Sterility In Soybean Research Articles

Identification of a Candidate restorer-of-fertility Gene Rf3 Encoding a Pentatricopeptide Repeat Protein for the Cytoplasmic Male Sterility in Soybean.

The cytoplasmic male sterility/restorer-of-fertility (CMS/Rf) system plays a vital role in high-efficiency hybrid seed production in crops, including soybean (Glycine max (L.) Merr.). The markers linked to fertility restoration and the restorer-of-fertility (Rf) genes are essential because they can facilitate the breeding of new CMS lines and production of commercial hybrid soybean seeds. To date, several soybean Rf genes have been mapped to various genetic loci in diverse genetic populations. However, the mapping range of restorer genes remains narrow, with relatively limited practical applicability. Therefore, in the present study, F2 and F3 segregating populations derived from the CMS line JLCMS5A crossed with the restorer line JLR2 were developed and used for Rf3 gene fine mapping. Genetic investigation indicated that the restorer line JLR2 was controlled by a single dominant gene, Rf3. By integrating bulk-segregant analysis and next-generation sequencing, a 4 Mb region on chromosome 9 was identified, which was most likely the target region harboring the candidate gene responsible for fertility restoration. This region was further narrowed down to 86.44 Kb via fine mapping in F2 and F3 populations using SSR, InDel, and dCAPS markers. This region contained 10 putative genes (Glyma.09G171100–Glyma.09G172000). Finally, Glyma.09G171200, which encodes a mitochondria-targeted pentatricopeptide repeat protein, was proposed as the potential candidate for Rf3 using sequence alignment and expression analysis in restorer and CMS lines. Based on single-nucleotide polymorphisms in Glyma.09G171200, a CAPS marker co-segregated with Rf3 named CAPS1712 was developed. Our results will be fundamental in the assisted selection and creation of potent lines for the production and rapid selection of novel restorer lines.

Confirmation of GmPPR576 as a fertility restorer gene of cytoplasmic male sterility in soybean.

In soybean, heterosis achieved through the three-line system has been gradually applied in breeding to increase yield, but the underlying molecular mechanism remains unknown. We conducted a genetic analysis using the pollen fertility of offspring of the cross NJCMS1A×NJCMS1C. All the pollen of F1 plants was semi-sterile; in F2, the ratio of pollen-fertile plants to pollen-semi-sterile plants was 208:189. This result indicates that NJCMS1A is gametophyte sterile, and the fertility restoration of NJCMS1C to NJCMS1A is a quality trait controlled by a single gene locus. Using bulked segregant analysis, the fertility restorer gene Rf in NJCMS1C was located on chromosome 16 between the markers BARCSOYSSR_16_1067 and BARCSOYSSR_16_1078. Sequence analysis of genes in that region showed that GmPPR576 was non-functional in rf cultivars. GmPPR576 has one functional allele in Rf cultivars but three non-functional alleles in rf cultivars. Phylogenetic analysis showed that the GmPPR576 locus evolved rapidly with the presence of male-sterile cytoplasm. GmPPR576 belongs to the RFL fertility restorer gene family and is targeted to the mitochondria. GmPPR576 was knocked out in soybean N8855 using CRISPR/Cas9. The T1 plants showed sterile pollen, and T2 plants produced few pods at maturity. The results indicate that GmPPR576 is the fertility restorer gene of NJCMS1A.

Male sterility in soybean: Occurrence, molecular basis and utilization

AbstractIn plants, male sterility (MS) is a specific breeding target trait. With the advancements in agriculture, utilization of heterosis breeding in hybrid production through MS lines has become the main breeding tool of various cross‐pollinated and even self‐pollinated crops. Soybean is an essential source of oil and protein; however, the low yield is a major factor limiting its development. Soybean MS mainly comprises cytoplasmic‐nuclear MS and nuclear/genic MS (NMS/GMS), which can effectively utilize heterosis to improve soybean yield. This review outlines the recent research progress on the development of new genetically MS lines, exploring the underlying molecular mechanism of MS, identification and cloning of MS and fertility restoration genes, and the application of MS lines. We further discussed and prospected the future developmental scenario direction of the soybean MS, based on the previous studies of other crops sterility system. Moreover, this review also provides comprehensive information for better application of MS to soybean breeding programme.

