Hybrid Males Research Articles

Avian Introgression Patterns are Consistent With Haldane's Rule.

According to Haldane’s Rule, the heterogametic sex will show the greatest fitness reduction in a hybrid cross. In birds, where sex is determined by a ZW system, female hybrids are expected to experience lower fitness compared to male hybrids. This pattern has indeed been observed in several bird groups, but it is unknown whether the generality of Haldane’s Rule also extends to the molecular level. First, given the lower fitness of female hybrids, we can expect maternally inherited loci (i.e., mitochondrial and W-linked loci) to show lower introgression rates than biparentally inherited loci (i.e., autosomal loci) in females. Second, the faster evolution of Z-linked loci compared to autosomal loci and the hemizygosity of the Z-chromosome in females might speed up the accumulation of incompatible alleles on this sex chromosome, resulting in lower introgression rates for Z-linked loci than for autosomal loci. I tested these expectations by conducting a literature review which focused on studies that directly quantified introgression rates for autosomal, sex-linked, and mitochondrial loci. Although most studies reported introgression rates in line with Haldane’s Rule, it remains important to validate these genetic patterns with estimates of hybrid fitness and supporting field observations to rule out alternative explanations. Genomic data provide exciting opportunities to obtain a more fine-grained picture of introgression rates across the genome, which can consequently be linked to ecological and behavioral observations, potentially leading to novel insights into the genetic mechanisms underpinning Haldane’s Rule.

Occasional paternal inheritance of the germline-restricted chromosome in songbirds

Songbirds have one special accessory chromosome, the so-called germline-restricted chromosome (GRC), which is only present in germline cells and absent from all somatic tissues. Earlier work on the zebra finch (Taeniopygia guttata castanotis) showed that the GRC is inherited only through the female line-like the mitochondria-and is eliminated from the sperm during spermatogenesis. Here, we show that the GRC has the potential to be paternally inherited. Confocal microscopy using GRC-specific fluorescent in situ hybridization probes indicated that a considerable fraction of sperm heads (1 to 19%) in zebra finch ejaculates still contained the GRC. In line with these cytogenetic data, sequencing of ejaculates revealed that individual males from two families differed strongly and consistently in the number of GRCs in their ejaculates. Examining a captive-bred male hybrid of the two zebra finch subspecies (T. g. guttata and T. g. castanotis) revealed that the mitochondria originated from a castanotis mother, whereas the GRC came from a guttata father. Moreover, analyzing GRC haplotypes across nine castanotis matrilines, estimated to have diverged for up to 250,000 y, showed surprisingly little variability among GRCs. This suggests that a single GRC haplotype has spread relatively recently across all examined matrilines. A few diagnostic GRC mutations that arose since this inferred spreading suggest that the GRC has continued to jump across matriline boundaries. Our findings raise the possibility that certain GRC haplotypes could selfishly spread through the population via occasional paternal transmission, thereby outcompeting other GRC haplotypes that were limited to strict maternal inheritance, even if this was partly detrimental to organismal fitness.

Strain differences in post-castration sexual and aggressive behavior in male mice

The degree to which male sexual behavior and territorial aggression are regulated by gonadal steroid hormones depends strongly on species and experience. While castration abolishes male sexual behavior in most laboratory rodents, approximately one third of B6D2F1 mice retain the full repertoire of male sexual behaviors long term (“maters”). It is not yet known whether maters retain other behaviors that typically rely on gonadal steroids to a greater extent than non-maters. In this study, we tested aggressive behavior in B6D2F1 males and males of each parental strain (C57BL/6J and DBA/2J) in the resident intruder paradigm before and after castration, as well as male sexual behavior after castration. Before castration, B6D2F1 residents displayed more attacks compared to DBA/2J males (p < 0.05). There was no difference in attack frequency between B6D2F1 and C57BL/6J males nor between DBA/2J and C57BL/6J males (p > 0.2). A greater proportion of hybrid males demonstrated intromissions and the ejaculatory reflex compared to males of either parental strain (p < 0.01). After castration, B6D2F1 residents attacked more than C57BL/6J males, but not DBA/2 J males (p < 0.05; p > 0.2). There was no difference in post-castration attack frequency between maters and non-maters (p > 0.7). Finally, residents that attacked during all 3 pre-castration resident intruder tests displayed more attacks post-castration than animals that attacked during 1 pre-castration test (p < 0.05). These data suggest that strain and experience influence the expression of aggressive behavior after castration and warrant future study in experience-induced transient increases in extragonadal testosterone.

