SSR screening and identification of walnut (Juglans regia) cultivars

Abstract:【Objective】The walnut(Juglans regia L.) is an important non-timber tree species that belongs to the family Juglandaceae and is extensively cultivated in China. In recent decades, with rapid progress in walnut breeding, a large number of new varieties were developed, promoting the rapid development of the walnut industry. However, some new varieties, due to being derived from several common ancestors,have a narrow genetic basis with small genetic differences and also small morphological differences, leading to difficulties in variety identification and in turn protection of plant breeders' right(PBR). Traditionally, morphology, pollen morphology, cytology and isoenzyme are the main techniques to identify walnut species and cultivars, but with the increase in the varieties and the decrease in genetic variation among them, distinguishing from different varieties is becoming more and more difficult using traditional techniques. Simple sequence repeat(SSR), or microsatellite, is a stretch of DNA consisting of tandem repeating 2-6 nucleotide units, which has been shown to be ubiquitous in most eukaryotic genomes. Currently SSR has been widely used in studies and practices for variety identification in many species. To identify walnut cultivars quickly, efficiently and reliably, we conducted this study to select a set of suitable SSR makers for walnut identification, which would benefit the protection of plant breeders' rights and the healthy development of the walnut industry.【Methods】22 pairs of primers(i.e. wga001, wga004, wga009,wga032, wga054, wga069, wga070, wga079, wga142, wga148, wga202, wga256, wga276, wga349, wga376,zmz05, zmz22, zmz27, zmz31, zmz35, zmz39, and zmz44) were selected from primers previously reported in J. nigra and J. regia, according to their polymorphism. The research materials used are five walnut cultivars of‘Chuanzao'‘(Chuanzao 1'‘,Chuanzao 2'‘,Shuangzao'‘,Zaofeng', and‘Shuling'), which are the improved hybrid cultivars breed by the Forestry College, Sichuan Agricultural University, and two native cultivars of Sichuan ‘(Shimianju'and‘Ludingzhi'). Three clones were sampled as three repeats of each cultivar. Genomic DNAs were extracted from dried leaves stored in silica gel by using the CTAB method with modifications. Primers were first synthesized as normal primers to do the product amplification and the agarose gel test. Then, the primers with clear product bands in gel were synthesized as fluorescence primers for PCR amplification and genotyping. PCR amplification was performed in a 25 μL volume, consisting of a 10×Taq Mix(with d NTP, Mg2+, loading buffer) 12.5 μL, primer F(10 nmol·L^-1) 1 μL, primer R(10 nmol·L^-1) 1 μL, genomic DNA(circa 50 ng·L^-1) 2 μL, and dd H2 O 8.5 μL. The reaction mixture was subjected to PCR amplification in a PTC-100(MJ) using a PCR program, 4 mins at 94 ℃, followed by 35 cycles of 94 ℃ for 30 s, 55 or 58 ℃ annealing temperature for 45 s, and 72 ℃ for 1 min, followed by 5 min at 72 ℃. PCR products were genotyped by using a genetic analyzer(ABI 3730), then the Gene Mapper 4.0was used to get the allele report. Fragment-size was determined according to the allele reports and adjusted manually. Gen Al Ex 6.0 was used to calculate allele number(Na), effective allele number(Ne), observed and expected heterozygosities(Ho & He), and fixed index(F). MEGA 6.2 was used for the unweighted pairgroup method analysis(UMPGA) with the genetic distances deriving from Gen Al Ex 6.0.【Results】Fifteen SSR loci(wga001, wga004, wga009, wga032, wga070, wga079, wga142, wga148, wga202, wga349, zmz05,zmz22, zmz35, zmz39, and zmz44) were found to have the clear, accurate and consensus information of alleles. Fragment-size of some alleles directly deriving from the automatic value had discrepancies among repeats, but some reasonably manual adjustments could remove these discrepancies and achieve the consensus values among repeats. Fifteen SSR loci produced seven identical profiles for the seven walnut cultivars. It was also found that only the loci wga032 could distinguish five cultivars ‘(Chuanzao 1'‘,Shuling',‘Zaofeng'‘,Shimianju'and‘Ludingzhi'), and the combination of wga001 and wga032 could distinguish‘Chuanzao 2'and‘Shuangzao', implying these loci could efficiently identify the walnut cultivars. For genetic diversity, fifteen SSR loci produced 2(wga009) to 8(wga032) alleles per locus with 64 alleles in total and 4.2 alleles on average. The effective allele number(Ne) is between 1.153 to 6.533 with 3.292 being the average. The observed heterozygosity(Ho) and the unbiased expected heterozygosity(He) range from 0.143 to 1.000 and 0.143 to 0.912 with the mean values of 0.648 and 0.704 respectively, exhibited a high polymorphism in the walnut germplasms. The genetic relationship of the seven cultivars deriving from the UPGMA dendrogram of using genetic distances based on all the fifteen loci were found to generally correspond to the original relationship among cultivars.【Conclusion】Accurately determining the fragment size of alleles is the first step when using SSR for identification within species, which could be achieved by means of manual adjustment based on comparison among repeats. Fifteen SSR loci screened from 22 loci exhibited favorable polymorphism and produced seven identical SSR profiles for these seven cultivars, demonstrating a satisfactory ability to identify the walnut cultivars. Furthermore, it was also found that even only one or two loci could distinguish these seven cultivars, implying the probability of a decreasing expenditure and consuming-time. As a result, these fifteen loci could assist in the identification of walnut cultivars of‘Chuanzao',and provide a reference to construct a more-broadly-used SSR identifying system for walnut cultivars. and also provide protection of plant breeder's rights(PBR) for walnuts.