A natural non-synonymous single nucleotide polymorphism in GmbHLH113 negates its inhibitory effect on root hair elongation in soybean.

Root hair length (RHL) is an important character that affects nutrient acquisition in plants. The regulatory network in soybean controlling RHL is yet to be fully understood. In this study, we identified a quantitative trait locus (QTL) regulating RHL. One candidate causal gene in this QTL (GmbHLH113), preferentially expressed in root hairs, was annotated as encoding a basic helix-loop-helix transcription factor. In wild soybeans, the allelic type of GmbHLH113 with a glycine in the 13th residue, which was associated with a reduction in RHL, was shown to localize in the nucleus and activate gene transcription. Another allelic type with a single nucleotide polymorphism that resulted in a glutamate in the 13th residue is fixed in cultivated soybeans, and it lost the ability to localize to the nucleus or negatively regulate RHL. The ectopic expression of GmbHLH113 from W05 in Arabidopsis root hairs resulted in shorter RHL and reduced phosphorus (P) accumulation in shoots. Hence, a loss-of-function allele in cultivated soybeans might have been selected during domestication due to its association with a longer RHL and improved nutrient acquisition.

Developing an SNP dataset for efficiently evaluating soybean germplasm resources using the genome sequencing data of 3,661 soybean accessions.

BACKGROUND: Single nucleotide polymorphism (SNP) markers play significant roles in accelerating breeding and basic crop research. Several soybean SNP panels have been developed. However, there is still a lack of SNP panels for differentiating between wild and cultivated populations, as well as for detecting polymorphisms within both wild and cultivated populations. RESULTS: This study utilized publicly available resequencing data from over 3,000 soybean accessions to identify differentiating and highly conserved SNP and insertion/deletion (InDel) markers between wild and cultivated soybean populations. Additionally, a naturally occurring mutant gene library was constructed by analyzing large-effect SNPs and InDels in the population. CONCLUSION: The markers obtained in this study are associated with numerous genes governing agronomic traits, thus facilitating the evaluation of soybean germplasms and the efficient differentiation between wild and cultivated soybeans. The natural mutant gene library permits the quick identification of individuals with natural mutations in functional genes, providing convenience for accelerating soybean breeding using reverse genetics.

The genome variation and developmental transcriptome maps reveal genetic differentiation of skeletal muscle in pigs.

Natural and artificial directional selections have resulted in significantly genetic and phenotypic differences across breeds in domestic animals. However, the molecular regulation of skeletal muscle diversity remains largely unknown. Here, we conducted transcriptome profiling of skeletal muscle across 27 time points, and performed whole-genome re-sequencing in Landrace (lean-type) and Tongcheng (obese-type) pigs. The transcription activity decreased with development, and the high-resolution transcriptome precisely captured the characterizations of skeletal muscle with distinct biological events in four developmental phases: Embryonic, Fetal, Neonatal, and Adult. A divergence in the developmental timing and asynchronous development between the two breeds was observed; Landrace showed a developmental lag and stronger abilities of myoblast proliferation and cell migration, whereas Tongcheng had higher ATP synthase activity in postnatal periods. The miR-24-3p driven network targeting insulin signaling pathway regulated glucose metabolism. Notably, integrated analysis suggested SATB2 and XLOC_036765 contributed to skeletal muscle diversity via regulating the myoblast migration and proliferation, respectively. Overall, our results provide insights into the molecular regulation of skeletal muscle development and diversity in mammals.

MicroRNA 4407 modulates nodulation in soybean by repressing a root-specific ISOPENTENYLTRANSFERASE (GmIPT3).