Applicability analysis and integrated optimization of an overhead airbag for full frontal crash

This research aims to analyze and optimize the performance of a driver side overhead airbag designed for the intelligent cockpit. First, the basic restraint system simulation model for 50 km/h full frontal crash was established and validated. Second, the restraint system model with overhead airbag for the intelligent cockpit was built based on the validated model, the kinematics and injury indexes for both Hybrid III 50th male and Hybrid III 05th female dummies were analyzed to evaluate the applicability of the overhead airbag for different size dummies. Finally, taking into account both Hybrid III 50th male dummy and Hybrid III 05th female dummy, the comprehensive weighed injury criterion (WIC) was set as the optimization goal. The DACE-Kriging model was constructed and the MOGA-II genetic algorithm was introduced for integrated optimization. The simulation results indicated that the overhead airbag could satisfy the requirements of different dummies among the full frontal crash. The optimized restraint system with overhead airbag had improved the overall protection performance for male and female dummies. The WIC values for Hybrid III 50th male dummy and Hybrid III 05th female dummy were reduced by 16.67% and 12.5% respectively. The comprehensive weighed injury criterion WIC was decreased by 15.52% after the integrated optimization.

Combining ability and gene action studies in cytoplasmic male sterility-based hybrids in cauliflower

Combining ability and gene action studies in cytoplasmic male sterility-based hybrids in cauliflower

Precocial male maturation contributes to the introgression of farmed Atlantic salmon into wild populations

The escape of domesticated Atlantic salmon Salmo salar from aquaculture facilities represents a continued threat to the genetic and demographic stability of wild salmon stocks. Escaped farm-origin salmon have been shown to hybridize with wild conspecifics, yet the long-term genetic impacts are generally unknown. Theoretically, life history variation, specifically precocial maturation of male hybrids, could fast-track introgression, but evidence for this has been sparse. Here, we used empirical and experimental data to examine the role of precocious male maturation in introgression. We examined hybrid class composition using juvenile sampling and genetic assignment over a 5 yr period following an escape event in southern Newfoundland, a region with high rates of natural male precocial maturation. Initially following the escape, the proportion of first-generation (F1) hybrids was high and then decreased annually, contrasting the proportion of backcross wild individuals, which increased over time. The presence and temporal distribution of backcross wild individuals supports the hypothesis that high rates of precocial maturation can fast-track the long-term impacts of escaped farmed salmon on wild populations via interbreeding. This was experimentally tested using lab-created wild, farmed, and F1 parr released and recaptured from a Newfoundland river to assess levels of precocious male maturation. Observed rates of maturation of F1 hybrids were high and similar to wild parr (77 vs. 73%, respectively), indicating that most F1 hybrids mature precocially. Our results suggest that rates of precocial male maturation should be explicitly considered in risk assessments evaluating the genetic impacts of escaped farmed salmon on wild populations.

Evaluation of Crossability between Nicotiana benthamiana and Nicotiana excelsior

Wild tobacco species in the Nicotiana section Suaveolentes are promising genetic resources to introduce their disease resistance to cultivated tobacco, Nicotiana tabacum. However, hybrid lethality is observed in hybrid seedlings from crosses between most Suaveolentes species and N. tabacum. In particular, N. benthamiana belonging to the section Suaveolentes produces only viable hybrids after crossing with N. tabacum. In the present study, crossability between N. benthamiana and N. excelsior (section Suaveolentes) was investigated to test the possible usefulness of N. benthamiana as the bridge parent to transfer desirable genes of N. excelsior to N. tabacum via bridge crossing. After reciprocal crosses using three accessions of N. benthamiana and N. excelsior each, several crossing barriers such as cross-incompatibility, seed abortion, and male and female hybrid sterility were observed. Although reciprocal hybrids between N. benthamiana and N. excelsior showed a high degree of chromosome pairing in meiosis, univalents and multivalents, as well as chromosome bridges and lagging chromosomes, were observed. These meiotic abnormalities were thought to cause hybrid sterility. The possible usefulness of reciprocal hybrids between N. benthamiana and N. excelsior is discussed.

