, some reports of low genetic diversity among walnuts populations have been published and unfortunately in recent years these reports has made more attention. Based on the results of several studies reporting low genetic diversity among walnuts, the following factors can be effective in this problem: natural disasters, human impacts such as deforestation and selection and propagation of superior genotypes, and sometimes walnuts self - pollination. In this respect, there is concern that if this trend of decreasing genetic diversity in the walnut population continues, this invaluable crop will be in danger of extinction. So we should think about a remedy. Finally, this investigation can be used as a start for conducting more researches in the region to maintain and manage this valuable crop germplasm and maximize genetic diversity for performing breeding programs in the future.

Keywords

Base pair; Cluster; Local varieties; Primer; Similarity

References

1- Aly M.M., Robert A., Fjellstrom G., McGranahan G.H. and Parfitt E. 1992. Origin of walnut somatic embryos determine by RFLP and Isozyme analysis. HortScience 27 (1): 61-63

2- 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. ScientiaHorticulturae. 111: 394-398

3- Christopoulos M. rouskas D. tsantili E. 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

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

5- Doyle, J.J. & Doyle, J.L. (1990). Isolation of plant DNA from fresh tissue. Focus,12, 13-15.

6- 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.

7- Fornari B., Malvolti M. E., Taurchini D.,Fineschi S., Beritognolo I., McCaglia E. And Cannata F. 2001. Isozym and organellar DNA analysis of genetic diversity in natural/naturalized European and Asiatic walnut (Juglans regia) populations. Acta Horticulturae. 544: 167-178

8- Foroni I., Woeste K., Monti L. M. and Rao R. 2006. Identification of ‘Sorrento’ walnut using simple sequence repeats (SSRs). Genetic Resources and Crop Evolution.85: 311-321

9- Haghjooyan R. 2002. Investigation of Toysarkan walnut genetic diversity and 4 walnut population in Iran with morphological and molecular markers. P.hD Thesis. Islamic Azad University, Science and Research Branch, Tehran (In persian)

10- Jia XD Wang T, Zhai M, Li YR, Guo ZR.2011. Genetic diversity and identification of Chinese-grown pecan using ISSR and SSR markers. Molecules. 2011 Dec 6;16(12):10078-92

11- Ji A., Wang Y., Wu G., Wu W., Yang H., Wang Q. 2014. Genetic Diversity and Population Structure of North China Mountain Walnut Revealed by ISSR. American Journal of Plant Sciences, 2014 (5): 3194-3202

12- Mahmoodi R., Rahmani F., Rezayi R. 2011. Investigation of Genetic Diversity Some Persian walnut Genotypes With ISSR Molecular Marker. Seventh Conference of Biotechnology Islamic Republic of Iran. (In Persian with English abstract)

13- Malvolti M. Paciucci M. Cannata F, Fineschi S.1993. Genetic variation in Italian population of Juglans Regia. L. Acta horticulture 311.

14- Moradi M.H., Rostamzadeh J., Rashidi A., Vahabi KH., Karimi F., Ahmadi M. 2006. Investigation of Genetic Diversity With ISSR Molecular Marker. Conference findings Agriculture and Natural Resources (West Country) - Iran, Sanandaj -2006 (In Persian with English abstract)

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

16- Orel G.,. Marchant A. D., McLeod J. A. and Richards G. D. 2003. Characterisation of 11 Juglandaceae genotypes based on morphology cpDNA and RAPD. HortScience. 38(6):1178-1183.

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

18- 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.

19- 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.

20- Tian L., xiang A., Li-SI Z, Hai Rong W, Qing L. 2011. ISSR Analysis of Genetic Diversity among Seedling Walnut (Juglans spp.)Populations. Journal of Plant Genetic Resources 2011-04

21- Vahdati K. 2000 .Walnut situation in Iran. Nucis-Newsletter. 9: 32-33.

22- Vahdati K. Mohseni S. Karimi R, Barzehkar R. Amiri R.Mozaffari M. and Woeste K 2014. Genetic diversity and gene flow of some Persian walnut populations in southeast of Iran revealed by SSR markers. Plant Systematics and Evolution

23- Wang H., Pei D., Gu R., Wang, B.2008. Genetic Diversity and Structure of Walnut Populations in Central and Southwestern China Revealed by Microsatellite Markers. Journal of the American Society for Horticultural Science. 133(2):197–203. 2008.

24- Wani N., Bhat A., Ahmad F., Akhter S., Razvi M., and Mir M. 2010. Molecular markers and their application in walnut improvment. International Journal of Current Research . 3:006-011