MicroRNAs (miRNAs) are important regulators of plant biological processes, including soybean nodulation. One miRNA, miR4407, was identified in soybean roots and nodules. However, the function of miR4407 in soybean is still unknown. MiR4407, unique to soybean, positively regulates lateral root emergence and root structures and represses a root-specific ISOPENTENYLTRANSFERASE (GmIPT3). By altering the expression of miR4407 and GmIPT3, we investigated the role of miR4407 in lateral root and nodule development. Both miR4407 and GmIPT3 are expressed in the inner root cortex and nodule primordia. Upon rhizobial inoculation, miR4407 was downregulated while GmIPT3 was upregulated. Overexpressing miR4407 reduced the number of nodules in transgenic soybean hairy roots while overexpressing the wild-type GmIPT3 or a miR4407-resistant GmIPT3 mutant (mGmIPT3) significantly increased the nodule number. The mechanism of miR4407 and GmIPT3 functions was also linked to autoregulation of nodulation (AON), where miR4407 overexpression repressed miR172c and activated its target, GmNNC1, turning on AON. Exogenous CK mimicked the effects of GmIPT3 overexpression on miR172c, supporting the notion that GmIPT3 regulates nodulation by enhancing root-derived CK. Overall, our data revealed a new miRNA-mediated regulatory mechanism of nodulation in soybean. MiR4407 showed a dual role in lateral root and nodule development.

Comparative analysis of the value of amide proton transfer-weighted imaging and diffusion kurtosis imaging in evaluating the histological grade of cervical squamous carcinoma.

BACKGROUND: Uterine cervical cancer (UCC) was the fourth leading cause of cancer death among women worldwide. The conventional MRI hardly revealing the microstructure information. This study aimed to compare the value of amide proton transfer-weighted imaging (APTWI) and diffusion kurtosis imaging (DKI) in evaluating the histological grade of cervical squamous carcinoma (CSC) in addition to routine diffusion-weighted imaging (DWI). METHODS: Forty-six patients with CSC underwent pelvic DKI and APTWI. The magnetization transfer ratio asymmetry (MTRasym), apparent diffusion coefficient (ADC), mean diffusivity (MD) and mean kurtosis (MK) were calculated and compared based on the histological grade. Correlation coefficients between each parameter and histological grade were calculated. RESULTS: The MTRasym and MK values of grade 1 (G1) were significantly lower than those of grade 2 (G2), and those parameters of G2 were significantly lower than those of grade 3 (G3). The MD and ADC values of G1 were significantly higher than those of G2, and those of G2 were significantly higher than those of G3. MTRasym and MK were both positively correlated with histological grade (r = 0.789 and 0.743, P <  0.001), while MD and ADC were both negatively correlated with histological grade (r = - 0.732 and - 0.644, P <  0.001). For the diagnosis of G1 and G2 CSCs, AUC (APTWI+DKI + DWI) > AUC (DKI + DWI) > AUC (APTWI+DKI) > AUC (APTWI+DWI) > AUC (MTRasym) > AUC (MK) > AUC (MD) > AUC (ADC), where the differences between AUC (APTWI+DKI + DWI), AUC (DKI + DWI) and AUC (ADC) were significant. For the diagnosis of G2 and G3 CSCs, AUC (APTWI+DKI + DWI) > AUC (APTWI+DWI) > AUC (APTWI+DKI) > AUC (DKI + DWI) > AUC (MTRasym) > AUC (MK) > AUC (MD > AUC (ADC), where the differences between AUC (APTWI+DKI + DWI), AUC (APTWI+DWI) and AUC (ADC) were significant. CONCLUSION: Compared with DWI and DKI, APTWI is more effective in identifying the histological grades of CSC. APTWI is recommended as a supplementary scan to routine DWI in CSCs.

Multi-omics analysis reveals the mechanism of seed coat color formation in Brassica rapa L.

