Genetic Diversity in Walnut (Juglans regia) from the Caucasus Nation of Azerbaijan

Analysis of genetic structure and differentiation using 12 microsatellite loci of six walnut populations, three each from the Greater Caucasus and the Talysh, revealed a moderate range of variation. The observed number of alleles ranged from 2 to 11 alleles with a mean of 5.6 alleles per locus. The populations differed significantly for the frequency and composition of alleles for ten out of twelve loci assayed, and the mean number of alleles per locus ranged from 4.0 to 4.6 with approximately 92% of the loci polymorphic. The mean observed and expected heterozygosity for different populations indicated that the populations conformed to Panmixia. The mean FST value indicates a significant departure from Hardy-Weinberg equilibrium, suggesting that there was significant differentiation among populations. The cluster analysis based on population pair-wise Nei’s unbiased genetic distances and the distance Wagner tree indicated that the Talysh populations are significantly different from and placed as a sister group to the Caucasus populations. INTRODUCTION The Caucasus nation of Azerbaijan is regarded as one of the centers of origin, diversity, and domestication of walnut. Walnut shows a wide range of adaptations to a multitude of environments created by the mountain ranges consisting of the Greater and Lesser Caucasus Mountains in the north and the Talysh Mountains in the south, where the diverse Tertiary relic Hyrcan flora presents a wide range of diversity in J. regia. Indigenous cultivars such as ‘Nazik-Qabiq’, ‘Davrish-Papaq’, ‘Katan Koynak’ and other local types were selected for shell thickness (‘Nazik-Qabiq’ meaning “paper shell”) and shape (‘Devrish-Papaq’ meaning “elongated shape”). Current practice of growing walnut in large plantations under the Forest Farming Systems is based on a narrow genetic base replacing many indigenous and folk forms recognized in natural walnut forests eroding the natural diversity. Azerbaijan has taken steps to deal with this crisis by establishing protected areas for in situ conservation representing the ecosystem diversity within the country and ex situ collections in gene banks to preserve the rich heritage of walnut germplasm for future generations. The Nakchivan province, which is geographically isolated from the rest of the country, contains diverse forms of walnut adapted to a gradient of environment from arid desert like conditions through medium to high rainfall mountainous terrains. The Araz experimental station under the Ministry of Agriculture in Ordubad, the southern border district of Nakchivan, maintains a collection of different forms representing the diversity of walnut in the province. The walnut in the Forest Farming areas can be grouped according to shell thickness and kernel percentage. About 70-80% of walnut populations have an average shell thickness of 0.5-2.0 mm and a kernel percentage of 45-55%, including J. regia f. semidura, J. regia f. tenera which have shell thickness of 0.5 mm and thinnest shell type with 55-66% kernel is a called ‘kachazi’ (paper shell) type (Table 1). According to local walnut experts, hard shell types which have more than 2.0 mm shell thickness are considered as J. regia L. var. dura. This variety is highly tolerant to environmental stress and resistant to insects (codling moth) and diseases (blight), which make it a good candidate for breeding. Overall, plant species show complex spatial and temporal diversity and adaptations to biotic and abiotic factors. Azerbaijan’s wide range of Proc. VIth Intl. Walnut Symposium Ed.: D.L. McNeil Acta Hort. 861, ISHS 2010 164 topographic and climatic conditions modulated by the Caucasus and Talish Mountain systems will lead to a maximum level of expression of this diversity. This diversity of Azerbaijan provides excellent sources of genetic resources for many plant species including walnut. MATERIALS AND METHODS Six walnut populations (Fig. 1), three each from the Caucasus and the Talysh Mountains (Fig. 1), were assayed for twelve microsatellite loci (Woeste et al., 2002; Table 2) using triplex polymerase chain reaction (PCR). The reaction mixture contained 1 μl of 10x PCR buffer, 2.5 mM MgCl2, 200 μM of each dNTP, 40 nM of each primer with forward primers labeled with 6FAM, HEX or NED fluorescent dye, 0.5 U Taq polymerase (New England Biolabs), 15-25 ng of template DNA, and dH2O to make up the final volume to 10 μl. The thermal cycler was set to run at 94°C for 5 min followed by 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 40 s, followed by a cycle of 7 min at 72°C. Amplified products were resolved using capillary electrophoresis on an ABI Prism 3100 genetic analyzer with the data collection software, version 1.2 (PE/Applied Biosystems). The data were further analyzed using Genescan, Version 3.1 and Genotyper, Version 2.5 and data were assembled as microsatellite genotypes. The multilocus SSR genotype data were analyzed for various within-group genetic variability measures such as mean number of alleles per locus and observed and expected levels of heterozygosities. Nei’s unbiased genetic identity (Nei, 1978) and Prevosti distance measures (Wright, 1978) were used to generate population pair-wise distance matrices. Genetic relationships among populations were examined using distance-based clustering methods such as the unweighted pair group method using arithmetic means (UPGMA) and the distance Wagner procedure (Swafford, 1981). The principal component analysis (PCA) was performed to compare the results of cluster analyses. Genetic differentiation within and among walnut populations was computed using F-statistics (Wright, 1978). Minimum Spanning Tree was superimposed on to 3D projection of populations to reveal distortions due to dimension reduction in PCA.