Genic Male Sterility Research Articles

Development of the PARMS Marker of the Dominant Genic Male Sterility (DGMS) Line and Its Utilization in Rapeseed (Brassica napus L.) Breeding.

The 8029AB line is a dominant genic male sterility (DGMS) two-type line in Brassica napus L., which can be used in a three-line approach for the seed production of rapeseed hybrids. Genetic analyses have demonstrated that the sterility of 8029A is controlled by a single dominant nuclear gene (BnMS5e) interacting with one recessive gene (BnMS5c). Six pairs of penta-primer amplification refractory mutation system (PARMS) markers were designed according to the sequence of BnMS5a, BnMS5c and BnMS5e. Two pairs of these PARMS markers were successfully identified and validated. The PARMS markers MS5-1Fc/MS5-1Ft/MS5-1R12 could distinguish BnMS5c from BnMS5a/BnMS5e, and the PARMS markers MS5-2Ft/MS5-2Fa/MS5-1R12 could genotype BnMS5a and BnMS5c/BnMS5e. The combination of these two pairs of PARMS markers could be used to identify the presence or absence of BnMS5a/BnMS5c/BnMS5e effectively. Consequently, marker-assisted selection can be carried out in the early generation to shorten the breeding period and improve the breeding efficiency.

Delayed callose degradation restores the fertility of multiple P/TGMS lines in Arabidopsis.

Photoperiod/temperature-sensitive genic male sterility (P/TGMS) is widely applied for improving crop production. Previous investigations using the reversible male sterile (rvms) mutant showed that slow development is a general mechanism for restoring fertility to P/TGMS lines in Arabidopsis. In this work, we isolated a restorer of rvms-2 (res3), as the male sterility of rvms-2 was rescued by res3. Phenotype analysis and molecular cloning show that a point mutation in UPEX1 l in res3 leads to delayed secretion of callase A6 from the tapetum to the locule and tetrad callose wall degradation. Electrophoretic mobility shift assay and chromatin immunoprecipitation analysis demonstrated that the tapetal transcription factor ABORTED MICROSPORES directly regulates UPEX1 expression, revealing a pathway for tapetum secretory function. Early degradation of the callose wall in the transgenic line eliminated the fertility restoration effect of res3. The fertility of multiple known P/TGMS lines with pollen wall defects was also restored by res3. We propose that the remnant callose wall may broadly compensate for the pollen wall defects of P/TGMS lines by providing protection for pollen formation. A cellular mechanism is proposed to explain how slow development restores the fertility of P/TGMS lines in Arabidopsis.

Use of CRISPR/Cas9-Based Gene Editing to Simultaneously Mutate Multiple Homologous Genes Required for Pollen Development and Male Fertility in Maize

Male sterility represents an important trait for hybrid breeding and seed production in crops. Although the genes required for male fertility have been widely studied and characterized in many plant species, most of them are single genic male-sterility (GMS) genes. To investigate the role of multiple homologous genes in anther and pollen developments of maize, we established the CRISPR/Cas9-based gene editing method to simultaneously mutate the homologs in several putative GMS gene families. By using the integrated strategies of multi-gene editing vectors, maize genetic transformation, mutation-site analysis of T0 and F1 plants, and genotyping and phenotyping of F2 progenies, we further confirmed gene functions of every member in ZmTGA9-1/-2/-3 family, and identified the functions of ZmDFR1, ZmDFR2, ZmACOS5-1, and ZmACOS5-2 in controlling maize male fertility. Single and double homozygous gene mutants of ZmTGA9-1/-2/-3 did not affect anther and pollen development, while triple homozygous gene mutant resulted in complete male sterility. Two single-gene mutants of ZmDFR1/2 displayed partial male sterility, but the double-gene mutant showed complete male sterility. Additionally, only the ZmACOS5-2 single gene was required for anther and pollen development, while ZmACOS5-1 had no effect on male fertility. Our results show that the CRISPR/Cas9 gene editing system is a highly efficient and convenient tool for identifying multiple homologous GMS genes. These findings enrich GMS genes and mutant resources for breeding of maize GMS lines and promote deep understanding of the gene family underlying pollen development and male fertility in maize.

