Identification of the domestication gene GmCYP82C4 underlying the major quantitative trait locus for the seed weight in soybean.

KEY MESSAGE: A major quantitative trait locus (QTL) for the hundred-seed weight (HSW) was identified and confirmed in the two distinct soybean populations, and the target gene GmCYP82C4 underlying this locus was identified that significantly associated with soybean seed weight, and it was selected during the soybean domestication and improvement process. Soybean is a major oil crop for human beings and the seed weight is a crucial goal of soybean breeding. However, only a limited number of target genes underlying the quantitative trait loci (QTLs) controlling seed weight in soybean are known so far. In the present study, six loci associated with hundred-seed weight (HSW) were detected in the first population of 573 soybean breeding lines by genome-wide association study (GWAS), and 64 gene models were predicted in these candidate QTL regions. The QTL qHSW_1 exhibits continuous association signals on chromosome four and was also validated by region association study (RAS) in the second soybean population (409 accessions) with wild, landrace, and cultivar soybean accessions. There were seven genes in qHSW_1 candidate region by linkage disequilibrium (LD) block analysis, and only Glyma.04G035500 (GmCYP82C4) showed specifically higher expression in flowers, pods, and seeds, indicating its crucial role in the soybean seed development. Significant differences in HSW trait were detected when the association panels are genotyped by single-nucleotide polymorphisms (SNPs) in putative GmCYP82C4 promoter region. Eight haplotypes were generated by six SNPs in GmCYP82C4 in the second soybean population, and two superior haplotypes (Hap2 and Hap4) of GmCYP82C4 were detected with average HSW of 18.27 g and 18.38 g, respectively. The genetic diversity of GmCYP82C4 was analyzed in the second soybean population, and GmCYP82C4 was most likely selected during the soybean domestication and improvement process, leading to the highest proportion of Hap2 of GmCYP82C4 both in landrace and cultivar subpopulations. The QTLs and GmCYP82C4 identified in this study provide novel genetic resources for soybean seed weight trait, and the GmCYP82C4 could be used for soybean molecular breeding to develop desirable seed weight in the future.

Genetic Dissection of Extreme Seed-Flooding Tolerance in a Wild Soybean PI342618B by Linkage Mapping and Candidate Gene Analysis.

Seed-flooding stress is one of major abiotic constraints that adversely affects soybean production worldwide. Identifying tolerant germplasms and revealing the genetic basis of seed-flooding tolerance are imperative goals for soybean breeding. In the present study, high-density linkage maps of two inter-specific recombinant inbred line (RIL) populations, named NJIRNP and NJIR4P, were utilized to identify major quantitative trait loci (QTLs) for seed-flooding tolerance using three parameters viz., germination rate (GR), normal seedling rate (NSR), and electrical conductivity (EC). A total of 25 and 18 QTLs were detected by composite interval mapping (CIM) and mixed-model-based composite interval mapping (MCIM), respectively, and 12 common QTLs were identified through both methods. All favorable alleles for the tolerance are notably from the wild soybean parent. Moreover, four digenic epistatic QTL pairs were identified, and three of them showed no main effects. In addition, the pigmented soybean genotypes exhibited high seed-flooding tolerance compared with yellow seed coat genotypes in both populations. Moreover, out of five identified QTLs, one major region containing multiple QTLs associated with all three traits was identified on Chromosome 8, and most of the QTLs within this hotspot were major loci (R2 > 10) and detectable in both populations and multiple environments. Based on the gene expression and functional annotation information, 10 candidate genes from QTL "hotspot 8-2" were screened for further analysis. Furthermore, the results of qRT-PCR and sequence analysis revealed that only one gene, GmDREB2 (Glyma.08G137600), was significantly induced under flooding stress and displayed a TTC tribasic insertion mutation of the nucleotide sequence in the tolerant wild parent (PI342618B). GmDREB2 encodes an ERF transcription factor, and the subcellular localization analysis using green fluorescent protein (GFP) revealed that GmDREB2 protein was localized in the nucleus and plasma membrane. Furthermore, overexpression of GmDREB2 significantly promoted the growth of soybean hairy roots, which might indicate its critical role in seed-flooding stress. Thus, GmDREB2 was considered as the most possible candidate gene for seed-flooding tolerance.

Global Transcriptome Profiling of Enterobacter Strain NRS-1 in Response to Hydrogen Peroxide Stress Treatment.

