Genetic Analysis and Fine Mapping of Spontaneously Mutated Male Sterility Gene in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Abstract

Chinese cabbage (Brassica rapa L. ssp. pekinensis), an important traditional vegetable indigenous to China, is a typical cross-pollinated Brassica crop exhibiting pronounced heterosis. However, its small flower organs make artificial pollination for hybrid seed production highly challenging. The use of male-sterile lines has emerged as a crucial approach in hybrid seed production. Therefore, understanding the genetic and molecular mechanisms underlying male sterility in Chinese cabbage holds profound theoretical and economic importance and is pivotal for advancing Chinese cabbage crossbreeding. Here, cytological comparative analysis of anthers from sterile line 366-2S and fertile line 366-2F revealed abnormalities in 366-2S during the late tetrad stage, including delayed tapetum degradation and the aggregation of tetrad microspores without separation, which prevented pollen production and caused male sterility. Construction of the F₂ segregating population, with 366-2S as the female parent and genetically diverse fertile material Y636-9 as the male parent, indicated that male sterility in 366-2S is controlled by a single recessive gene. Using bulked segregant analysis sequencing and kompetitive allele-specific polymerase chain reaction (KASP) technology, the sterile gene was mapped to 65 kb between the PA11 and PA13 markers, with 11 genes in the candidate region. Functional annotation, expression, and sequence variation analyses identified BraA09g012710.3C, encoding acyl-CoA synthetase 5, as a candidate gene for 366-2S male sterility. Quantitative real-time polymerase chain reaction analysis revealed minimal expression of BraA09g012710.3C in 366-2S but high expression in the flower buds of 366-2F. Further analysis of candidate gene DNA sequences identified a large deletion encompassing BraA09g012710.3C, BraA09g012720.3C, BraA09g012730.3C, and BraA09g012740.3C in sterile line 366-2S (A09: 7452347-7479709). Cloning and verification of the other three deleted genes in the F₂ population via agarose gel electrophoresis confirmed their presence in F₂ sterile individuals, indicating that their deletion was not associated with male sterility, underscoring BraA09g012710.3C as the key gene driving male sterility in 366-2S.

Keywords: Chinese cabbage; acyl-CoA synthetase 5; genic male sterility; map-based cloning; phenylpropanoid biosynthesis.

Figure 1

Phenotypic observations and anther scanning electron microscopy of the 366-2F (fertile) and 366-2S (sterile) lines. (A) (a,b): Floret at the anthesis stage, exhibiting normal flower organs, in fertile line 366-2F. (e,f): Floret at the anthesis stage, exhibiting shorter filaments and anthers without pollen, in sterile line 366-2S. (c,d): 366-2F anther rectification and pollen grain staining. (g,h): 366-2S anther rectification and pollen grain staining. (a,b,e,f): bar = 5 mm; (c,g): bar = 1 mm; and (d,h): bar = 50 µm. (B) (a): 366-2F anther at the indehiscent stage. (b): Anatomical diagram of 366-2F anther at the indehiscent stage. (c): 366-2F anther anatomy at the flowering stage. (d): 366-2S anther at the indehiscent stage. (e): Anatomical diagram of 366-2S anthers at the indehiscent stage. (f): 366-2S anther anatomy at the flowering stage. (a,d): bar = 1 mm; (b,c,e,f): bar = 200 μm.

Figure 2

DAPI staining of pollen microspores and anther paraffin sections. (A). (a–e): Microspore development process of fertile line 366-2F. (f–j): Microspore development process of sterile line 366-2S. Bar = 50 µm. (B). (a–e): Microspore development of 366-2F pollen. (f–j): Pollen microspore development process of 366-2S, with abnormal development beginning in the tetrad stage. T: tapetum; MMC: microspore mother cell; Td: tetrad; and M: microspore. Bar = 20 µm.

Figure 3

Fine mapping of the nuclear sterility gene. (A) Single-nucleotide polymorphism (SNP)-index analysis of fertility in the F₂ population. (B) Initial physical location map of the sterility gene. (C) Fine mapping of the nuclear sterility gene. (D) Details of candidate genes in the interval.

Figure 4

DNA sequence analysis of candidate genes. (A) Visualization analysis results of candidate interval sequencing reads. (B) Amplification results of full-length and deletion fragments of BraA09g02710.3C in 366-2S and 366-2F. (C) Missing fragment pattern. Note: 366-2-FU stands for parent material 366-2, Y636-9-MU stands for parent material Y636-9.

Figure 5

Expression level of BraA09g012710.3C and electrophoretic verification of three deleted genes. (A) Relative expression levels of BraA09g012710.3C in different tissues of the 366-2S and 366-2F lines. (B) Breakpoint histograms of 366-2S and 366-2F at the early and late FPKM values. (C) Results of agarose gel electrophoresis validation of the expression of BraA09g012720.3C, BraA09g012730.3C, and BraA09g012740.3C in F₂ sterile individuals. Note: Y636-9 is a fertile parent; 366-2S is a sterile parent; F₂-Sterility is the sterile F₂ generation; and FPKM is fragments per kilobase of transcript per million mapped reads. Note: the seven numbers in the Figure, 263, 438, 286, 108, 130, 6, and 288 represent sterile single plant numbers, and these seven plants were selected by us based on different combinations of the three genes.

Figure 6

Phylogenetic tree of the BrACOS5 gene. Note: At, Arabidopsis thaliana; Bn, Brassica napus; Br, Brassica rapa; Zm, Zea mays; Os, Oryza sativa.

Figure 7

BraA09g012710.3C enrichment analysis and mapping of the metabolic pathway in which it is located. (A) Bubble map of Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of differentially expressed genes (DEGs). (B) Bubble map of Gene Ontology (GO) enrichment of DEGs. (C) Phenylpropane metabolic pathway map.