TMT-based comparative proteomic analysis of the male-sterile mutant ms01 sheds light on sporopollenin production and pollen development in wucai (Brassica campestris L.)

A spontaneous male-sterile mutant ms01 was discovered from the excellent high-generation inbred line ‘hx12-6-3’ in wucai. Compared with wild-type ‘hx12-6-3’, ms01 displayed complete male sterility with degenerated stamens and no pollen. In this study, cytological observation revealed that the tapetum of the anthers of ms01 had degraded in advance, and microspore development had stagnated in the mononuclear stage, ultimately resulting in completely aborted pollen. Genetic analysis indicated that the sterility of ms01 was controlled by a single recessive nuclear gene. In the differential proteomic analysis of ‘hx12-6-3’ and ms01 flower buds using a tandem mass tags-based approach, a comparison of two stages (stage a and stage e) revealed 1272 differentially abundant proteins (DAPs). The abnormal variation of the anther cuticle, pollen coat, and sporopollenin production were effected by lipid metabolism and phenylpropanoid biosynthesis in the mutant ms01. Further analysis elucidated that pollen development was associated with amino acid metabolism, protein synthesis and degradation, carbohydrate metabolism, flavonoid biosynthesis and glutathione metabolism. These results provide novel insights into the molecular mechanism of GMS (genic male sterility) in wucai. Significancems01, as the first indentified spontaneous male-sterile mutant in wucai, plays a significant role in the initial study of GMS (genic male sterility). In our study, the key DAPs related to anther and pollen development were obtained by TMT-based comparative proteomic analysis. We found that the abnormal accumulation of H2O2 might induce premature degradation of the tapetum, causing anther metabolism disorder and pollen abortion. This process involved multiple DAPs and formed a complex regulatory network that generated a series of physiological metabolic alterations, ultimately leading to male sterility. Our results provide a theoretical foundation for further research on the complex anther and pollen development process.

Divergence of X-linked trans regulatory proteins and the misexpression of gene targets in sterile Drosophila pseudoobscura hybrids

BackgroundThe genetic basis of hybrid incompatibilities is characterized by pervasive cases of gene interactions. Sex chromosomes play a major role in speciation and X-linked hybrid male sterility (HMS) genes have been identified. Interestingly, some of these genes code for proteins with DNA binding domains, suggesting a capability to act as trans-regulatory elements and disturb the expression of a large number of gene targets. To understand how interactions between trans- and cis-regulatory elements contribute to speciation, we aimed to map putative X-linked trans-regulatory elements and to identify gene targets with disrupted gene expression in sterile hybrids between the subspecies Drosophila pseudoobscura pseudoobscura and D. p. bogotana.ResultsWe find six putative trans-regulatory proteins within previously mapped X chromosome HMS loci with sequence changes that differentiate the two subspecies. Among them, the previously characterized HMS gene Overdrive (Ovd) had the largest number of amino acid changes between subspecies, with some substitutions localized within the protein’s DNA binding domain. Using an introgression approach, we detected transcriptional responses associated with a sterility/fertility Ovd allele swap. We found a network of 52 targets of Ovd and identified cis-regulatory effects among target genes with disrupted expression in sterile hybrids. However, a combined analysis of polymorphism and divergence in non-coding sequences immediately upstream of target genes found no evidence of changes in candidate regulatory proximal cis-elements. Finally, peptidases were over-represented among target genes.ConclusionsWe provide evidence of divergence between subspecies within the DNA binding domain of the HMS protein Ovd and identify trans effects on the expression of 52 gene targets. Our results identify a network of trans-cis interactions with possible effects on HMS. This network provides molecular evidence of gene × gene incompatibilities as contributors to hybrid dysfunction.