Microbes are often subjected to oxidative stress in nature that badly affects their growth rate and viability. Although the response of microbes against oxidative stress has been characterized at the chemical, physiological, and molecular levels, the mechanism of gene-regulation network adaptations of bacteria in response to oxidative stress remains largely unknown. In this study, transcriptomic profiling of glyphosate-tolerant Enterobacter strain NRS-1 was analyzed under 9 mM H2O2 stress using RNA-seq and qRT-PCR. The lag period in the growth of NRS-1 was very short compared with wild-type strain under H2O2 treatment. A total of 113 genes are identified as differentially expressed genes (DEGs) under H2O2 that include 38 upregulated and 75 downregulated transcripts. But not any genes regulated by major oxidative regulons, viz., oxyR, soxR, rpoS, perR, ohrR, and σв, have been reported in DEGs, hence potentially reflecting that specific changes have occurred in NRS-1 for adaptation to oxidative stress. Based on the functions of the DEGs, six elements namely formate dehydrogenase, processes associated with iron ions, repair programs, multidrug resistance, antioxidant defense, and energy generation (mqo, sdhC) might have contributed for stress tolerance in NRS-1. These elements are proposed to form a molecular network explaining gene response of NRS-1 to stress, and ensure global cell protection and growth recovery of NRS-1. These findings enrich the view of gene regulation in bacteria in response to H2O2 oxidative stress.

Complex gene response of herbicide-resistant Enterobacter strain NRS-1 under different glyphosate stresses.

Knowledge of biological evolution and genetic mechanisms is gained by studying the adaptation of bacteria to survive in adverse environmental conditions. In this regard, transcriptomic profiling of a glyphosate-tolerant Enterobacter strain NRS-1 was studied under four different treatments to investigate the gene-regulatory system for glyphosate tolerance. A total of 83, 83, 60 and 74 genes were up-regulated and 108, 87, 178 and 117 genes down-regulated under 60-NPG, 110-NPG, NaCl (355 mM) and HCl (pH 4.46) stress treatments, respectively. Complex gene network was identified to be involved in regulating tolerance to glyphosate. This study revealed that NRS-1 has gained glyphosate tolerance at the cost of osmotic and acidic resistance. The 25 differentially expressed genes are reported to may have partly changed the function for providing resistance to glyphosate directly, among them genes metK, mtbK, fdnG and wzb that might detoxify/degrade the glyphosate. However, under 110-NPG condition, NRS-1 might have utilized economical and efficient ways by depressing its metabolism and activity to pass through this stress. Hence, the present study provides insights into the genes involved in glyphosate tolerance, which can be effectively utilized to engineer herbicide-resistant crop varieties after their proper validation to manage weed growth.

Knockdown of Decoy Receptor 3 Impairs Growth and Invasiveness of Hepatocellular Carcinoma Cell Line of HepG2.

BACKGROUND: Decoy receptor 3 (DcR3) binds to Fas ligand (FasL) and inhibits FasL-induced apoptosis. The receptor is overexpressed in hepatocellular carcinoma (HCC), and it is associated with the growth and metastatic spread of tumors. DcR3 holds promises as a new target for the treatment of HCC, but little is known regarding the molecular mechanisms underlying the oncogenic properties of DcR3. The present work, therefore, examined the role of DcR3 in regulating the growth and invasive property of liver cancer cell HepG2. METHODS: HepG2 cells were stably transfected with lentivirus-based short hairpin RNA vector targeting DcR3. After the knockdown of DcR3 was confirmed, cell proliferation, clone formation, ability of migrating across transwell membrane, and wound healing were assessed in vitro. Matrix metalloproteinase-9 (MMP 9) and vascular epithelial growth factor (VEGF)-C and D expressions of the DcR3 knockdown were also studied. Comparisons between multiple groups were done using one-way analysis of variance (ANOVA), while pairwise comparisons were performed using Student's t test. P< 0.05 was regarded statistically significant. RESULTS: DcR3 was overexpressed in HepG2 compared to other HCC cell lines and normal hepatocyte Lo-2. Stable knockdown of DcR3 slowed down the growth of HepG2 (P < 0.05) and reduced the number of clones formed by 50% compared to those without DcR3 knockdown (P < 0.05). The knockdown also reduced the migration of HepG2 across transwell matrix membrane by five folds compared to the control (P < 0.05) and suppressed the closure of scratch wound (P < 0.05). In addition, the messenger RNA levels of MMP 9, VEGF-C, and VEGF-D were significantly suppressed by DcR3 knockdown by 90% when compared with the mock control (P < 0.05). CONCLUSIONS: Loss of DcR3 impaired the growth and invasive property of HCC cell line of HepG2. Targeting DcR3 may be a potential therapeutic approach for the treatment of HCC.

Marker-assisted breeding for transgressive seed protein content in soybean [Glycine max (L.) Merr].