Mutation in a PHD-finger protein MS4 causes male sterility in soybean

BackgroundMale sterility has tremendous scientific and economic importance in hybrid seed production. Identification and characterization of a stable male sterility gene will be highly beneficial for making hybrid seed production economically feasible. In soybean, eleven male-sterile, female-fertile mutant lines (ms1, ms2, ms3, ms4, ms5, ms6, ms7, ms8, ms9, msMOS, and msp) have been identified and mapped onto various soybean chromosomes, however the causal genes responsible for male sterility are not isolated. The objective of this study was to identify and functionally characterize the gene responsible for the male sterility in the ms4 mutant.ResultsThe ms4 locus was fine mapped to a 216 kb region, which contains 23 protein-coding genes including Glyma.02G243200, an ortholog of Arabidopsis MALE MEIOCYTE DEATH 1 (MMD1), which is a Plant Homeodomain (PHD) protein involved in male fertility. Isolation and sequencing of Glyma.02G243200 from the ms4 mutant line showed a single base insertion in the 3rd exon causing a premature stop codon resulting in truncated protein production. Phylogenetic analysis showed presence of a homolog protein (MS4_homolog) encoded by the Glyma.14G212300 gene. Both proteins were clustered within legume-specific clade of the phylogenetic tree and were likely the result of segmental duplication during the paleoploidization events in soybean. The comparative expression analysis of Ms4 and Ms4_homologs across the soybean developmental and reproductive stages showed significantly higher expression of Ms4 in early flowering (flower bud differentiation) stage than its homolog. The functional complementation of Arabidopsis mmd1 mutant with the soybean Ms4 gene produced normal stamens, successful tetrad formation, fertile pollens and viable seeds, whereas the Ms4_homolog was not able to restore male fertility.ConclusionsOverall, this is the first report, where map based cloning approach was employed to isolate and characterize a gene responsible for the male-sterile phenotype in soybean. Characterization of male sterility genes may facilitate the establishment of a stable male sterility system, highly desired for the viability of hybrid seed production in soybean. Additionally, translational genomics and genome editing technologies can be utilized to generate new male-sterile lines in other plant species.

Genome-wide analysis of DNA methylation to identify genes and pathways associated with male sterility in soybean

DNA methylation is an epigenetic modification, which is important for gene expression regulation. Although genome-wide DNA methylation studies have been reported in several plant species, the difference in the methylation pattern between the cytoplasmic male sterile (CMS) line and its maintainer in soybean remains unclear. We compared genome-wide DNA methylation between the soybean CMS line NJCMS1A and its maintainer NJCMS1B using methylated DNA immunoprecipitation combined with high-throughput sequencing (MeDIP-seq) technology. The results showed that the methylation level was higher in transposable elements (TEs) than promoter and intron; however, the methylation levels varied among different types of TEs with the highest level for long terminal repeats (LTRs) and the lowest for transcription start sites (TSS) and transcription termination sites (TTS). We observed 178 differentially methylated genes (DMGs) between NJCMS1A and NJCMS1B, including 156 hypo-methylated and 22 hyper-methylated genes in NJCMS1A compared to NJCMS1B. Gene Ontology (GO) analysis showed that 114 DMGs were annotated to one or more GO categories, among which four GO terms were significantly enriched. KEGG pathway analysis showed that 18 DMGs were significantly enriched in 10 metabolism pathways, including homologous recombination, purine metabolism, proteasome, non-homologous end-joining, and pyrimidine metabolism. Further analysis indicated that the male sterility of NJCMS1A might be caused by the altered DNA methylation and disturbed metabolism pathways of some key DMGs, involved in carbohydrate and energy metabolism, transcriptional regulation, male gametophyte development, encoding mitochondrion protein, etc. However, the relationship between the DMGs and the CMS of NJCMS1A still need further research.