Combined Transcriptome and Proteome Analysis of Anthers of AL-type Cytoplasmic Male Sterile Line and Its Maintainer Line Reveals New Insights into Mechanism of Male Sterility in Common Wheat.

Cytoplasmic male sterility (CMS) plays an essential role in hybrid seeds production. In wheat, orf279 was reported as a CMS gene of AL-type male sterile line (AL18A), but its sterility mechanism is still unclear. Therefore, transcriptomic and proteomic analyses of the anthers of AL18A and its maintainer line (AL18B) were performed to interpret the sterility mechanism. Results showed that the electron transport chain and ROS scavenging enzyme expression levels changed in the early stages of the anther development. Biological processes, i.e., fatty acid synthesis, lipid transport, and polysaccharide metabolism, were abnormal, resulting in pollen abortion in AL18A. In addition, we identified several critical regulatory genes related to anther development through combined analysis of transcriptome and proteome. Most of the genes were enzymes or transcription factors, and 63 were partially homologous to the reported genic male sterile (GMS) genes. This study provides a new perspective of the sterility mechanism of AL18A and lays a foundation to study the functional genes of anther development.

Disruption of the bHLH transcription factor Abnormal Tapetum 1 causes male sterility in watermelon

Although male sterility has been identified as a useful trait for hybrid vigor utilization and hybrid seed production, its underlying molecular mechanisms in Cucurbitaceae species are still largely unclear. Here, a spontaneous male-sterile watermelon mutant, Se18, was reported to have abnormal tapetum development, which resulted in completely aborted pollen grains. Map-based cloning demonstrated that the causal gene Citrullus lanatus Abnormal Tapetum 1 (ClATM1) encodes a basic helix-loop-helix (bHLH) transcription factor with a 10-bp deletion and produces a truncated protein without the bHLH interaction and functional (BIF) domain in Se18 plants. qRT–PCR and RNA in situ hybridization showed that ClATM1 is specifically expressed in the tapetum layer and in microsporocytes during stages 6–8a of anther development. The genetic function of ClATM1 in regulating anther development was verified by CRISPR/Cas9-mediated mutagenesis. Moreover, ClATM1 was significantly downregulated in the Se18 mutant, displaying a clear dose effect at the transcriptional level. Subsequent dual-luciferase reporter, β-glucuronidase (GUS) activity, and yeast one-hybrid assays indicated that ClATM1 could activate its own transcriptional expression through promoter binding. Collectively, ClATM1 is the first male sterility gene cloned from watermelon, and its self-regulatory activity provides new insights into the molecular mechanism underlying anther development in plants.

Identification of miRNAs and their target genes in genic male sterility lines in Brassica napus by small RNA sequencing

BackgroundBrassica napus is the third leading source of edible oil in the world. Genic male sterility (GMS) lines provide crucial material for harnessing heterosis for rapeseed. GMS lines have been used successfully for rapeseed hybrid production in China. MicroRNAs (miRNAs) play crucial regulatory roles in various plant growth, development, and stress response processes. However, reports on miRNAs that regulate the pollen development of GMS lines in B. napus are few.ResultsIn this study, 12 small RNA and transcriptome libraries were constructed and sequenced for the flower buds from the fertile and sterile lines of two recessive GMS (RGMS) lines, namely, “6251AB” and “6284AB”. At the same time, 12 small RNA and transcriptome libraries were also constructed and sequenced for the flower buds from the fertile and sterile lines of two dominant GMS (DGMS) lines, namely, “4001AB” and “4006AB”. Based on the results, 46 known miRNAs, 27 novel miRNAs on the other arm of known pre-miRNAs, and 44 new conserved miRNAs were identified. Thirty-five pairs of novel miRNA-3p/miRNA-5p were found. Among all the identified miRNAs, fifteen differentially expressed miRNAs with over 1.5-fold change between flower buds of sterile and fertile lines were identified, including six differentially expressed miRNAs between “4001A” and “4001B”, two differentially expressed miRNAs between “4006A” and “4006B”, four differentially expressed miRNAs between “6251A” and “6251B”, and ten differentially expressed miRNAs between “6284A” and “6284B”. The correlation analysis of small RNA and transcriptome sequencing was conducted. And 257 candidate target genes were predicted for the 15 differentially expressed miRNAs. The results of 5′ modified RACE indicated that BnaA09g48720D, BnaA09g11120D, and BnaCnng51960D were cleaved by bna-miR398a-3p, bna-miR158-3p and bna-miR159a, respectively. Among the differentially expressed miRNAs, miR159 was chosen to analyze its function. Overexpression of bna-miR159 in Arabidopsis resulted in decreased seed setting rate, and shortened siliques, illustrating that miR159 may regulate the fertility and silique development in rapeseed.ConclusionsOur findings provide an overview of miRNAs that are potentially involved in GMS and pollen development. New information on miRNAs and their related target genes are provided to exploit the GMS mechanism and reveal the miRNA networks in B. napus.

