细胞质雄性不育 CMS-D8 棉花恢复基因 Rf 的物理图谱和 InDel 标记开发。
Physical mapping and InDel marker development for the restorer gene Rf in cytoplasmic male sterile CMS-D8 cotton.
机构信息
State Key Laboratory of Cotton Biology, Institute of Cotton Research of Chinese Academy of Agricultural Sciences, 38 Huanghe Dadao, Anyang, 455000, Henan, China.
Western Agricultural Research Center of Chinese Academy of Agricultural Sciences, 195 Ningbian East Road, Changji, 831100, Xinjiang, China.
出版信息
BMC Genomics. 2021 Jan 6;22(1):24. doi: 10.1186/s12864-020-07342-y.
BACKGROUND
Cytoplasmic male sterile (CMS) with cytoplasm from Gossypium Trilobum (D8) fails to produce functional pollen. It is useful for commercial hybrid cotton seed production. The restore line of CMS-D8 containing Rf gene can restore the fertility of the corresponding sterile line. This study combined the whole genome resequencing bulked segregant analysis (BSA) with high-throughput SNP genotyping to accelerate the physical mapping of Rf locus in CMS-D8 cotton.
METHODS
The fertility of backcross population ((sterile line×restorer line)×maintainer line) comprising of 1623 individuals was investigated in the field. The fertile pool (100 plants with fertile phenotypes, F-pool) and the sterile pool (100 plants with sterile phenotypes, S-pool) were constructed for BSA resequencing. The selection of 24 single nucleotide polymorphisms (SNP) through high-throughput genotyping and the development insertion and deletion (InDel) markers were conducted to narrow down the candidate interval. The pentapeptide repeat (PPR) family genes and upregulated genes in restore line in the candidate interval were analysed by qRT-PCR.
RESULTS
The fertility investigation results showed that fertile and sterile separation ratio was consistent with 1:1. BSA resequencing technology, high-throughput SNP genotyping, and InDel markers were used to identify Rf locus on candidate interval of 1.48 Mb on chromosome D05. Furthermore, it was quantified in this experiment that InDel markers co-segregated with Rf enhanced the selection of the restorer line. The qRT-PCR analysis revealed PPR family gene Gh_D05G3391 located in candidate interval had significantly lower expression than sterile and maintainer lines. In addition, utilization of anther RNA-Seq data of CMS-D8 identified that the expression level of Gh_D05G3374 encoding NB-ARC domain-containing disease resistance protein in restorer lines was significantly higher than that in sterile and maintainer lines.
CONCLUSIONS
This study not only enabled us to precisely locate the restore gene Rf but also evaluated the utilization of InDel markers for marker assisted selection in the CMS-D8 Rf cotton breeding line. The results of this study provide an important foundation for further studies on the mapping and cloning of restorer genes.
背景
细胞质雄性不育（CMS）与棉属三叶棉（D8）的细胞质结合，无法产生功能性花粉。它对商业杂交棉花种子生产很有用。含有 Rf 基因的 CMS-D8 恢复系能够恢复相应不育系的育性。本研究结合全基因组重测序分离群体分析（BSA）和高通量 SNP 基因分型，加速 CMS-D8 棉花 Rf 基因的物理图谱定位。
方法
在田间调查包含 1623 个个体的回交群体（不育系×恢复系）的育性。构建 BSA 重测序的可育池（100 株具有可育表型的 F 池）和不育池（100 株具有不育表型的 S 池）。通过高通量基因分型选择 24 个单核苷酸多态性（SNP）并开发插入缺失（InDel）标记，缩小候选区间。对候选区间内恢复系的五肽重复（PPR）家族基因和上调基因进行 qRT-PCR 分析。
结果
育性调查结果表明，可育和不育的分离比例符合 1:1。BSA 重测序技术、高通量 SNP 基因分型和 InDel 标记用于鉴定染色体 D05 上 1.48 Mb 候选区间的 Rf 基因。此外，本实验定量分析表明，与 Rf 共分离的 InDel 标记增强了恢复系的选择。qRT-PCR 分析表明，候选区间内的 PPR 家族基因 Gh_D05G3391 的表达水平明显低于不育系和保持系。此外，利用 CMS-D8 的花药 RNA-Seq 数据，鉴定出恢复系中编码 NB-ARC 结构域含有抗病蛋白的 Gh_D05G3374 的表达水平明显高于不育系和保持系。
结论
本研究不仅精确定位了恢复基因 Rf，还评估了 InDel 标记在 CMS-D8 Rf 棉花育性系标记辅助选择中的利用。本研究结果为进一步研究恢复基因的作图和克隆提供了重要基础。
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