KEYMESSAGE: Multi-omics analysis of the transcriptome, metabolome and genome identified major and minor loci and candidate genes for seed coat color and explored the mechanism of flavonoid metabolites biosynthesis in Brassica rapa. Yellow seed trait is considered an agronomically desirable trait with great potential for improving seed quality of Brassica crops. Mechanisms of the yellow seed trait are complex and not well understood. In this study, we performed an integrated metabolome, transcriptome and genome-wide association study (GWAS) on different B. rapa varieties to explore the mechanisms underlying the seed coat color formation. A total of 2,499 differentially expressed genes and 116 differential metabolites between yellow and black seeds with strong association with the flavonoid biosynthesis pathway was identified. In addition, 330 hub genes involved in the seed coat color formation, and the most significantly differential flavonoids biosynthesis were detected based on weighted gene co-expression network analysis. Metabolite GWAS analysis using the contents of 42 flavonoids in developing seeds of 159 B. rapa lines resulted in the identification of 1,626 quantitative trait nucleotides (QTNs) and 37 chromosomal intervals, including one major locus on chromosome A09. A combination of QTNs detection, transcriptome and functional analyses led to the identification of 241 candidate genes that were associated with different flavonoid metabolites. The flavonoid biosynthesis pathway in B. rapa was assembled based on the identified flavonoid metabolites and candidate genes. Furthermore, BrMYB111 members (BraA09g004490.3C and BraA06g034790.3C) involved in the biosynthesis of taxifolin were functionally analyzed in vitro. Our findings lay a foundation and provide a reference for systematically investigating the mechanism of seed coat color in B. rapa and in the other plants.

QTL analyses of soybean root system architecture revealed genetic relationships with shoot-related traits.

KEY MESSAGE: The genetic basis of soybean root system architecture (RSA) and the genetic relationship between shoot and RSA were revealed by integrating data from recombinant inbred population grafting and QTL mapping. Variations in root system architecture (RSA) affect the functions of roots and thus play vital roles in plant adaptations and agricultural productivity. The aim of this study was to unravel the genetic relationship between RSA traits and shoot-related traits in soybean. This study characterized RSA variability at seedling stage in a recombinant inbred population, derived from a cross between cultivated soybean C08 and wild soybean W05, and performed high-resolution quantitative trait locus (QTL) mapping. In total, 34 and 41 QTLs were detected for RSA-related and shoot-related traits, respectively, constituting eight QTL clusters. Significant QTL correspondence was found between shoot biomass and RSA-related traits, consistent with significant correlations between these phenotypes. RSA-related QTLs also overlapped with selection regions in the genome, suggesting the cultivar RSA could be a partial consequence of domestication. Using reciprocal grafting, we confirmed that shoot-derived signals affected root development and the effects were controlled by multiple loci. Meanwhile, RSA-related QTLs were found to co-localize with four soybean flowering-time loci. Consistent with the phenotypes of the parental lines of our RI population, diminishing the function of flowering controlling E1 family through RNA interference (RNAi) led to reduced root growth. This implies that the flowering time-related genes within the RSA-related QTLs are actually contributing to RSA. To conclude, this study identified the QTLs that determine RSA through controlling root growth indirectly via regulating shoot functions, and discovered superior alleles from wild soybean that could be used to improve the root structure in existing soybean cultivars.

Integration of multi-omics data reveals cis-regulatory variants that are associated with phenotypic differentiation of eastern from western pigs.

BACKGROUND: The genetic mechanisms that underlie phenotypic differentiation in breeding animals have important implications in evolutionary biology and agriculture. However, the contribution of cis-regulatory variants to pig phenotypes is poorly understood. Therefore, our aim was to elucidate the molecular mechanisms by which non-coding variants cause phenotypic differences in pigs by combining evolutionary biology analyses and functional genomics. RESULTS: We obtained a high-resolution phased chromosome-scale reference genome with a contig N50 of 18.03 Mb for the Luchuan pig breed (a representative eastern breed) and profiled potential selective sweeps in eastern and western pigs by resequencing the genomes of 234 pigs. Multi-tissue transcriptome and chromatin accessibility analyses of these regions suggest that tissue-specific selection pressure is mediated by promoters and distal cis-regulatory elements. Promoter variants that are associated with increased expression of the lysozyme (LYZ) gene in the small intestine might enhance the immunity of the gastrointestinal tract and roughage tolerance in pigs. In skeletal muscle, an enhancer-modulating single-nucleotide polymorphism that is associated with up-regulation of the expression of the troponin C1, slow skeletal and cardiac type (TNNC1) gene might increase the proportion of slow muscle fibers and affect meat quality. CONCLUSIONS: Our work sheds light on the molecular mechanisms by which non-coding variants shape phenotypic differences in pigs and provides valuable resources and novel perspectives to dissect the role of gene regulatory evolution in animal domestication and breeding.

