Fertility results and statistical analysis
As Figure 1 shows for the male sterile dual-purpose line of the sweet pepper AB91, the anthers of the fertile plant are full and bright yellow, the stigma is lower than the anther, and the whole anther is covered in pollen; however, the sterile plant anther is small and lavender, the stigma is higher than the anther, the anther is shriveled, and there is no pollen. A total of 473 plants of self-crossing F2 generation of the sweet pepper AB91 dual-purpose fertility plant (Msms) were planted in a field, and fertility identification was performed at the flowering stage. As the investigation results for the F2 groups implies, there were 359 fertile plants and 114 sterile plants; the ratio between fertile and sterile plants was in line with the expected theoretical value 3:1(χ2=0.101﹤χ20.05=0.751).The fertile plants (Msms, MsMs) of the male sterile F2 generation of sweet peppers were singly seeded and planted separately; the group size for each single plant ranges from 30 to 40. The fertile plant (Msms) and pure dominant fertile plant (MsMs) of sweet pepper nuclear male sterile hybrid were filtered out according to the fertility separation of eachlineof F3 generation. There were 110 pure dominant fertile plants and 249 heterozygous fertile plants in F2.
Bulked‑segregant analysis and data analysis
MsMs and msms pools were sequenced by the bulked‑segregant analysis sequencing (BSA-Seq) technique; 417.04 million and 467.1 million filtered reads were obtained from the MsMs and msms pools, respectively. The MsMs-pool comparison ratio was 99.31%, the average coverage depth for the reference genome (excluding the N region) was 19.26X, and the 1X coverage degree (at least one base got covered) was 92.78%. The msms-pool comparison ratio was 99.03%, the average coverage depth for the reference genome (excluding the N region) was 21.55X, and the 1X coverage degree (at least one base got covered) was 92.95%. According to the results in Table 1, the comparison results are normal and can be used for subsequent mutation detection and correlation analysis.
In order to reduce the impact of sequencing errors and comparison errors, the SNP index and InDel index polymorphism sites were filtered out, and the number of polymorphic marker sites was 11,348,482 after filtration. By making a difference between the SNP index and InDel index for two descendants by choosing a 95% confidence level. The window larger than the threshold was selected as the candidate interval, and the numbers of polymorphism marker sites selected out for SNP and InDel were 27,541 and 1,865 respectively. For the candidate site extraction ANNOVAR annotation results, stop loss, and stop gain were selected with the priority, non-synonymous mutation, or alternative splicing site selected as the candidate gene. The number of SNP candidate genes filtered out from the fertile and sterile gene pools was 35(InDel has no annotated candidate genes above),and 33 candidate genes of them locatedat chromosome #5 (Figure 2).
Functional annotations of genes
The 33 genes in the correlated region are compared to the databases of NR, SwissProt[19]. GO[20] and KEGG[21]used BLAST software to identify 10 candidate genes and 11 candidate sites connecting to sweet pepper nuclear infertility, respectively, and they are speculated to be related to putative pentatricopeptide repeat-containing protein., galactinol synthase, glucosyltransferase, zeatin O-xylosyltransferase, threonine-protein kinase, histone acetyltransferase, U-box domain-containing protein, cell division cycle, and apoptosis regulator protein. All these candidate gene sites are located at chromosome #5. Hence, the candidate gene controlling the sweet pepper nuclear male sterile AB91 is determined at chromosome #5(Table2).
Mass spectrometry analysis
A mass spectrometry genotyping technique was used to detect the 11 mutation sites obtained by BSA-sequencing a population of 222 self-crossed F2 generation of sweet pepper nuclear male sterile dual-purpose AB91 fertile plant(Msms). Both sites 19194137 and 34599677 did not exhibit complete cosegregation with msc2. Therefore, the two corresponding genes Capana05g000617 and Capana05g000896 were excluded. The remaining 9 locations showed co-segregation, but it was slightly different from the msc2 phenotype detection in the field; the mutation site 28594037 had the highest similarity with msc2, and the accuracy rate (the number consistent with the msc2 phenotype detection in the field/F2 generation total population size) was 99.5%, and the corresponding gene for this site is Capana05g000747. Hence, the Capana05g000747 gene was determined to be the most powerful candidate gene for msc2(Figure 3).
Expression pattern of Fertile and sterile plants in different periods
The qRT-PCR were used to detect the changes of msc2 expression in fertile and sterile strains of sweet pepper male sterile AB91 at three stages of microspore tetrad prophase, microspore tetrad and microspore tetrad anaphase. Gene of msc2 were expressed in the three stages,and the expression of sterile plants were higher than fertile plants, However, the expression of sterile plants was significantly higher than fertile plants in the tetraspore prophase stage (p<0.05) (Fig 4). Therefore, it can be presumed that msc2 is a specific gene. The mutation of bases was significantly affected, and the mutation occurred in the prophase of tetraspore, which was earlier than the cytological observation of tetraspore [8].
Sequence analysis of male sterility gene msc2 for sweet pepper line AB91
The gene msc2 of sweet pepper male sterile AB91 is composed of eight exons and seven introns. The cDNA sequence is 1350bp in length and encodes 450 amino acids. The mutation site of the sterility gene is located at the 6th exon of Capana05g000747, the base C is mutated into A, and the amino acid is changed from alanine to serine, which causes a change in the fertility expression of the sterile plant of sweet pepper nuclear male sterile dual-purpose line AB91, leading to male sterility(Figure 5).
Three-dimensional protein structure prediction
The protein sequences of fertile and sterile plants candidate gene Capana05g000747 were analyzed by the online software Zhang Lab QUARK based on the protein structure. To construct the three-dimensional protein structure of the fertile and sterile plants candidate gene Capana05g000747 in the sweet pepper male sterile line AB91. Three-dimensional structures revealed the site “c” base mutation of Capana05g000747 gene lead to significant differences(figure 6). Therefore, it can be concluded that the base "c" mutation of gene Capana05g000747 may lead to pollen abortion.
Homology of nuclear male sterility gene msc2 in sweet pepper AB91 with other species
For a further functional study of msc2, we performed a sequence alignment and homology relationship analysis for the genemsc2 and other species, which showed that msc2 gene was highly conserved and at the same branch as tomato, potato, and tobacco, indicating that the their homology relationship is very close and msc2 is more likely to have the same effect as a close homology. However, the function of gene msc2 in tomato, potato, and tobacco sequences is predicted, and its function was found to be similar to the At2g02148 gene, which encodes the pentatricopeptide repeats (PPR) protein. This finding preliminarily implies that the msc2 gene has a connection with the PPR protein(Figure 7).