Fine mapping of a male sterility gene ms-3 in a novel cucumber (Cucumis sativus L.) mutant

Abstract

The cucumber male sterility gene ms - 3 was fine mapped in a 76 kb region harboring an MMD1 -like gene Csa3M006660 that may be responsible for the male sterile in cucumber. A cucumber (Cucumis sativus L.) male sterile mutant (ms-3) in an advanced-generation inbred line was identified, and genetic analysis revealed that the male sterility trait was controlled by a recessive nuclear gene, ms-3, which was stably inherited. Histological studies suggested that the main cause of the male sterility was defective microsporogenesis, resulting in no tetrad or microspores being formed. Bulked segregant analysis (BSA) and genotyping of an F₂ population of 2553 individuals were employed used to fine map ms-3, which was delimited to a 76 Kb region. In this region, a single non-synonymous SNP was found in the Csa3M006660 gene locus, which was predicted to result in an amino acid change. Quantitative RT-PCR analysis of Csa3M006660 was consistent with the fact that it plays a role in the early development of cucumber pollen. The protein encoded by Csa3M006660 is predicted to be homeodomain (PHD) finger protein, and the high degree of sequence conservation with homologs from a range of plant species further suggested the importance of the ms-3 non-synonymous mutation. The data presented here provide support for Csa3M006660 as the most likely candidate gene for Ms-3.

Fig. 1

Flowers of male sterile and fertile cucumber plants. a-1 Sterile stamen and anther, ×80; a-2 male sterile pollen, ×200; b-1 fertile stamen and anther, ×80; b-2 fertile pollen, ×200; c comparison of sterile stamen (left) and fertile stamen (right) in stage I, ×20; d comparison of sterile stamen (left) and fertile stamen (right) in stage II, ×20; e comparison of sterile stamen (left) and fertile stamen (right) in stage III, ×20; f comparison of sterile stamen (left) and fertile stamen (right) in stage IV, ×20; g comparison of sterile stamen (left) and fertile stamen (right) in stage V, ×20

Fig. 2

Microstructural analysis of microspore development in cucumber male sterile and fertile lines. a–f Microspore development in male fertile plants; g–l microspore development in male sterile plants. PS pollen sac, T tapetum, Ms microsporocyte, Tds tetrads, Msp microspore, MP mature pollen; ×500

Fig. 3

Δ(SNP-index) graph of MF (male fertile) and MS (male sterile) pool from the bulked segregant analysis (BSA). The X-axis represents the chromosome position. The Y-axis represents the Δ(SNP-index) value. The spots represent Δ(SNP-index) values calculated by the formula: [Δ(SNP-index) = SNP-index_Largest-SNP-index_Smallest]. The black line corresponds to the fitted value of the Δ(SNP-index) and the red line represents the confidence value (99%). Two candidate regions (region 1 166,710–564,531, size 397 Kb; and region 2 1,954,776–2,371,279, size 416 Kb) above the confidence value were identified on cucumber chromosome 3

Fig. 4

Fine mapping of the cucumber ms-3 gene. Single-nucleotide polymorphism (SNP) markers were selected from the region (166,710–2,371,279), and genotyping in an F2 population of 2553 individuals delimited ms-3 to a 76 Kb region with flanking marker T785241C and T861262G, respectively, with two and four recombinants. One non-synonymous mutation was detected in Csa3M006660 between WT and ms-3

Fig. 5

Expression analysis of the Csa3M006660 gene in cucumber organs. Csa3M006660 expression in a roots, stems, leaves, and young flower buds of wild type (WT); and b different stages of flower bud development: stage I (shorter than 3 mm), stage II (3–6 mm), stage III (6–9 mm), stage IV (9–12 mm), stage V (12–15 mm), and stage VI (longer than 15 mm). Expression levels were determined using q-PCR, and values were normalized using UBI-ep as the reference gene. Error bars indicate standard deviation from three biological replicates

Fig. 6

Phylogenetic analysis of Ms-3 and its homologs in 25 other plant species. Evolutionary relationships were inferred using the neighbor-joining method. The ID numbers refer to the gene IDs in the NCBI database and the names of the species are given. The bootstrap test values (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic relationships