Abstract
BackgroundCytoplasmic 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 Rf2 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 Rf2 locus in CMS-D8 cotton.MethodsThe 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.ResultsThe 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 Rf2 locus on candidate interval of 1.48 Mb on chromosome D05. Furthermore, it was quantified in this experiment that InDel markers co-segregated with Rf2 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.ConclusionsThis study not only enabled us to precisely locate the restore gene Rf2 but also evaluated the utilization of InDel markers for marker assisted selection in the CMS-D8 Rf2 cotton breeding line. The results of this study provide an important foundation for further studies on the mapping and cloning of restorer genes.
Highlights
Cytoplasmic male sterile (CMS) with cytoplasm from Gossypium Trilobum (D8) fails to produce functional pollen
A total of 1623 BC1F1 plants were classified as 850 fertile and 773 sterile plants, and the ratio of the number of fertile plants (850) to the number of sterile plants (773) fit a 1:1 segregation (χ2= 3.6531 < χ2(0.05,1) = 3.84), confirming that fertility restoration is conditioned by one dominant restorer gene, Rf2
We developed insertion and deletion (InDel) markers based on InDel variations and used these markers to locate the Rf2 gene in a 1.48 Mb region
Summary
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 Rf2 gene can restore the fertility of the corresponding sterile line. The cytoplasmic male sterility (CMS) system plays an important role in utilization of crop heterosis. The CMS system avoids the removal of anthers, thereby enabling the generation of dramatically superior F1 progenies through hybrid technology. These offsprings display significant advantages over their parents and existing popular cultivars in terms of yield, stress tolerance, adaptability, etc. The CMS phenomenon exists in more than 150 plants and is used for hybrid breeding of crops, such as maize [6, 7], rice [8, 9], pepper [10] and sorghum [11]
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