Transcriptomic analysis reveals candidate genes for male sterility in Prunus sibirica.

BackgroundThe phenomenon of male sterility widely occurs in Prunus sibirica and has a serious negative impact on yield. We identified the key stage and cause of male sterility and found differentially expressed genes related to male sterility in Prunus sibirica, and we analyzed the expression pattern of these genes. This work aimed to provide valuable reference and theoretical basis for the study of reproductive development and the mechanisms of male sterility in Prunus sibirica.MethodThe microstructures of male sterile flower buds and male fertile flower buds were observed by paraffin section. Transcriptome sequencing was used to screen genes related to male sterility in Prunus sibirica. Quantitative real-time PCR analysis was performed to verify the transcriptome data.ResultsAnther development was divided into the sporogenous cell stage, tetrad stage, microspore stage, and pollen maturity stage. Compared with male fertile flower buds, in the microspore stage, the pollen sac wall tissue in the male sterile flower buds showed no signs of degeneration. In the pollen maturity stage, the tapetum and middle layer were not fully degraded, and anther development stopped. Therefore, the microspore stage was the key stage for anther abortion , and the pollen maturity stage was the post stage for anther abortion. A total of 4,108 differentially expressed genes were identified by transcriptome analysis. Among them, 1,899 were up-regulated, and 2,209 were down-regulated in the transcriptome of male sterile flower buds. We found that “protein kinase activity”, “apoptosis process”, “calcium binding”, “cell death”, “cytochrome c oxidase activity”, “aspartate peptidase activity”, “cysteine peptidase activity” and other biological pathways such as “starch and sucrose metabolism” and “proteasome” were closely related to male sterility in Prunus sibirica. A total of 331 key genes were preliminarily screened.ConclusionThe occurrence of male sterility in Prunus sibirica involved many biological processes and metabolic pathways. According to the results of microstructure observations, related physiological indexes determination and transcriptome analysis, we reveal that the occurrence of male sterility in Prunus sibirica may be caused by abnormal metabolic processes such as the release of cytochrome c in the male sterile flower buds, the imbalance of the antioxidant system being destroyed, and the inability of macromolecular substances such as starch to be converted into soluble small molecules at the correct stage of reproductive development, resulting in energy loss. As a result, the tapetum cannot be fully degraded, thereby blocking anther development, which eventually led to the formation of male sterility.

Comparative analysis of morphology, cytology, and mitochondrial transcriptome of sterile male flowers of rubber tree

AbstractRubber tree [Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg.] is the exclusive commercial source of natural rubber. Male‐sterile (MS) germplasm has proven to be valuable for rubber tree hybrid breeding, but its male sterility mechanism remains unclear. Here, morphological observations of a new MS clone Reyan93‐114 showed shriveled anthers, short filaments, and withered male flowers. Transmission electron microscopy revealed the tapetal cells of the anthers were hypertrophic and vacuolated. Microspore protoplasts reduced in size, and the microspore wall had obvious defects: the incomplete intine and the insufficiently deposited exine. Organelles had disappeared in both tapetal cells and microspores. Comparative mitochondrial transcriptome analysis identified three differentially expressed genes in the MS clones: orf126, orf51, and atp8. In particular, orf126 and orf51 were unique insertions in the MS mitochondrial genomes. The former was an unknown open reading frame, and the latter was a fusion gene that contained a portion of atp9. These two genes should be studied as candidate male sterility genes in rubber tree. These results clarified the cytological causes of male sterility and expanded to understand the relationship between mitochondrial genes and male sterility in rubber tree.