B6D2F1 mice that retain sexual behavior long term after castration outperform those that cease in the radial arm maze

In rodents, gonadal steroids play a critical yet variable role in behaviors such as social interaction and cognitive performance. Gonadal steroids organize sex differences observed in spatial working memory, while the absence of activational effects induced by castration generally impedes spatial learning and memory. Although male sexual behavior is typically inhibited following castration, a significant proportion of gonadectomized B6D2F1 hybrid males retains the complete repertoire of male reproductive behavior. In a prior study, amyloid precursor protein and tau, proteins involved in cognitive behavior, facilitated steroid-independent male sex behavior in B6D2F1 hybrid male mice. We used this strain to investigate the relationship between gonadal steroid-independent male sexual behavior and cognition. After identifying “maters” (animals retaining steroid-independent male sex behavior) and “non-maters,” we tested spatial memory in an 8-arm radial arm maze. Although neither group demonstrated a decrease in errors as a function of time, maters committed fewer errors compared to non-maters overall (p < 0.05). Maters also completed the maze more quickly than non-maters (p < 0.05). We measured mRNA expression of APP and MAPT as well as LEPR and D2R to probe potential roles of metabolism and motivation. Uniquely among maters, increased relative expression of D2R and LEPR in the hippocampus was associated with a longer latency to complete the maze during the last 3 or across all trials, respectively. These data demonstrate that maters outperform non-maters in the radial arm maze, warranting further study of potential differences in acquisition of spatial memory tasks or learning strategy between these groups.

Comprehensive Analysis of mRNA, lncRNA, circRNA, and miRNA Expression Profiles and Their ceRNA Networks in the Longissimus Dorsi Muscle of Cattle-Yak and Yak

Cattle-yak, as the hybrid offspring of cattle (Bos taurus) and yak (Bos grunniens), demonstrates obvious heterosis in production performance. Male hybrid sterility has been focused on for a long time; however, the mRNAs and non-coding RNAs related to muscle development as well as their regulatory networks remain unclear. The phenotypic data showed that the production performance (i.e., body weight, withers height, body length, and chest girth) of cattle-yak was significantly better than that of the yak, and the economic benefits of the cattle-yak were higher under the same feeding conditions. Then, we detected the expression profiles of the longissimus dorsi muscle of cattle-yak and yak to systematically reveal the molecular basis using the high-throughput sequencing technology. Here, 7,126 mRNAs, 791 lncRNAs, and 1,057 circRNAs were identified to be differentially expressed between cattle-yaks and yaks in the longissimus dorsi muscle. These mRNAs, lncRNA targeted genes, and circRNA host genes were significantly enriched in myoblast differentiation and some signaling pathways related to muscle development (such as HIF-1 signaling pathway and PI3K-Akt signaling pathway). We constructed a competing endogenous RNA (ceRNA) network and found that some non-coding RNAs differentially expressed may be involved in the regulation of muscle traits. Taken together, this study may be used as a reference tool to provide the molecular basis for studying muscle development.

Aberrant levels of DNA methylation and H3K9 acetylation in the testicular cells of crossbred cattle-yak showing infertility.