KEY MESSAGE: After two cycles of marker-assisted breeding on three loci, lines with transgressive segregation of 8.22-9.32 % protein content were developed based on four original soybean parents with 35.35-44.83 % protein content. Marker-assisted breeding has been an innovative approach in conventional breeding, which is to be further demonstrated, especially for quantitative traits. A study on continuous transgressive breeding for seed protein content (SPC) in soybean using marker-assisted procedures is reported here. The SPC of the recombinant inbred line (RIL) population XG varied in 38.04-47.54 % under five environments with P 1 of 35.35 %, P 2 of 44.34 % and total heritability of 89.11 %. A transgressive segregant XG30 with SPC 45.53 % was selected for further improvement. The linkage mapping of XG showed its genetic constitution composed of five additive QTL (32.16 % of phenotypic variation or PV) and two pairs of epistatic QTL (2.96 % PV) using 400 SSR markers with the remnant heritability 53.99 % attributed to the undetected collective of minor QTL. Another transgressive segregant WT133 with SPC 48.39 % was selected from the RIL population WT (44.83 % SPC for both parents). XG30 and WT133 were genotyped on the three major additive QTL (Prot-08-1, Prot-14-1 and Prot-19-2) as A 2 A 2 B 2 B 2 L 1 L 1 and A 1 A 1 B 1 B 1 L 2 L 2 , respectively. From WT133×XG30, surprising transgressive progenies were obtained, among which the recombinants with all three positive alleles A 2 _B 2 _L 2 _ performed the highest SPC, especially that of Prot-08-1. The five F 2-derived superior families showed their means higher than the high parent value in F 2:3 and F 2:4 and more transgressive effect in F 2:5:6, with the highest as high as 54.15 %, or 4.82 and 9.32 % more than WT133 and its original high parent, respectively. This study demonstrated the efficiency of marker-assisted procedure in breeding for transgressive segregation of quantitative trait.

Identification of regulated genes conferring resistance to high concentrations of glyphosate in a new strain of Enterobacter.

Glyphosate is a widely used herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity. Most plants and microbes are sensitive to glyphosate. However, transgenic-resistant crops that contain a modified epsps obtained from the resistant microbes have been commercially successful and therefore, new resistance genes and their adaptive regulatory mechanisms are of great interest. In this study, a soil-borne, glyphosate-resistant bacterium was selected and identified as Enterobacter. The EPSPS in this strain was found to have been altered to a resistant one. A total of 42 differentially expressed genes (DEGs) in the glyphosate were screened using microarray techniques. Under treatment, argF, sdhA, ivbL, rrfA-H were downregulated, whereas the transcripts of speA, osmY, pflB, ahpC, fusA, deoA, uxaC, rpoD and a few ribosomal protein genes were upregulated. Data were verified by quantitative real-time PCR on selected genes. All transcriptional changes appeared to protect the bacteria from glyphosate and associated osmotic, acidic and oxidative stresses. Many DEGs may have the potential to confer resistance to glyphosate alone, and some may be closely related to the shikimate pathway, reflecting the complex gene interaction network for glyphosate resistance.

Genome-wide identification and transcription analysis of soybean carotenoid oxygenase genes during abiotic stress treatments.

Carotenoid oxygenase is a key enzyme in carotenoid metabolism leading to the synthesis of two phytohormones, abscisic acid (ABA) and strigolactone, as well as norisoprenoids. Few studies have analyzed inter-relationship of the metabolic networks of these three substances. In this present paper, soybean carotenoid oxygenase genes were identified to reveal their phylogenetic relationships, and the transcriptional response of these genes to four abiotic stresses (NaCl, PEG, high and low temperature) and ABA treatment were investigated to characterize their potential roles in plant resistance. Positive selection was found in the branches of carotenoid cleavage dioxygenase (CCD1), CCD8 and NCED (9-cis-epoxycarotenoid oxygenase), indicating an adaptive evolution in these clades. In soybean eight carotenoid oxygenase genes were identified. The transcriptional responses of almost all of them under stress and ABA conditions were significantly altered when assessed by quantitative polymerase chain reaction. Notably, CCD1 and CCD4, previously known as the key genes in norisoprenoids metabolism, showed especially strong responses to the abiotic stresses and ABA treatment. Furthermore, transcription levels of CCD7 and CCD8, key genes for the strigolactone pathway, highly increased during ABA treatment providing further evidence that ABA is involved in regulating strigolactone metabolism. All of the carotenoid oxygenase genes in soybean are involved in plant abiotic stress physiology, and ABA is presumed to be a core regulatory substance. These findings provide some insights into the mechanisms that underlie the regulation of tolerance response to abiotic stresses in soybean.

[Genetic analysis and RAPD marker of the genes for brachytic stem trait in soybean].

Three crosses between NG94-156 (brachytic stem) and three varieties (normal stem) were made, and F2 segregative population and two recombined inbred line populations(F(7:8)) were obtained. Genetic analysis indicated that the brachytic stem of NG94-156 was controlled by two duplicate recessive genes. In searching for RAPD marker linked to the genes controlling brachytic stem, 260 RAPD primers were applied to screen four parents of three combinations and RIL. Polymorphic bands revealed by the primer S-506 exhibited the best repeatability among all primers. Linkage analysis indicated the genetic distance between S-506(1600) and brachytic stem gene was 6.94 cM.