葡萄无核性状的SSR新分子标记开发及应用

【目的】开发葡萄无核性状的SSR新分子标记,对葡萄杂种后代进行早期无核性状鉴定,为分子标记辅助育种奠定基础。【方法】对母本‘红地球’与父本‘森田尼无核’及F1代杂交群体的133份葡萄材料进行RAD-seq,将测序结果比对到参考基因组上;运用SAMtools软件生成Ca SFS软件所需的pileup和glf文件,过滤获得亲本之间的有效SNP集;采用窗口滑动的方法,确定每个窗口的基因型,选择以每15个SNP为一个窗口,每次滑动一个SNP,得到每个个体的基因型并生成bin图;对生成的bin图采用拟测交的方式利用Joinmap软件进行连锁分析,构建‘红地球’与‘森田尼无核’遗传连锁图谱;用Window QTLCartographer2.0软件进行QTL定位,在定位区间通过Perl程序语言找到符合SSR特征序列的35个SSR分子标记,用Primer premier5.0软件设计35个SSR分子标记引物对,通过HRM技术筛选亲本之间在无核性状上存在差异的SSR分子标记,并在131株F1代杂交群体及65个葡萄品种的自然群体中检测无核分型正确率、无核检测率、无核保持率。【结果】在‘红地球’与‘森田尼无核’构建的遗传连锁图谱上,将葡萄无核性状定位在chr18号染色体上,定位区间26 835 846—26 960 426,对无核的贡献率为77.9%,LOD阈值为26.3。亲本之间在无核性状上存在差异的SSR分子标记为Vv SD10,其在111 bp等位点能对葡萄无核性状进行鉴定,利用分子标记Vv SD10上的111 bp等位点通过HRM技术在葡萄F1代杂交群体及自然群体中进行葡萄无核性状的鉴定。结果表明,分子标记Vv SD10在F1代杂交群体及自然群体中的无核分型正确率分别为97%、94%,其无核检测率均为56%,无核保持率分别为77%、85%。【结论】分子标记Vv SD10在遗传群体及自然群体中的无核分型均能提供较高的准确信息,为无核葡萄的分子标记辅助育种奠定了基础。

Development and Application of SSR New Molecular Marker for Seedless Traits in Grape

【Objective】The objective of this study is to develop a new SSR molecular marker for seedless traits in grape, and to identify the early seedless traits of grape hybrids, and to lay the foundation for molecular marker assisted selection breeding. 【Method】RAD-seq was carried out in 133 grape materials including the female parent ‘Red Globe’, the male parent ‘Centennial Seedless’ and their F₁ hybrids, and the sequencing results were compared to the reference genome. SAMtools software was used to generate CaSFS software required for pileup and GLF files, and the effective SNP sets between parents were obtained by filtration. A window sliding method was used to determine the genotype of each window, and selected one window per 15 SNP and slided one SNP at a time. The genotype of each individual was obtained and the bin generated. Linkage analysis was carried out based on Joinmap software for generated bin, and genetic linkage map was constructed between ‘Red Globe’ and ‘Centennial Seedless’. The QTL loci was mapped using WindowQTLCartographer2.0 software. 35 SSR molecular markers consistent with the SSR characteristic sequence were identified by Perl programming language in the QTL mapping interval, and then 35 pairs of SSR molecular marker primers were designed. The molecular marker was screened in the differences with the seedless traits between parents by HRM technology, and the accuracy of seedless typing, the rate of seedless detection and the rate of seedless holding were detected in 131 F₁ hybrid population and natural population including 65 grape varieties.【Result】On the genetic linkage map between ‘Red Globe’ and ‘Centennial Seedless’, the seedless traits of grape were mapped on chromosome 18, and the mapping interval was 26 835 846-26 960 426, and the contribution rate to seedless was 77.9%, and the LOD threshold was 26.3. The molecular marker VvSD10 was screened in the differences with the seedless traits between parents, and there was a 111 bp loci in seedless traits of grape. Identification of seedless traits in grape F₁ hybrid population and natural population by HRM technology using 111 bp loci on VvSD10 marker, the results indicated that in F1 hybrid population and natural population, the correct typing rate of molecular marker VvSD10 was 97% and 94%, respectively, and the rate of seedless detection of both populations was 56%, the rate of seedless holding was 77% and 85%, respectively. 【Conclusion】Seedless typing in grape by Molecular marker VvSD10 could provide high accurate information in both genetic and natural populations and laid the foundation for molecular marker assisted breeding program in seedless grape.