Although the interspecies hybridization of bovids, such as cattle-yak (Bos taurus × Bos grunniens), has heterosis benefits, the infertility of hybrid males affects the maintenance of dominant traits in subsequent generations. To achieve reproductive capacity, male germ cell development requires coordinated changes in gene expression, including DNA methylation and generalized histone modifications. Although gene expression-related mechanisms underlying hybrid male sterility have been investigated recently, information on the cell types and stage-specific controls remains limited. Here, we used immunohistochemistry and image analyses to evaluate the 5-methylcytosine (5MC) and acetyl-histone H3 Lys9 (AcK9) expression in all spermatogonia and testicular somatic cell types to determine their roles in cattle-yak spermatogenesis. Testicular tissues from yak (1-3years old) and backcrossed hybrids (2years old) were used. In yak, the AcK9 expression levels increased in all cell types during maturation, but the 5MC expression levels did not change until reaching 3years when they increased in all testicular cell types, except spermatogonia. Cattle-yak hybrids showed higher 5MC expression levels and different AcK9 expression levels in all cell types compared to the same-aged yak. These results suggested that both gene modulation by AcK9 and constant levels of DNA methylation are required for spermatogenesis during maturation in yak. Therefore, inappropriate expression levels of both AcK9 and DNA methylation might be the major factors for disruption of normal germ cell development in cattle-yak. Additionally, various modulations occurred depending on the cell type. Further experiments are needed to identify the stage-specific gene expression modulations in each cell type in yak and cattle-yak to potentially solve the infertility issue in crossbreeding.

Stage-specific disruption of X chromosome expression during spermatogenesis in sterile house mouse hybrids.

Hybrid sterility is a complex phenotype that can result from the breakdown of spermatogenesis at multiple developmental stages. Here, we disentangle two proposed hybrid male sterility mechanisms in the house mice, Mus musculus domesticus and M. m. musculus, by comparing patterns of gene expression in sterile F1 hybrids from a reciprocal cross. We found that hybrid males from both cross directions showed disrupted X chromosome expression during prophase of meiosis I consistent with a loss of meiotic sex chromosome inactivation (MSCI) and Prdm9-associated sterility, but that the degree of disruption was greater in mice with an M. m. musculus X chromosome consistent with previous studies. During postmeiotic development, gene expression on the X chromosome was only disrupted in one cross direction, suggesting that misexpression at this later stage was genotype-specific and not a simple downstream consequence of MSCI disruption which was observed in both reciprocal crosses. Instead, disrupted postmeiotic expression may depend on the magnitude of earlier disrupted MSCI, or the disruption of particular X-linked genes or gene networks. Alternatively, only hybrids with a potential deficit of Sly copies, a Y-linked ampliconic gene family, showed overexpression in postmeiotic cells, consistent with a previously proposed model of antagonistic coevolution between the X- and Y-linked ampliconic genes contributing to disrupted expression late in spermatogenesis. The relative contributions of these two regulatory mechanisms and their impact on sterility phenotypes await further study. Our results further support the hypothesis that X-linked hybrid sterility in house mice has a variable genetic basis, and that genotype-specific disruption of gene regulation contributes to overexpression of the X chromosome at different stages of development. Overall, these findings underscore the critical role of epigenetic regulation of the X chromosome during spermatogenesis and suggest that these processes are prone to disruption in hybrids.

Rapid Divergence of Key Spermatogenesis Genes in nasuta-Subgroup of Drosophila.

The crosses between closely relatedDrosophilaspecies usually produce sterile hybrid males with spermatogenesis disrupted at post-meiotic phase, especially in sperm individualization stage than the pre-meiotic stage. This is possibly due to the rapid interspecies divergence of male sex and reproduction-related genes. Here we annotated 11 key spermatogenesis genes in 35 strains of species belonging tonasuta-subgroup ofDrosophila, where many interspecies crosses produce sterile males. We characterized the divergence and polymorphism in the protein coding regions by employing gene-wide, codon-wide, and lineage-specific selection analysis to test the mode and strength of selection acting on these genes.Our analysis showed signature of positive selection at bag of marbles (bam)andbenign gonial cell neoplasma (bgcn)despite the selection constrains and the absence of endosymbiont infection which could potentially drive rapid divergence due to an arms race whileroughex (rux)showed lineage-specific rapid divergence in frontal sheen complex ofnasuta-subgroup.cookie monster (comr)showed rapid divergence consistent with the possibility of meiotic arrest observed in sterile hybrids ofDrosophilaspecies. Rapid divergence observed atdon juan (dj) and Mst98Ca-likewas consistent with fused sperm-tail abnormality observed in the hybrids ofDrosophila nasutaandDrosophila albomicans. These findings highlight the potential role of rapid nucleotide divergence in bringing about hybrid incompatibility in the form of male sterility; however, additional genetic manipulation studies can widen our understanding of hybrid incompatibilities. Furthermore, our study emphasizes the importance of young species belonging tonasuta-subgroup ofDrosophilain studying post-zygotic reproductive isolation mechanisms.

