利用2b-RAD测序结合HRM分析技术开发与梨矮生性状相关的DNA分子标记

【目的】果树的矮生性状是一种重要的农艺性状,对果树的集约化栽培具有重要意义。来自西洋梨实生变异品种‘Le Nain Vert’的矮生性状受控于一个单显性基因Pc Dw,目前关于该基因的序列信息等还不清楚。本研究的目的是开发与其紧密连锁的DNA分子标记,为鉴定该基因提供依据。【方法】根据分离群体分组分析的原理,以‘矮生梨’ב茌梨’和‘2-3’ב绿宝石’2个F1杂交分离群体为试材,应用IIB型限制性内切酶的RAD技术(Restriction association site DNA,2b-RAD)对2对矮生型/普通型对比基因池进行基因组测序分析,在对比基因池间筛选出单核苷酸多态性(Single nucleotide polymorphism,SNP)位点,从中筛查出位于Pc Dw定位染色体上的SNPs,用高分辨率熔解曲线分析技术(High-resolution melting analysis,HRM)在群体上进一步检测验证,以确定其与Pc Dw位点的连锁关系。【结果】对4个样本(即4个对比基因池)的2b-RAD标签测序文库的测序结果共产生67 186 260条reads,平均每个样本测序reads数为16 796 565。将原始reads进行质量过滤后的统计结果表明,每个样本获得平均unique标签数目为86 810,平均测序深度为77×,该测序深度能够达到准确分型的标准。SOAP软件定位结果表明,4个测序文库中含有酶切位点的高质量reads占测序原始reads的70%以上,表明测序质量较好。在来自2个不同群体的矮生型基因池与普通型基因池间进行对比分析,初步筛选出SNP位点1 317个,其中有8个位于PcDw的定位染色体scaffold00074上。用HRM技术对这8个SNP标记在群体上的进一步检测结果表明,在‘矮生梨’ב茌梨’群体上有2个SNP标记、在‘2-3’ב绿宝石’群体上有4个SNP标记表现出与Pc Dw位点共分离的特性,根据其扩增子的熔解曲线形状差异,可有效区分矮生型和普通型表型。在来自‘矮生梨’ב茌梨’群体的215个杂种后代和来自‘2-3’ב绿宝石’群体的168个杂种后代中,未发现有标记与性状的重组类型。【结论】2b-RAD测序技术与HRM分析技术相结合,进行果树重要农艺性状分子标记的开发,是一种行之有效的方法。基于这一策略,本研究鉴定获得了4个与西洋梨矮生性状单显性基因Pc Dw连锁的SNP标记。

Development of DNA Molecular Markers for the Dwarf Trait in Pear Through the Method of 2b-RAD Sequencing and HRM Analysis

【Objective】As an important agronomic trait, the dwarf character of tree is significant to fruit intensive culture. The dwarf traits of pear originated from variety ‘Le Nain Vert’, a chance seedling of Pyrus communis, is determined by a dominant gene PcDw, which sequence information is still unknown. Development of DNA molecular markers tightly linked to the PcDw locus could provide important information for identifying of this gene.【Method】Using 2 different F₁ populations obtained from ‘Aishengli’בChili’ and ‘2-3’בLvbaoshi’, respectively, as plant materials, 2 pairs of bulks for dwarf type/standard type were sequenced through IIB restriction association site DNA (2b-RAD) technology, and then single nucleotide polymorphism (SNP) markers were detected between the dwarf and standard bulks based on the principle of bulked segregant analysis. After that, those SNPs located on the PcDw mapped chromosome were selected and tested on the whole population to confirm their linkage relationship to the PcDw gene by high-resolution melting (HRM) analysis.【Result】A total of 67 186 260 reads were produced from the 2b-RAD sequencing of the 4 samples (2 pairs of bulks). That means the average reads per sample was 16 796 565. Statistical analysis of the filtered raw reads showed that the average unique tags per sample were 86 810, and the average sequencing depth was 77× which was enough for accurate genotyping. For the 4 libraries, mapping through SOAP software indicated that the high quality reads containing the restriction site accounted for more than 70% of the raw reads, which means the high quality of sequencing. Totally 1 317 polymorphic SNP markers were screened between the dwarf bulks and standard bulks derived from the 2 different populations, and 8 of them were mapped on the PcDw-localized chromosome scaffold00074. Further detection of the 8 SNPs through HRM analysis showed that 2 and 4 SNP markers co-segregated with the PcDw locus were identified from population ‘Aishengli’בChili’ and population ‘2-3’בLvbaoshi’, respectively. According to the different shapes of the melting curves of amplicons, the dwarf/normal phenotype could be distinguished effectively. The recombinants for each marker and the target trait were not found in both of the two populations, which contained 215 and 168 progenies, respectively. 【Conclusion】The combination of 2b-RAD sequencing and HRM analysis technology is an efficient method for exploiting molecular markers of important agronomic traits in fruit trees. In this study, a total of 4 SNP markers co-segregated with the PcDw locus were identified based on this strategy.