[Glucosides of chaenomeles speciosa attenuate ischemia/reperfusion-induced brain injury by regulating NF-κB P65/TNF-α in mouse model].

OBJECTIVE: To investigate the effect and mechanism of glucosides of chaenomeles speciosa (GCS) on ischemia/reperfusion-induced brain injury in mouse model. METHODS: Fifty 8-week C57BL/C mice were randomly divided into five groups with 10 in each group:sham group, model group, GCS 30 mg/kg group, GCS 60 mg/kg group and GCS 90 mg/kg group, and the GCS was administrated by gavage (once a day) for 14 d. HE staining was performed to investigate the cell morphology; the Zea-Longa scores were measured for neurological activity; TUNEL staining was performed to investigate the cell apoptosis; ELISA was used to detected the oxidative stress and inflammation; Western Blot was performed to investigate the key pathway and neurological functional molecules. RESULTS: Compared with the sham group, the brain tissues in model group were seriously damaged, presenting severe cell apoptosis, oxidative stress and inflammation, associated with increased NF-κB P65 and TNF-α levels as well as decreased myelin associate glycoprotein (MAG) and oligodendrocyte-myelin glycoprotein (OMgp)levels (all P<0.01). Compared with the model group, the brain tissues in GCS groups were ameliorated, and cell apoptosis, oxidative stress and inflammation were inhibited, associated with decreased NF-κB P65 and TNF-α levels as well as increased MAG and OMgp levels (all P<0.01), which were more markedly in GCS 60 mg/kg group. CONCLUSIONS: GCS can inhibit the NF-κB P65 and TNF-α, reduce the oxidative stress and inflammation, decrease the cell apoptosis in mouse ischemia/reperfusion-induced brain injury model, and 60 mg/kg GCS may be the optimal dose.

Functional identification and regulatory analysis of Δ6-fatty acid desaturase from the oleaginous fungus Mucor sp. EIM-10.

OBJECTIVES: To enlarge the diversity of the desaturases associated with PUFA biosynthesis and to better understand the transcriptional regulation of desaturases, a Δ6-desaturase gene (Md6) from Mucor sp. and its 5'-upstream sequence was functionally identified in Saccharomyces cerevisiae. RESULTS: Expression of the Δ6-fatty acid desaturase (Md6) in S. cerevisiae showed that Md6 could convert linolenic acid to γ-linolenic acid. Computational analysis of the promoter of Md6 suggested it contains several eukaryotic fundamental transcription regulatory elements. In vivo functional analysis of the promoter showed the 5'-upstream sequence of Md6 could initiate expression of GFP and Md6 itself in S. cerevisiae. A series deletion analysis of the promoter suggested that sequence between -919 to -784 bp (relative to start site) named as eMd6 is the key factor for high activity of Δ6-desaturase. The activity of Δ6-desaturase was increased by 2.8-fold and 2.5-fold when the eMd6 sequence was placed upstream of -434 with forward or reverse orientations respectively. CONCLUSION: To our best knowledge, the native promoter of Md6 from Mucor is the strongest promoter for Δ6-desaturase reported so far and the sequence between -919 to -784 bp is an enhancer for Δ6-desaturase activity.

Whole-genome sequencing of cultivated and wild peppers provides insights into Capsicum domestication and specialization.

