Enhancers located in introns are abundant and play a major role in the regulation of gene expression in mammalian species. By contrast, the functions of intronic enhancers in plants have largely been unexplored and only a handful of plant intronic enhancers have been reported. We performed a genome-wide prediction of intronic enhancers in Arabidopsis thaliana using open chromatin signatures based on DNase I sequencing. We identified 941 candidate intronic enhancers associated with 806 genes in seedling tissue and 1,271 intronic enhancers associated with 1,069 genes in floral tissue. We validated the function of 15 of 21 (71%) of the predicted intronic enhancers in transgenic assays using a reporter gene. We also created deletion lines of three intronic enhancers associated with two different genes using CRISPR/Cas. Deletion of these enhancers, which span key transcription factor binding sites, did not abolish gene expression but caused varying levels of transcriptional repression of their cognate genes. Remarkably, the transcriptional repression of the deletion lines occurred at specific developmental stages and resulted in distinct phenotypic effects on plant morphology and development. Clearly, these three intronic enhancers are important in fine-tuning tissue- and development-specific expression of their cognate genes.

Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is one of the most fatal pests of soybean (Glycine max (L.) Merr.) worldwide and causes huge loss of soybean yield each year. Multiple sources of resistance are urgently needed for effective management of SCN via the development of resistant cultivars. The aim of the present study was to investigate the genetic architecture of resistance to SCN HG Type 0 (race 3) and HG Type 1.2.3.5.7 (race 4) in landraces and released elite soybean cultivars mostly from China. A total of 440 diverse soybean landraces and elite cultivars were screened for resistance to SCN HG Type 0 and HG Type 1.2.3.5.7. Exactly 131 new sources of SCN resistance were identified. Lines were genotyped by SNP markers detected by the Specific Locus Amplified Fragment Sequencing (SLAF-seq) approach. A total of 36,976 SNPs were identified with minor allele frequencies (MAF) > 4 % that were present in 97 % of all the genotypes. Genome-wide association mapping showed that a total of 19 association signals were significantly related to the resistance for the two HG Types. Of the 19 association signals, eight signals overlapped with reported QTL including Rhg1 and Rhg4 genes. Another eight were located in the linked regions encompassing known QTL. Three QTL were found that were not previously reported. The average value of female index (FI) of soybean accessions with resistant alleles was significantly lower than those with susceptible alleles for each peak SNP. Disease resistance proteins with leucine rich regions, cytochrome P450s, protein kinases, zinc finger domain proteins, RING domain proteins, MYB and WRKY transcription activation families were identified. Such proteins may participate in the resistant reaction to SCN and were frequently found in the tightly linked genomic regions of the peak SNPs. GWAS extended understanding of the genetic architecture of SCN resistance in multiple genetic backgrounds. Nineteen association signals were obtained for the resistance to the two Hg Types of SCN. The multiple beneficial alleles from resistant germplasm sources will be useful for the breeding of cultivars with improved resistance to SCN. Analysis of genes near association signals may facilitate the recognition of the causal gene(s) underlying SCN resistances.

The hundred seed weight (HSW) is one of the yield components of soybean [Glycine max (L.) Merrill] and is especially critical for various soybean food types. In this study, a representative sample consisting of 185 accessions was selected from Northeast China and analysed in three tested environments to determine the quantitative trait nucleotide (QTN) of HSW through a genome-wide association study (GWAS). A total of 24,180 single nucleotide polymorphisms (SNPs) with minor allele frequencies greater than 0.2 and missing data less than 3% were utilized to estimate linkage disequilibrium (LD) levels in the tested association panel. Thirty-four association signals were identified as associated with HSW via GWAS. Among them, nineteen QTNs were novel, and another fifteen QTNs were overlapped or located near the genomic regions of known HSW QTL. A total of 237 genes, derived from 31 QTNs and located near peak SNPs from the three tested environments in 2015 and 2016, were considered candidate genes, were related to plant growth regulation, hormone metabolism, cell, RNA, protein metabolism, development, starch accumulation, secondary metabolism, signalling, and the TCA cycle, some of which have been found to participate in the regulation of HSW. A total of 106 SNPs from 16 candidate genes were significantly associated with HSW in soybean. The identified loci with beneficial alleles and candidate genes might be valuable for the molecular network and MAS of HSW.

Soybean [Glycine max (L.) Merr.] is an important oil crop that provides valuable resources for human consumption, animal feed, and biofuel. Through the transcriptome analysis in our previous study, GmLecRlk (Glyma.07G005700) was identified as a salt-responsive candidate gene in soybean. In this study, qRT-PCR analysis showed that the GmLecRlk gene expression level was significantly induced by salt stress and highly expressed in soybean roots. The pCAMBIA3300-GmLecRlk construct was generated and introduced into the soybean genome by Agrobacterium rhizogenes. Compared with the wild type (WT), GmLecRlk overexpressing (GmLecRlk-ox) soybean lines had significantly enhanced fresh weight, proline (Pro) content, and catalase (CAT) activity, and reduced malondialdehyde (MDA) and H₂O₂ content under salt stress. These results show that GmLecRlk gene enhanced ROS scavenging ability in response to salt stress in soybean. Meanwhile, we demonstrated that GmLecRlk gene also conferred soybean salt tolerance when it was overexpressed alone in soybean hairy root. Furthermore, the combination of RNA-seq and qRT-PCR analysis was used to determine that GmLecRlk improves the salt tolerance of soybean by upregulating GmERF3, GmbHLH30, and GmDREB2 and downregulating GmGH3.6, GmPUB8, and GmLAMP1. Our research reveals a new mechanism of salt resistance in soybean, which exposes a novel avenue for the cultivation of salt-resistant varieties.

Soybean is sensitive to drought stress, and increasing tolerance to drought stresses is an important target for improving the performance of soybean in the field. The genetic mechanisms underlying soybean’s drought tolerance remain largely unknown. Via a genome-wide association study (GWAS) combined with linkage analysis, we identified 11 single-nucleotide polymorphisms (SNPs) and 22 quantitative trait locus (QTLs) that are significantly associated with soybean drought tolerance. One of these loci, namely qGI10-1, was co-located by GWAS and linkage mapping. The two intervals of qGI10-1 were differentiated between wild and cultivated soybean. A nuclear factor Y transcription factor, GmNFYB17, was located in one of the differentiated regions of qGI10-1 and thus selected as a candidate gene for further analyses. The analysis of 29 homologous genes of GmNFYB17 in soybean showed that most of the genes from this family were involved in drought stress. The over-expression of GmNFYB17 in soybean enhanced drought resistance and yield accumulation. The transgenic plants grew better than control under limited water conditions and showed a lower degree of leaf damage and MDA content but higher RWC, SOD activity and proline content compared with control. Moreover, the transgenic plants showed a fast-growing root system, especially regarding a higher root–top ratio and more branching roots and lateral roots. The better agronomic traits of yield were also found in GmNFYB17 transgenic plants. Thus, the GmNFYB17 gene was proven to positively regulate drought stress resistance and modulate root growth in soybean. These results provide important insights into the molecular mechanisms underlying drought tolerance in soybean.