Banding data show hummingbirds have high rates of hybridization

Abstract We estimate hybridization rates among hummingbirds using nearly a million banding records from the United States and Canada. Annually from 2006 to 2019, an average of 44,600 individual hummingbirds and 14 hybrids were banded. Nearly all reports of hybrids come from localities west of the Mississippi, where multiple species breed in sympatry, whereas only Ruby-throated Hummingbird (Archilochus colubris) breeds east of the Mississippi. Adult male hybrids comprise 62% of all hybrids banded, a significantly greater fraction than “regular” adult males, which are 29% of all birds banded (excluding Ruby-throated Hummingbird). We infer that this excess of adult male hybrids is caused by ascertainment bias: banders more often misidentify female hybrids as parental species because females mostly lack species-specific showy sexual ornaments of male hummingbirds, making them harder to identify, rather than Haldane’s rule of reduced survivorship of the heterogametic sex. Also influencing the apparent hybridization rate are banders, a few of whom seek out or avoid hybrids. After considering these biases, the data suggest that, in areas of the United States and Canada with &amp;gt;1 species, approximately 1 hummingbird in a thousand (0.1%) is an F1 hybrid.

Revisiting the hybridization processes in the Triatoma brasiliensis complex (Hemiptera, Triatominae): Interspecific genomic compatibility point to a possible recent diversification of the species grouped in this monophyletic complex.

Triatomines are hematophagous insects of great epidemiological importance, since they are vectors of the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. Triatoma brasiliensis complex is a monophyletic group formed by two subspecies and six species: T. b. brasiliensis, T. b. macromelasoma, T. bahiensis, T. juazeirensis, T. lenti, T. melanica, T. petrocchiae and T. sherlocki. The specific status of several species grouped in the T. brasiliensis complex was confirmed from experimental crossing and analysis of reproductive barriers. Thus, we perform interspecific experimental crosses between T. lenti and other species and subspecies of the T. brasiliensis complex and perform morphological analysis of the gonads and cytogenetic analysis in the homeologous chromosomes of the hybrids of first generation (F1). Besides that, we rescue all the literature data associated with the study of reproductive barriers in this monophyletic complex of species and subspecies. For all crosses performed between T. b. brasiliensis, T. b. macromelasoma, T. juazeirensis and T. melanica with T. lenti, interspecific copulas occurred (showing absence of mechanical isolation), hybrids were obtained, none of the male hybrids presented the phenomenon of gonadal dysgenesis and 100% pairing between the chromosomes homeologous of the hybrids was observed. Thus, we demonstrate that there are no pre-zygotic reproductive barriers installed between T. lenti and the species and subspecies of the T. brasiliensis complex. In addition, we demonstrate that the hybrids obtained between these crosses have high genomic compatibility and the absence of gonadal dysgenesis. These results point to reproductive compatibility between T. lenti and species and subspecies of the T. brasiliensis complex (confirming its inclusion in the complex) and lead us to suggest a possible recent diversification of the taxa of this monophyletic group.

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.

