核桃品种微卫星标记的筛选与鉴别

SSR screening and identification of walnut(Juglans regia) cultivars

【目的】筛选合适的微卫星标记,以便精确、快速、高效、可靠地鉴定核桃品种。【方法】从已报道的核桃微卫星引物遴选出22对引物,对5个川早系列核桃品种和2个四川乡土核桃进行鉴别。【结果】经过筛选和比对重复,有15个位点获得了清晰、准确、一致的等位基因信息。15个位点共获得了64个等位基因,平均等位基因数为4.2个,平均表观杂合度为0.648;平均无偏期望杂合度为0.704,表现出了很高的多态性。少数几个位点(wga001、wga032、wga148、wga004与wga079组合、wga349与zmz39组合,wga349与zmz22组合,wga202与zmz05组合,zmz35与zmz44组合等)就可以有效地对供试核桃品种进行鉴定。【结论】15个筛选出的微卫星位点表现出很好的品种鉴别能力,为川早系列核桃品种的鉴定提供了技术支持,也为建立应用更为广泛的核桃品种微卫星鉴别体系提供了一定的借鉴。

[Objective 1The 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）. Tradition- ally, morphology, pollen morphology, cytology and isoenzyme are the main techniques to identify walnut species and cuhivars, 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 andJ. regia, according to their polymorphism. The research materials used are five walnut culti- vars 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 eultivar. 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 dNTP, Mg^2＋, loading buffer） 12.5 μL, primer F（10 nmol. L^-1） 1 μL, primer R （10 nmol. L^-1） 1μL, genomic DNA （circa 50 ng. L^-1） 2 μL, and ddH20 8.5 μL. The reaction mixture was subjected to PCR amplification in a PTC- 100 （MJ） using a PCR program, 4 rains at 94 ℃, followed by 35 cycles of 94 ℃ for 30 s, 55 or 58 ℃ annealing temperature for 45 s, and 72 ℃ for I min, followed by 5 min at 72 ℃. PCR products were genotyped by using a genetic analyzer （ABI 3730）, then the GeneMapper 4.0 was used to get the allele report. Fragment-size was determined according to the allele reports and adjusted manually. GenAlEx 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 GenAlEx 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 cultivats. 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 cuhivars. 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 poly morphism in the walnut germplasms. The genetic relationship of the seven cuhivars 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. [ConclusionlAccurately 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 cuhivars. 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.