An insertion mutation located on putative enhancer regions of the MYB26-like gene induces inhibition of anther dehiscence resulting in novel genic male sterility in radish (Raphanus sativus L.).

The online version contains supplementary material available at 10.1007/s11032-021-01254-9.

Fertility regulation and molecular design hybrid breeding in crops

<p indent=0mm>Fertility regulation affects crop yield as well as heterosis utilization. Here we summarize the genetic basis and molecular regulatory mechanism of male sterility and hybrid sterility in crops. The advances include cytoplasmic male sterility and its application in three-line hybrid breeding, photoperiod- and temperature-sensitive genic male sterility and its application in two-line hybrid breeding, biotechnology-based male sterility system and its application by molecular design breeding technology, and hybrid fertility and its application in distant hybrid breeding. On this basis, we discuss the bottleneck and strategies for exploring mechanisms of crop fertility regulation and molecular design hybrid breeding in China, with future trend predicted.

Genic male sterility increases rice drought tolerance

Plant fertility and resistance to stress environments are antagonistic to each other. At booting stage, fertility is often sacrificed for survive in rice under abiotic stress. However, the relationship between fertility and resistance at molecular level remains elusive. Here, we identified a transcription factor, OsAlfin like 5, which regulates the OsTMS5 and links both the drought stress response and thermosensitive genic male sterility. The OsAL5 overexpression plants (OE-OsAL5) became sensitive to temperature owning to the OsTMS5 that the OE-OsAL5 plants were fertile under low temperature (23 °C) and sterile under high temperature (28 °C). Significantly, the survival rate of OE-OsAL5 lines was higher than that of the wide-type (WT) under drought stress. Further experiments confirmed that the OsAL5 regulated both of the OsTMS5 and the down-stream drought-related genes by binding to the ‘GTGGAG’ element in vivo, revealing that the OsAL5 participated both in the drought stress response and thermosensitive genic male sterility in rice. These findings open up the possibility of breeding elite TGMS lines with strong drought tolerance by manipulating the expression of OsAL5.

Mapping and Analysis of a Novel Genic Male Sterility Gene in Watermelon (Citrullus lanatus).

Seed production is critical for watermelon production, which mostly involves first-generation hybrid varieties. However, watermelon hybrid seed production currently requires complex procedures, including artificial isolation and pollination. Therefore, the development and use of a male-sterile system to generate watermelon hybrids can simplify the process. The scarcity of male-sterile watermelon germplasm resources necessitates the use of molecular breeding methods. Unfortunately, the genes responsible for male sterility in watermelon have not been cloned. Thus, the genetic basis of the male sterility remains unknown. In this study, two DNA pools derived from male-sterile and normal plants in the F2 population were used for whole-genome resequencing. The Illumina high-throughput sequencing resulted in 62.99 Gbp clean reads, with a Q30 of 80% after filtering. On the basis of the SNP index association algorithm, eight candidate regions (0.32 Mb) related to specific traits were detected on chromosome 6. Expression pattern analyses and watermelon transformation studies generated preliminary evidence that Cla006625 encodes a pollen-specific leucine-rich repeat protein (ClaPEX1) influencing the male sterility of watermelon. The identification and use of genic male sterility genes will promote watermelon male sterility research and lay the foundation for the efficient application of seed production technology.