As an economic crop, pepper satisfies people's spicy taste and has medicinal uses worldwide. To gain a better understanding of Capsicum evolution, domestication, and specialization, we present here the genome sequence of the cultivated pepper Zunla-1 (C. annuum L.) and its wild progenitor Chiltepin (C. annuum var. glabriusculum). We estimate that the pepper genome expanded ∼0.3 Mya (with respect to the genome of other Solanaceae) by a rapid amplification of retrotransposons elements, resulting in a genome comprised of ∼81% repetitive sequences. Approximately 79% of 3.48-Gb scaffolds containing 34,476 protein-coding genes were anchored to chromosomes by a high-density genetic map. Comparison of cultivated and wild pepper genomes with 20 resequencing accessions revealed molecular footprints of artificial selection, providing us with a list of candidate domestication genes. We also found that dosage compensation effect of tandem duplication genes probably contributed to the pungent diversification in pepper. The Capsicum reference genome provides crucial information for the study of not only the evolution of the pepper genome but also, the Solanaceae family, and it will facilitate the establishment of more effective pepper breeding programs.

An ultra-high-density map as a community resource for discerning the genetic basis of quantitative traits in maize.

BACKGROUND: To safeguard the food supply for the growing human population, it is important to understand and exploit the genetic basis of quantitative traits. Next-generation sequencing technology performs advantageously and effectively in genetic mapping and genome analysis of diverse genetic resources. Hence, we combined re-sequencing technology and a bin map strategy to construct an ultra-high-density bin map with thousands of bin markers to precisely map a quantitative trait locus. RESULTS: In this study, we generated a linkage map containing 1,151,856 high quality SNPs between Mo17 and B73, which were verified in the maize intermated B73 × Mo17 (IBM) Syn10 population. This resource is an excellent complement to existing maize genetic maps available in an online database  (iPlant, http://data.maizecode.org/maize/qtl/syn10/ ). Moreover, in this population combined with the IBM Syn4 RIL population, we detected 135 QTLs for flowering time and plant height traits across the two populations. Eighteen known functional genes and twenty-five candidate genes for flowering time and plant height trait were fine-mapped into a 2.21-4.96 Mb interval. Map expansion and segregation distortion were also analyzed, and evidence for inadvertent selection of early flowering time in the process of mapping population development was observed. Furthermore, an updated integrated map with 1,151,856 high-quality SNPs, 2,916 traditional markers and 6,618 bin markers was constructed. The data were deposited into the iPlant Discovery Environment (DE), which provides a fundamental resource of genetic data for the maize genetic research community. CONCLUSIONS: Our findings provide basic essential genetic data for the maize genetic research community. An updated IBM Syn10 population and a reliable, verified high-quality SNP set between Mo17 and B73 will aid in future molecular breeding efforts.

A low-density SNP array for analyzing differential selection in freshwater and marine populations of threespine stickleback (Gasterosteus aculeatus).

BACKGROUND: The threespine stickleback (Gasterosteus aculeatus) has become an important model species for studying both contemporary and parallel evolution. In particular, differential adaptation to freshwater and marine environments has led to high differentiation between freshwater and marine stickleback populations at the phenotypic trait of lateral plate morphology and the underlying candidate gene Ectodysplacin (EDA). Many studies have focused on this trait and candidate gene, although other genes involved in marine-freshwater adaptation may be equally important. In order to develop a resource for rapid and cost efficient analysis of genetic divergence between freshwater and marine sticklebacks, we generated a low-density SNP (Single Nucleotide Polymorphism) array encompassing markers of chromosome regions under putative directional selection, along with neutral markers for background. RESULTS: RAD (Restriction site Associated DNA) sequencing of sixty individuals representing two freshwater and one marine population led to the identification of 33,993 SNP markers. Ninety-six of these were chosen for the low-density SNP array, among which 70 represented SNPs under putatively directional selection in freshwater vs. marine environments, whereas 26 SNPs were assumed to be neutral. Annotation of these regions revealed several genes that are candidates for affecting stickleback phenotypic variation, some of which have been observed in previous studies whereas others are new. CONCLUSIONS: We have developed a cost-efficient low-density SNP array that allows for rapid screening of polymorphisms in threespine stickleback. The array provides a valuable tool for analyzing adaptive divergence between freshwater and marine stickleback populations beyond the well-established candidate gene Ectodysplacin (EDA).

