Mapping and Preliminary Analysis of ABORTED MICROSPORES (AMS) as the Candidate Gene Underlying the Male Sterility (MS-5) Mutant in Melon (Cucumis melo L.)

Abstract

Melon is an important agricultural and economic vegetable crop worldwide. The genetic male sterility mutant (ms-5) has a recessive nuclear gene that controls the male sterility germplasm. Male sterility could reduce the cost of F₁ seed production in melon, but heterozygous fertile plants should be removed before pollination. In this study, bulked segregant analysis combined with specific length amplified fragment sequencing was applied to map the single nuclear male sterility recessive gene. A 30-kb candidate region on chromosome 9 located on scaffold 000048 and spanning 2,522,791 to 2,555,104 bp was identified and further confirmed by cleavage amplified polymorphic sequence markers based on parental line resequencing data and classical mapping of 252 F₂ individuals. Gene prediction indicated that six annotated genes are present in the 30-kb candidate region. Quantitative RT-PCR revealed significant differences in the expression level of the LOC103498166 ABORTED MICROSPORES (AMS) gene in male-sterile lines (ms-5) and male-fertile (HM1-1) lines during the 2-mm (tetrad) and 5-mm (the first pollen mitosis) periods, and negative regulation of the AMS candidate gene transcription factor was also detected. Sequencing and cluster analysis of the AMS transcription factor revealed five single-nucleotide polymorphisms between the parental lines. The data presented herein suggest that the AMS transcription factor is a possible candidate gene for single nuclear male sterility in melon. The results of this study will help breeders to identify male-sterile and -fertile plants at seeding as marker-assisted selection methods, which would reduce the cost of seed production and improve the use of male-sterile lines in melon.

Keywords: AMS gene; SLAF-seq; fine mapping; genetic male sterility; melon.

FIGURE 1

Flower structure and SEM (scanning electron microscopy) of ms5 and HM1-1. (A) ms5 is shown on the left with a black background. (B) HM1-1 is shown on the right. (C) Pollen of ms5 is bright yellow with smooth flower buds. (D) HM1-1 is orange (on the right) with full pollen on the surface of buds. (E) Pollen viability of ms5 detected using acetocarmine. (F) Pollen viability of HM1-1 detected using acetocarmine.

FIGURE 2

Specific length amplified fragment (SLAF) marker distribution on chromosomes. Abscissa: length of chromosome, each yellow bar denotes one chromosome, and darker color indicates more SLAF markers in each window. ordinate: chromosome ID.

FIGURE 3

Location of predicted candidate genes on the genetic map of melon chromosome 9 using F₂ populations. (A) ΔSNP polt. The X-axis represents the chromosome position, and the Y-axis represents the ΔSNP-index value. The black line shows the average values of ΔSNP-index; the red dotted line is the ΔSNP value (0); the threshold value was 4.30, with an imaginary black line, calculated by Loess regression. The peak region contains 22 SLAF markers. (B) The genetic map generated using Joinmap 4.0 software. (C) Linked markers to male sterility 5 (ms5), and the candidate region is 1.75 Mb; two markers linked to ms5 are BSA3-3 and BSA16, at genetic distances of 0.1 and 0.3 cM, respectively. Fourteen predicted genes were screened, but only six were annotated; the candidate genes are LOC103498166, LOC103498167, LOC103498168, LOC103498194, LOC103498169, and LOC103498170.

FIGURE 4

Relative expression of six candidate genes in 2- and 5-mm stage flower buds diameter of ms5 and HM1-1. The melon β-actin gene was used as a control. ^∗∗Significantly different expression compared with 2-mm buds in fertile plants (HM1-1). (A) Cucumis melo transcription factor ABORTED MICROSPORES (LOC103498166) gene expression. (B) Cucumis melo geranylgeranyl transferase type-1 subunit beta (LOC103498167) gene expression. (C) Cucumis melo uncharacterized LOC103498168 (LOC1034981) gene expression. (D) Cucumis melo RING-H2 finger protein ATL52-like (LOC103498194) gene expression. (E) Cucumis melo serine/threonine-protein phosphatase 7-like (LOC103498169) gene expression. (F) Cucumis melo heat shock cognate 70 kDa protein 2-like (LOC103498170) gene expression.

FIGURE 5

Cluster analysis of candidate and other genes at the AMS locus. The transcription factor AMS Cucumis melo. L is the candidate gene.

FIGURE 6

Gene expression verified in F₂ plants and BSA16 marker amplification in different generations. (A) AMS gene expression in F₂ male-sterile and -fertile pools. (B) Lined marker amplified in F₂ homozygous male-sterile and male-fertile plants, and heterozygous F₂ male-fertile plants. P₁: ms5; P₂: HM1-1; F₁: F₁ plants from the cross of ms5 and HM1-1; 1–8: male-sterile plants of the F₂ population; 9–16: male-fertile heterozygous plants in the F₂ population; 17–24: male-sterile homozygous plants in the F₂ population. M: the reference markers. ^∗∗ means significant gene expression level based on fs pool (2 mm).