Comparative transcriptome analysis reveals differential gene expression in sterile and fertile rubber tree varieties during flower bud differentiation

Plant male sterility (MS) is an important agronomic trait that provides an efficient tool for hybridization and heterosis utilization of crops. Based on phenotypic and cytological observations, our study performed a multi-comparison transcriptome analysis strategy on multiple sterile and fertile rubber tree varieties using RNA-seq. Compared with the male-fertile varieties, a total of 1590 differentially expressed genes (DEGs) were detected in male-sterile varieties, including 970 up-regulated and 620 down-regulated transcripts in sterile varieties. Key DEGs were further assessed focusing on anther development, microsporogenesis and plant hormone metabolism. Twenty DEGs were selected randomly to validate transcriptome data using quantitative real-time PCR (qRT-PCR). Eleven key genes were subjected to expression pattern analysis using qRT-PCR and fluorescence in situ hybridization. Among them, nine genes, i.e., A6, GAI1, ACA7, TKPR1, CYP704B1, XTH26, MS1, MS35 and MYB33, that regulate callose metabolism, pollen wall formation, tapetum and microspores development were identified as candidate male-sterile genes. These findings provide insights into the molecular mechanism of male sterility in rubber tree.

A cellular mechanism underlying the restoration of thermo/photoperiod-sensitive genic male sterility

During anther development, the transformation of the microspore into mature pollen occurs under the protection of first the tetrad wall and later the pollen wall. Mutations in genes involved in this wall transition often lead to microspore rupture and male sterility; some such mutants, such as the reversible male sterile (rvms) mutant, are thermo/photoperiod-sensitive genic male sterile (P/TGMS) lines. Previous studies have shown that slow development is a general mechanism of P/TGMS fertility restoration. In this study, we identified restorer of rvms-2 (res2), which is an allele of QUARTET 3 (QRT3) encoding a polygalacturonase that shows delayed degradation of the tetrad pectin wall. We found that MS188, a tapetum-specific transcription factor essential for pollen wall formation, can activate QRT3 expression for pectin wall degradation, indicating a non-cell-autonomous pathway involved in the regulation of the cell wall transition. Further assays showed that a delay in degradation of the tetrad pectin wall is responsible for the fertility restoration of rvms and other P/TGMS lines, whereas early expression of QRT3 eliminates low temperature restoration of rvms-2 fertility. Taken together, these results suggest a likely cellular mechanism of fertility restoration in P/TGMS lines, that is, slow development during the cell wall transition of P/TGMS microspores may reduce the requirement for their wall protection and thus support their development into functional pollens, leading to restored fertility.

Role of MS1 homolog Ntms1 gene of tobacco infertility.

The sterile line is the basis of crop heterosis utilization. To broaden the sources of male sterility in tobacco, the Ntms1 (Nicotiana tabacum L. ms1) gene was cloned from the tobacco variety K326 by homologous cloning based on the Cams1 (Capsicum annuum L. ms1) gene sequence of male-sterility genes in pepper. The protein structure and physicochemical properties of the two genes were determined by bioinformatics analysis, and the function of the Ntms1 gene was verified by the CRISPR/Cas9 system. The results showed that the sequences of Ntms1 and Cams1 were 85.25% similar, and plant homeodomains were found in both genes; the physical and chemical properties were also very similar. It is speculated that the Ntms1 gene had the same function as the Cams1 gene in controlling male sterility. Compared to the wild-type plants, the filaments of the Ntms1 knockout mutant plants were shorter, and the stamen was shorter than the pistil. The anthers did not develop fully and had few viable pollen grains; the tapetum and the anther wall had developed abnormally, and the anther chamber was severely squeezed. The malondialdehyde content in the mutant plants was significantly higher than that in the wild-type plants, while self-fertility was significantly lower in the mutant plants. The results showed that the Ntms1 gene plays an important role in regulating fertility in tobacco.

Genetic Mapping of ms1s, a Recessive Gene for Male Sterility in Common Wheat.