Genomic footprints of speciation in Atlantic eels (Anguilla anguilla and A. rostrata).

The importance of speciation-with-geneflow scenarios is increasingly appreciated. However, the specific processes and the resulting genomic footprints of selection are subject to much discussion. We studied the genomics of speciation between the two panmictic, sympatrically spawning sister species; European (Anguilla anguilla) and American eel (A. rostrata). Divergence is assumed to have initiated more than 3 Ma, and although low gene flow still occurs, strong postzygotic barriers are present. Restriction-site-associated DNA (RAD) sequencing identified 328 300 SNPs for subsequent analysis. However, despite the presence of 3757 strongly differentiated SNPs (FST > 0.8), sliding window analyses of FST showed no larger genomic regions (i.e. hundreds of thousands to millions of bases) of elevated differentiation. Overall FST was 0.041, and linkage disequilibrium was virtually absent for SNPs separated by more than 1000 bp. We suggest this to reflect a case of genomic hitchhiking, where multiple regions are under directional selection between the species. However, low but biologically significant gene flow and high effective population sizes leading to very low genetic drift preclude accumulation of strong background differentiation. Genes containing candidate SNPs for positive selection showed significant enrichment for gene ontology (GO) terms relating to developmental processes and phosphorylation, which seems consistent with assumptions that differences in larval phase duration and migratory distances underlie speciation. Most SNPs under putative selection were found outside coding regions, lending support to emerging views that noncoding regions may be more functionally important than previously assumed. In total, the results demonstrate the necessity of interpreting genomic footprints of selection in the context of demographic parameters and life-history features of the studied species.

Chromosome-level genome assembly and annotation of rare and endangered tropical bivalve, Tridacna crocea.

Tridacna crocea is an ecologically important marine bivalve inhabiting tropical coral reef waters. High quality and available genomic resources will help us understand the population structure and genetic diversity of giant clams. This study reports a high-quality chromosome-scale T. crocea genome sequence of 1.30 Gb, with a scaffold N50 and contig N50 of 56.38 Mb and 1.29 Mb, respectively, which was assembled by combining PacBio long reads and Hi-C sequencing data. Repetitive sequences cover 71.60% of the total length, and a total of 25,440 protein-coding genes were annotated. A total of 1,963 non-coding RNA (ncRNA) were determined in the T. crocea genome, including 62 micro RNA (miRNA), 58 small nuclear RNA (snRNA), 83 ribosomal RNA (rRNA), and 1,760 transfer RNA (tRNA). Phylogenetic analysis revealed that giant clams diverged from oyster about 505.7 Mya during the evolution of bivalves. The genome assembly presented here provides valuable genomic resources to enhance our understanding of the genetic diversity and population structure of giant clams.

Haplotype-resolved assembly of a pig genome using single-sperm sequencing.

Single gamete cell sequencing together with long-read sequencing can reliably produce chromosome-level phased genomes. In this study, we employed PacBio HiFi and Hi-C sequencing on a male Landrace pig, coupled with single-sperm sequencing of its 102 sperm cells. A haplotype assembly method was developed based on long-read sequencing and sperm-phased markers. The chromosome-level phased assembly showed higher phasing accuracy than methods that rely only on HiFi reads. The use of single-sperm sequencing data enabled the construction of a genetic map, successfully mapping the sperm motility trait to a specific region on chromosome 1 (105.40-110.70 Mb). Furthermore, with the assistance of Y chromosome-bearing sperm data, 26.16 Mb Y chromosome sequences were assembled. We report a reliable approach for assembling chromosome-level phased genomes and reveal the potential of sperm population in basic biology research and sperm phenotype research.

Comparative genomic analyses reveal the genetic basis of the yellow-seed trait in Brassica napus.

