A Semi-Quantitative Bulked Segregant Analysis Preliminarily Localizes a Maize Male-Sterility Gene

Bulked segregant analysis (BSA) assists in map-based cloning of mutant genes. However, a traditional BSA requires many high-density markers for successful linkage analysis which is labor-intensive and time-consuming. In this study, a semi-quantitative DNA analysis program was optimized and combined it with BSA, resulting in a semi-quantitative BSA (sq-BSA). The sq-BSA approach allowed evaluation of the proportions of marker-defined individuals (dominant or recessive marker types) in bulks. The sq-BSA method was used to map a male-sterility (ms) gene, ms2016, in maize. Forty polymorphic markers were screened from one-third of each chromosome (from the head or tail) for mapping. Among these markers, seven were identified as candidate gene-linked markers, of which four markers (bnlg1046, umc1563, umc1171 and umc1722) were located on chromosome 5. Using group validation, ms2016 was anchored on chromosome 5 and was most closely linked to bnlg1046. Furthermore, four new InDel markers located near bnlg1046 were screened to map the preliminary location of ms2016. The ms2016 gene was mapped to an 8.7 Mb interval flanked by the InDel polymorphic markers I5-3 (chr5:14588060) and I5-12 (chr5:23308445). Thus, this improved BSA method (sq-BSA) requires only a small number of molecular markers to quickly localize a target gene, representing a high-efficiency tool for mutant gene mapping. © 2021 Friends Science Publishers

Genetic Analysis and Fine Mapping of a Spontaneously Mutated Male Sterility Gene in Brassica rapa ssp. chinensis

Male sterility has been widely used in hybrid seed production in Brassica, but not in B. rapa ssp. chinensis, and genetic models of male sterility for this subspecies are unclear. We discovered a spontaneous mutant in B. rapa ssp. chinensis. A series of progeny tests indicated that male sterility in B. rapa ssp. chinensis follows a three-allele model with BrMsa, BrMsb, and BrMsc. The male sterility locus has been mapped to chromosome A07 in BC1 and F2 populations through genotyping by sequencing. Fine mapping in a total of 1,590 F2 plants narrowed the male sterility gene BrMs to a 400 kb region, with two SNP markers only 0.3 cM from the gene. Comparative gene mapping shows that the Ms gene in B. rapa ssp. pekinensis is different from the BrMs gene of B. rapa ssp. chinensis, despite that both genes are located on chromosome A07. Interestingly, the DNA sequence orthologous to a male sterile gene in Brassica napus, BnRf, is within 400 kb of the BrMs locus. The BnRf orthologs of B. rapa ssp. chinensis were sequenced, and one KASP marker (BrMs_indel) was developed for genotyping based on a 14 bp indel at intron 4. Cosegregation of male sterility and BrMs_indel genotypes in the F2 population indicated that BnRf from B. napus and BrMs from B. rapa are likely to be orthologs. The BrMs_indel marker developed in this study will be useful in marker-assisted selection for the male sterility trait.