The utilization of heterosis is an important way to improve wheat yield, and the production of wheat hybrid seeds mainly relies on male-sterile lines. Male sterility in line 15 Fan 03 derived from a cross of 72,180 and Xiaoyan 6 is controlled by a single recessive gene. The gene was mapped to the distal region of chromosome 4BS in a genetic interval of 1.4 cM and physical distance of 6.57 Mb between SSR markers Ms4BS42 and Ms4BS199 using an F2 population with 1205 individuals. Sterile individuals had a deletion of 4.57 Mb in the region presumed to carry the Ms1 locus. The allele for sterility was therefore named ms1s. Three CAPS markers were developed and verified from the region upstream of the deleted fragment and can be used for ms1s marker-assisted selection in wheat hybrid breeding. This work will enrich the utilization of male sterility genetic resources.

Development and utilization of the functional co-dominant KASP marker for thermo-sensitive genic male sterility in rice Oryza sativa L.

Thermo-sensitive genic male sterility (TGMS) is an important genetic means of two-line hybrid rice breeding. Pollen fertility in TGMS lines is regulated by a single point mutation in TGMS genes. Based on single nucleotide polymorphisms (SNPs) and insertion-deletion mutations, Kompetitive Allele-Specific PCR (KASP) markers were developed and utilized in rice molecular breeding via high-throughput detection, which saves time and money. In this study, we converted the SNPs on TGMS genes (including p/tms12-1and tms9-1) to functional co-dominant KASP markers and bred the resultant two-line hybrid rice. We differentiated the TGMS lines carrying p/tms12-1 or tms9-1 from other TGMS lines using the KASP assay. Pei’ai64S and Hua201S (containing p/tms12-1) had a homozygous GG genotype generating a blue signal. HengnongS-1 (containing tms9-1) had a homozygous CC genotype generating a red signal. The KASP assay for tms9-1 was identified as a recessive Mendelian trait. Seed purity was tested using the KASP marker for the two-line hybrid varieties of Liangyoupeijiu and Hualiangyou1206, which was consistent with findings by the dCAPS marker. Moreover, new TGMS lines were generated by pyramiding tms12-1 and tms9-1 gene under the same genetic background. Therefore, the KASP marker to detect the TGMS genes developed in this study can be widely used in two-line hybrid rice breeding. It provides a visually convenient toolkit for breeders to select individual target plants using high-throughput screening in two-line rice breeding.

A single nucleotide polymorphism in an R2R3 MYB transcription factor gene triggers the male sterility in soybean ms6 (Ames1)

Key messageIdentification and functional analysis of the male sterile gene MS6 in Glycine max.Soybean (Glycine max (L.) Merr.) is an important crop providing vegetable oil and protein. The male sterility-based hybrid breeding is a promising method for improving soybean yield to meet the globally growing demand. In this research, we identified a soybean genic male sterile locus, MS6, by combining the bulked segregant analysis sequencing method and the map-based cloning technology. MS6, highly expressed in anther, encodes an R2R3 MYB transcription factor (GmTDF1-1) that is homologous to Tapetal Development and Function 1, a key factor for anther development in Arabidopsis and rice. In male sterile ms6 (Ames1), the mutant allele contains a missense mutation, leading to the 76th leucine substituted by histidine in the DNA binding domain of GmTDF1-1. The expression of soybean MS6 under the control of the AtTDF1 promoter could rescue the male sterility of attdf1 but ms6 could not. Additionally, ms6 overexpression in wild-type Arabidopsis did not affect anther development. These results evidence that GmTDF1-1 is a functional TDF1 homolog and L76H disrupts its function. Notably, GmTDF1-1 shows 92% sequence identity with another soybean protein termed as GmTDF1-2, whose active expression also restored the fertility of attdf1. However, GmTDF1-2 is constitutively expressed at a very low level in soybean, and therefore, not able to compensate for the MS6 deficiency. Analysis of the TDF1-involved anther development regulatory pathway showed that expressions of the genes downstream of TDF1 are significantly suppressed in ms6, unveiling that GmTDF1-1 is a core transcription factor regulating soybean anther development.