Yellow-seed trait is a desirable breeding characteristic of rapeseed (Brassica napus) that could greatly improve seed oil yield and quality. However, the underlying mechanisms controlling this phenotype in B. napus plants are difficult to discern because of their complexity. Here, we assemble high-quality genomes of yellow-seeded (GH06) and black-seeded (ZY821). Combining in-depth fine mapping of a quantitative trait locus (QTL) for seed color with other omics data reveal BnA09MYB47a, encoding an R2R3-MYB-type transcription factor, as the causal gene of a major QTL controlling the yellow-seed trait. Functional studies show that sequence variation of BnA09MYB47a underlies the functional divergence between the yellow- and black-seeded B. napus. The black-seed allele BnA09MYB47aZY821, but not the yellow-seed allele BnA09MYB47aGH06, promotes flavonoid biosynthesis by directly activating the expression of BnTT18. Our discovery suggests a possible approach to breeding B. napus for improved commercial value and facilitates flavonoid biosynthesis studies in Brassica crops.

Genome sequencing and population resequencing provide insights into the genetic basis of domestication and diversity of vegetable soybean.

Vegetable soybean is one of the most important vegetables in China, and the demand for this vegetable has markedly increased worldwide over the past two decades. Here, we present a high-quality de novo genome assembly of the vegetable soybean cultivar Zhenong 6 (ZN6), which is one of the most popular cultivars in China. The 20 pseudochromosomes cover 94.57% of the total 1.01 Gb assembly size, with contig N50 of 3.84 Mb and scaffold N50 of 48.41 Mb. A total of 55 517 protein-coding genes were annotated. Approximately 54.85% of the assembled genome was annotated as repetitive sequences, with the most abundant long terminal repeat transposable elements. Comparative genomic and phylogenetic analyses with grain soybean Williams 82, six other Fabaceae species and Arabidopsis thaliana genomes highlight the difference of ZN6 with other species. Furthermore, we resequenced 60 vegetable soybean accessions. Alongside 103 previously resequenced wild soybean and 155 previously resequenced grain soybean accessions, we performed analyses of population structure and selective sweep of vegetable, grain, and wild soybean. They were clearly divided into three clades. We found 1112 and 1047 genes under selection in the vegetable soybean and grain soybean populations compared with the wild soybean population, respectively. Among them, we identified 134 selected genes shared between vegetable soybean and grain soybean populations. Additionally, we report four sucrose synthase genes, one sucrose-phosphate synthase gene, and four sugar transport genes as candidate genes related to important traits such as seed sweetness and seed size in vegetable soybean. This study provides essential genomic resources to promote evolutionary and functional genomics studies and genomically informed breeding for vegetable soybean.

The Poly-Glutamate Motif of GmMATE4 Regulates Its Isoflavone Transport Activity.

Multidrug and toxic compound extrusion (MATE) transporters in eukaryotes have been characterized to be antiporters that mediate the transport of substrates in exchange for protons. In plants, alkaloids, phytohormones, ion chelators, and flavonoids have been reported to be the substrates of MATE transporters. Structural analyses have been conducted to dissect the functional significance of various motifs of MATE proteins. However, an understanding of the functions of the N- and C-termini has been inadequate. Here, by performing phylogenetic analyses and protein sequence alignment of 14 representative plant species, we identified a distinctive N-terminal poly-glutamate motif among a cluster of MATE proteins in soybean. Amongst them, GmMATE4 has the most consecutive glutamate residues at the N-terminus. A subcellular localization study showed that GmMATE4 was localized at the vacuolar membrane-like structure. Protein charge prediction showed that the mutation of the glutamate residues to alanine would reduce the negative charge at the N-terminus. Using yeast as the model, we showed that GmMATE4 mediated the transport of daidzein, genistein, glycitein, and glycitin. In addition, the glutamate-to-alanine mutation reduced the isoflavone transport capacity of GmMATE4. Altogether, we demonstrated GmMATE4 as an isoflavone transporter and the functional significance of the N-terminal poly-glutamate motif of GmMATE4 for regulating the isoflavone transport activity.