Differential proteomics analysis to identify proteins and pathways associated with male sterility of soybean using iTRAQ-based strategy

To further elucidate the molecular mechanism of cytoplasmic male sterility (CMS) in soybean, a differential proteomic analysis was completed between the CMS line NJCMS1A and its maintainer NJCMS1B using iTRAQ-based strategy. As a result, 180 differential abundance proteins (DAPs) were identified, of which, 60 were down-regulated and 120 were up-regulated in NJCMS1A compared with NJCMS1B. Bioinformatic analysis showed that 167 DAPs were annotated in 41 Gene Ontology functional groups, 106 DAPs were classified into 20 clusters of orthologous groups of protein categories, and 128 DAPs were enrichment in 53 KEGG pathways. Fifteen differential level proteins/genes with the same expression pattern were identified in the further conjoint analysis of DAPs and the previously reported differential expression genes. Moreover, multiple reaction monitoring test, qRT-PCR analysis and enzyme activity assay validated that the iTRAQ results were reliable. Based on functional analysis of DAPs, we concluded that male sterility in NJCMS1A might be related to insufficiencies in energy supply, unbalance of protein synthesis and degradation, disruption of flavonoid synthesis, programmed cell death, abnormalities of substance metabolism, etc. These results might facilitate our understanding of the molecular mechanisms behind CMS in soybean. Biological significanceSoybean is an important global crop that provides protein and oil. Heterosis is a significantly potential approach to increase the yield of soybean. Cytoplasmic male sterility (CMS) plays a vital role in the production of hybrid seeds. However, the genetic and molecular mechanisms of male sterility in soybean still need to be further elucidated. In the present paper, a differential proteomic analysis was carried out and the results showed that several key proteins involved in key pathways were associated with male sterility in soybean. This work provides a new insight to understand the genetic and molecular mechanisms underlying CMS in soybean.

Day Temperature Influences the Male‐Sterile Locus ms9 in Soybean

ABSTRACTCultivated soybean [Glycine max (L.) Merr.] is a major source of oil and protein in global food production. As demand for soy‐based products increases, it is essential for breeders to find ways to increase yield beyond conventional methods. Alternative methods of cultivar development and improvement must be identified. Our objectives were to determine if the ms9 locus conferring male sterility in soybean was environmentally sensitive and, if it were, to determine which environments could be used in hybrid seed production. This was accomplished through characterization of pollen phenotypes and selfed seed set among progeny of soybean genotype T359H (Genetic Type Collection number T359H), a heterozygous maintainer line for the ms9 locus. T359H was evaluated in seven environmental regimens in growth chamber experiments. We used a split‐plot design to test the effect of temperature regimens and male‐sterile phenotypic classifications within chambers. As day temperature increased from 30 to 35°C, selfed seed set on male‐sterile, female‐fertile plants decreased. Conversely, night temperature affected neither fertile nor male‐sterile, female‐fertile selfed seed set. This suggests that ms9 can be used in hybrid seed production in controlled environments where day temperature is 35°C during the flowering. These temperature conditions and their effect on ms9 may provide an alternative method to produce hybrid seed in soybean cultivar development programs.

Inheritance and fine mapping of a restorer-of-fertility (Rf) gene for the cytoplasmic male sterility in soybean

The cytoplasmic male sterility (CMS) line FuCMS5A and its restorer line FuHui9 were crossed to produce a segregating F2 population for pollen fertility assay and the genetic mapping of restorer-of-fertility (Rf) gene. Results showed that the individual F2 plants were fertile or semi-fertile based on their pollen fertility characteristics. The average ratios of viable pollen were 96.90% and 50.00% for each class of individuals. The segregation of F2 plants showed a good fit to a 1:1 ratio, which reflects a typical heredity pattern of gametophytic CMS with fertility restorer being controlled by a single dominant gene. Using bulk segregation analysis (BSA) and genetic mapping, the Rf gene was mapped on molecular linkage group J (chromosome 16), between the simple sequence repeat (SSR) makers BARCSOYSSR-16-1064 and BARCSOYSSR-16-1082 with the distances of 0.59 and 0.83cM, respectively. Four SSR markers (BARCSOYSSR-16-1070, Sctt011, BARCSOYSSR-16-1076 and BARCSOYSSR-16-1077) were cosegregating with this Rf gene in the mapping population. These makers will greatly facilitate the maker assisted selection procedures in CMS breeding programs and it lays a foundation for further map-base cloning of the Rf gene.

Fine mapping of foxglove aphid (Aulacorthum solani) resistance gene Raso1 in soybean and its effect on tolerance to Soybean dwarf virus transmitted by foxglove aphid.

