基于EST-SSR标记的越橘栽培种和几个中国野生种的遗传结构分析

Genetic structure analysis of the cultivated blueberry(Vaccinium spp.) species and wild species in China based on EST-SSR markers

【目的】遗传结构分析能够提供个体的血统来源及组成信息,是遗传关系分析的重要手段。研究越橘栽培种和中国野生种的遗传结构差异,以期为我国越橘育种中野生越橘资源的发掘、利用提供遗传背景依据。【方法】筛选EST-SSR引物并对越橘属84份种质资源的基因组DNA进行PCR扩增,利用ABI 3730xl DNA遗传分析仪进行毛细管电泳分离,统计条带并计算遗传多样性指数,利用STRUCTURE软件构建遗传结构图并进行基于Nie’s遗传距离的聚类分析和主成分分析。【结果】1)从54对EST-SSR引物中筛选到8对高多态性引物,共检测到101个等位变异,平均值12.63,有效等位基因数(Ne)、Shannon’s多态性信息指数(I)、观察杂合度(Ho)、期望杂合度(He)平均值分别为8.711、2.262、0.523和0.860,多态性信息含量(PIC)为0.640~0.915,平均值为0.845,所选引物多态性高。2)贝叶斯遗传结构分析表明,当K=4时,ΔK最大,供试材料被划分为4个类群,类群S1主要是红豆越橘(Vaccinium vitis-idaea)和蔓越橘(V.macrocarpon),以红果类型为特征;类群S2主要是笃斯越橘(V.uliginosum),以抗寒为特征;类群S3主要包括南高丛越橘(V.corymbosum SHB)和兔眼越橘(V.ashei),以低需冷量为特征;类群S4主要包括北高丛越橘(V.corymbosum NHB)和半高丛越橘(V.corymbosum HHB),以高需冷量为特征,表明越橘种质资源内部存在明显的遗传结构差异,4个类群与材料的抗寒性、低温需冷量特性等有明显的对应关系。3)N-J聚类分析和主成分分析结果表明,野生越橘与引进的栽培品种之间亲缘关系较远。4)供试84份材料中Q值≥0.6的品种占91.67%,绝大多数供试越橘资源遗传背景较单一。【结论】从国外引进的越橘栽培种和我国野生种间存在一定程度的基因交流,不存在绝对的生殖隔离,可以通过杂交的方式获得变异植株,实现越橘种质创新和遗传基础拓宽,应加大我国野生越橘资源的发掘和利用。

【Objective】Blueberries, belonging to the genus Vaccinium of family Ericaceae, including highbush blueberries（Vaccinium corymbosum）, rabbiteye blueberries（V. ashei） and low-bush blueberries（V.augustifolium） are the most recognized commercially cultivated species in the world. Some wild species of this genus are unique to cold resistance, drought resistance and fruit nutritions, and are valuable resources for the germplasm innovation and also broaden the genetic basis of the blueberry. An understanding of the genetic structural differences, relationship and genetic backgrounds among cultivated blueberries and wild species have crucial meanings, and guide the breeding programs accompanied by genetic exploration and utilization of the wild blueberry species.【Methods】Conducted a total of 84 tests including 58 cultivat-ed varieties from abroad and 26 wild types from China. DNA was extracted from the young leaves of theplants using a CTAB method with modification. PCR reactions were carried out in a final volume of 15 μLcontaining 3 μL template DNA（20 mg·L^（-1） ）, 0.2 μL forward primer（2.0 μmol·L^（-1） ）, 1.0 μL M13（labeledwith 6-FAM, NED, VIC or PET）, 1.2 μL reverse primer（2.0 μmol·L^（-1） ）, 7.5 μL Mix-Taq（CWBIO, China）and 2.1 μL H2O（CWBIO, China）. PCR amplification conditions were as follows： 94 °C for 5 min, 35 cy-cles of 95 °C for 30 s, 52 °C for 45 s and 72 °C for 45 s, and a final extension at 72 °C for 10 min. All reac-tions were conducted using a thermal cycler（Veriti 96, Applied Biosystems, Foster City, USA）. The PCRproducts were separated by capillary electrophoresis using a ABI3730 xl DNA Analyzer（Applied Biosys-tems, Foster City, USA）. After PCR amplification confirmation with 2.0% agarosegel, nest-PCR multiplexsets were made based on fluorescence-labeled M13 primers. For nest-PCR multiplexing, 1 μL of 6-FAM, VIC-, NED-and 0.5 μL PET labeled PCR products representing different SSRs were combined in13 μL H2 O 1 μL mixed product which was then added to 7 μL Hi-Di formamide containing 0.07 μL Gen-e Scan？ 500 LIZ？as the internal size standard, denatured for 7 min at 95 °C, quick chilled on ice for 5min, and loaded on a ABI3730 xl DNA Analyzer. The fragment analysis was performed by using Gene Map-per v4.0 software（Applied Biosystems, Foster City, USA）. The genetic structure was investigated by usingsoftware STRUCTURE and Principal Coordinate Analysis（PCA） and a neighbor-joining tree clusterwhich was analyzed based on Nie＇s genetic distance and applied through software Power Marker.【Results】1） Totally, 101 polymorphic alleles were obtained with 8 EST-SSR primer pairs, which were screenedfrom a total of 54 EST-SSR primer pairs, with an average of 12.63 alleles per locus. The average of effec-tive alleles（Ne）, Shannon＇s index（I）, observed heterozygosity（Ho） and expected heterozygosity（He） perlocus were 8.711, 2.262, 0.523 and 0.860,respectively, and the polymorphic information content（PIC）ranged from 0.640 to 0.915 with an average of 0.845, indicating a high polymorphism. 2） Genetic structureanalysis based on the Bayesian method indicated that ΔK reached maximum when K=4, all the 84 testedblueberry accessions were clustered into 4 groups： S1, which is given priority to lingonberry（V. vitisidaea） and cranberry（V. macrocarpon） and characterized by red fruit; S2, which is given priority to bog bil-berry（V. uliginosum） and characterized by being highly cold-resistant; S3, which is given priority to south-ern highbush（V. corymbosum SHB） and rabbiterry（V. ashei） blueberry, and characterized by having a lowchilling requirement; S4, which is given priority to northern highbush（V. corymbosum NHB） and half-highbush（V. corymbosum HHB） blueberries and characterized by having a high chilling requirement, theabove results showed an obvious genetic structure within Vaccinium spp. 3） Both the results of neighbor-joining tree cluster analysis and principal component analysis（PCA） identified large genetic differencesthat existed between the cultivated species and wild species of China; 4） The Q value of the 84 tested blue-berry accessions higher than 0.6 accounted for 91.67%, which were considered an individual assigned un-equivocally to a cluster, with single genetic components.【Conclusion】Obvious genetic structure and dis-tantly relationships existed between the cultivated species and wild species of China, however, there was alimited amount of cultivated varieties with an ancestry of wild species（with S1 or S2 components） and thisindicates that there was introgression between the cultivated variety and wild species. This shows that youcan obtain variant plants through hybridization to innovated germplasm and broaden the genetic basis forblueberries, which also should enhance the exploitation and utilization of wild blueberry resources in China.