Fertility investigation in F1 hybrid and karyotype analysis of backcross progeny from Misgurnus anguillicaudatus and Paramisgurnus dabryanus

Dojo loach (Misgurnus anguillicaudatus) (DD, 2n = 50) and large-scale loach (Paramisgurnus dabryanus) (PP, 2n = 48) are freshwater fish species widely distributed across East Asia. Hybrids of these two loach species have disrupted gonadal development, but few studies have reported the actual fertility and chromosome number in gametes of reciprocal hybrid loaches (DD × PP and PP × DD, assigned as DP and PD, 2n = 49). In this study, selfing and backcrossing experiments between M. anguilicaudatus and P. dabryanus were conducted. The fertilization, hatching, deformity, and survival rates were examined along with the embryonic development and growth performance to analyze the actual fertility of the hybrid loach. The karyotypes of viable backcross offspring were studied to indirectly assess the chromosome number in eggs of DP and PD hybrids. The failure in fertilization among crosses using male hybrid proved that the male hybrid was sterile. The moderate fertilization rates (11.49%‐–49.16%), high hatching rates (82.78%–86.31%), high survival rates (78.67%–81.33%), and the normal embryonic development among crosses using female hybrid revealed that the female hybrid was fertile, albeit with relatively high deformity rates (12.68%–27.45%). The karyotype analysis of viable backcross offspring showed 49 chromosomes for DP × DD (assigned as DPD) and PD × DD (assigned as PDD) hybrids, and 48 chromosomes for DP × PP (assigned as DPP) and PD × PP (assigned as PDP) hybrids. This indicated that fertile gametes produced by female hybrid loach contained 24 chromosomes. This study provides valuable information for loach hybrid breeding and in-depth research on the precise mechanism of meiosis in these hybrid loach.

Evaluation of male industrial hybrids of silkworm

Evaluation of male industrial hybrids of silkworm

Altering the Binding Properties of PRDM9 Partially Restores Fertility across the Species Boundary.

Sterility or subfertility of male hybrid offspring is commonly observed. This phenomenon contributes to reproductive barriers between the parental populations, an early step in the process of speciation. One frequent cause of such infertility is a failure of proper chromosome pairing during male meiosis. In subspecies of the house mouse, the likelihood of successful chromosome synapsis is improved by the binding of the histone methyltransferase PRDM9 to both chromosome homologs at matching positions. Using genetic manipulation, we altered PRDM9 binding to occur more often at matched sites, and find that chromosome pairing defects can be rescued, not only in an intersubspecific cross, but also between distinct species. Using different engineered variants, we demonstrate a quantitative link between the degree of matched homolog binding, chromosome synapsis, and rescue of fertility in hybrids between Mus musculus and Mus spretus. The resulting partial restoration of fertility reveals additional mechanisms at play that act to lock-in the reproductive isolation between these two species.

Genetic, morphometric, and molecular analyses of interspecies differences in head shape and hybrid developmental defects in the wasp genus Nasonia.

Males in the parasitoid wasp genus Nasonia have distinct, species-specific, head shapes. The availability of fertile hybrids among the species, along with obligate haploidy of males, facilitates analysis of complex gene interactions in development and evolution. Previous analyses showed that both the divergence in head shape between Nasonia vitripennis and Nasonia giraulti, and the head-specific developmental defects of F2 haploid hybrid males, are governed by multiple changes in networks of interacting genes. Here, we extend our understanding of the gene interactions that affect morphogenesis in male heads. Use of artificial diploid male hybrids shows that alleles mediating developmental defects are recessive, while there are diverse dominance relationships among other head shape traits. At the molecular level, the sex determination locus doublesex plays a major role in male head shape differences, but it is not the only important factor. Introgression of a giraulti region on chromsome 2 reveals a recessive locus that causes completely penetrant head clefting in both males and females in a vitripennis background. Finally, a third species (N. longicornis) was used to investigate the timing of genetic changes related to head morphology, revealing that most changes causing defects arose after the divergence of N. vitripennis from the other species, but prior to the divergence of N. giraulti and N. longicornis from each other. Our results demonstrate that developmental gene networks can be dissected using interspecies crosses in Nasonia, and set the stage for future fine-scale genetic dissection of both head shape and hybrid developmental defects.

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.