Soybean dwarf virus (SbDV) causes serious dwarfing, yellowing and sterility in soybean (Glycine max). The soybean cv. Adams is tolerant to SbDV infection in the field and exhibits antibiosis to foxglove aphid (Aulacorthum solani), which transmits SbDV. This antibiosis (termed “aphid resistance”) is required for tolerance to SbDV in the field in segregated progenies of Adams. A major quantitative trait locus, Raso1, is reported for foxglove aphid resistance. Our objectives were to fine map Raso1 and to reveal whether Raso1 alone is sufficient to confer both aphid resistance and SbDV tolerance. We introduced Raso1 into cv. Toyomusume by backcrossing and investigated the degree of aphid antibiosis to foxglove aphid and the degree of tolerance to SbDV in the field. All Raso1-introduced backcross lines showed aphid resistance. Interestingly, only one Raso1-introduced backcross line (TM-1386) showed tolerance to SbDV in the field. The results demonstrated Raso1 alone is sufficient to confer aphid resistance but insufficient for SbDV tolerance. Tolerance to SbDV was indicated to require additional gene(s) to Raso1. Additionally, Raso1 was mapped to a 63-kb interval on chromosome 3 of the Williams 82 sequence assembly (Glyma1). This interval includes a nucleotide-binding site–leucine-rich repeat encoding gene and two other genes in the Williams 82 soybean genome sequence.

Cloning and Characterization of a Novel Gene GmMF1 in Soybean ( Glycine max L. Merr.)

Abstract Cytoplasmic male sterility plays an important role in utilization of crop heterosis. Screening of soybean for novel genes related to male sterility in soybean could provide a basis for studying the molecular mechanism of male sterility in plants. In this study, gene differential expressions between the cytoplasmic male-sterile line NJCMS1A and its maintainer line NJCMS1B in soybean were analyzed using cDNA-AFLP. A differentially expressed fragment, GmMF-T4A15, was isolated from large flower buds of NJCMS1B. By searching the soybean genomic library and PCR amplification, the cDNA full-length sequence of 1 311 bp was obtained and named GmMF1. The expression characteristics of GmMF1 were studied by semiquantitative real-time PCR and real-time quantitative PCR. The results showed that GmMF1 was expressed highly in flower buds of NJCMS1B. The deduced protein contains 436 amino acids and shows high similarity to members of the DUF620 protein family with unknown functions in other plant species. It is predicted that the protein encoded by GmMF1 is localized in the nucleus.

Molecular mapping of a fertility restorer gene for cytoplasmic male sterility in soybean

With 2 figures and 2 tables Abstract In this study, we report the mapping of the Rf locus in soybean by microsatellite simple sequence repeat (SSR) genetic markers. A cross was made between cytoplasmic male sterility (CMS) line JLCMS82A and restorer line JIHUI 1 based on the DNA polymorphisms revealed by 109 SSR markers. A F2 population derived from a single F1 plant containing 103 individuals was used for mapping the Rf locus. The Rf gene of JIHUI 1 gametophytically restores male fertility to JLCMS82A. Fertile and semi-fertile DNA bulks and parental DNAs were screened with 219 SSR markers, and Satt215 which was previously mapped to soybean LG J, was found linked to the Rf gene. Five additional polymorphic SSR markers from LG J were used for analysis and a regional linkage map around the Rf locus was established. SSR markers, Sctt011 and Satt547, flanked the Rf locus at 3.6 cM and 5.4 cM, respectively. The availability of these SSR markers will facilitate the selection of restorer lines in hybrid soybean breeding.

Male Sterility in Soybean (Glycine max). I. Phenotypic Expression of the ms2 Mutant

The effects of a nuclear male-sterile mutant (ms2) of soybean, Glycine max (L.) Merr., on anther development were analyzed by means of light- and electron-microscopy. The structure of microspore mother cells (MMCs) in male-sterile plants was identical to that of male-fertile plants. Meiosis was completed, and tetrads of microspores formed. Microspores degenerated after the deposition of primexine and probacullae. The sheath of callose surrounding microspores did not dissolve. No structural abnormalities of the microspores were detected before the onset of degeneration. The tapetal and anther wall layers were characterized by aberrant development. Tapetal abnormalities included premature vacuolation, a persistent inner tangential cell wall, failure to differentiate normal concentrations of endoplasmic reticulum and dictyosomes, disruption of plastids, and premature degeneration. Malfunction of the tapetal layer preceded, and may have induced, microspore degeneration. Gross anther morphology was not influenced until advanced stages of development.