Key messageGmDW1 encodes an ent-kaurene synthase (KS) acting at the early step of the biosynthesis pathway for gibberellins (GAs) and regulates the development of plant height in soybean.AbstractPlant height is an important component of plant architecture, and significantly affects crop breeding practices and yield. Here, we report the characterization of an EMS-induced dwarf mutant (dw) of the soybean cultivar Zhongpin 661 (ZDD23893). The dw mutant displayed reduced plant height and shortened internodes, both of which were mainly attributed to the longitudinally decreased cell length. The bioactive GA1 (gibberellin A1) and GA4 (gibberellin A4) were not detectable in the stem of dw, and the dwarf phenotype could be rescued by treatment with exogenous GA3. Genetic analysis showed that the dwarf trait of dw was controlled by a recessive nuclear gene. By combining linkage analysis and mapping-by-sequencing, we mapped the GmDW1 gene to an approximately 460-kb region on chromosome (Chr.) 8, containing 36 annotated genes in the reference Willliams 82 genome. Of these genes, we identified two nonsynonymous single nucleotide polymorphisms (SNPs) that are present in the encoding regions of Gmdw1 and Glyma.08G165100 in dw, respectively. However, only the SNP mutation (T>A) at nucleotide 1224 in Gmdw1 cosegregated with the dwarf phenotype. GmDW1 encodes an ent-kaurene synthase, and was expressed in various tissues including root, stem, and leaf. Further phenotypic analysis of the allelic variations in soybean accessions strongly indicated that GmDW1 is responsible for the dwarf phenotype in dw. Our results provide important information for improving our understanding of the genetics of soybean plant height and crop breeding.Electronic supplementary materialThe online version of this article (10.1007/s00122-017-3044-8) contains supplementary material, which is available to authorized users.

Molecular characterization of sequence flanking exogenous fragment insertion is essential for safety assessment and labeling of genetically modified organism (GMO). In this study, the T-DNA insertion sites and flanking sequences were identified in two newly developed transgenic glyphosate-tolerant soybeans GE-J16 and ZH10-6 based on whole genome sequencing (WGS) method. More than 22.4 Gb sequence data (∼21 × coverage) for each line was generated on Illumina HiSeq 2500 platform. The junction reads mapped to boundaries of T-DNA and flanking sequences in these two events were identified by comparing all sequencing reads with soybean reference genome and sequence of transgenic vector. The putative insertion loci and flanking sequences were further confirmed by PCR amplification, Sanger sequencing, and co-segregation analysis. All these analyses supported that exogenous T-DNA fragments were integrated in positions of Chr19: 50543767–50543792 and Chr17: 7980527–7980541 in these two transgenic lines. Identification of genomic insertion sites of G2-EPSPS and GAT transgenes will facilitate the utilization of their glyphosate-tolerant traits in soybean breeding program. These results also demonstrated that WGS was a cost-effective and rapid method for identifying sites of T-DNA insertions and flanking sequences in soybean.

Soybean is an important economic crop and a typical short-day crop, sensitive to photoperiod, and has narrow geographical adaptative region, which limit the creation of transgenic materials and reduce the breeding efficiency of new varieties. In addition, the genetic transformation efficiency of soybean is lower than that of many other crops, and the available receptor genotypes are limited. In this study, Agrobacterium-mediated transformation were used to introduce the CRISPR/Cas9 expression vector into soybean cultivar Jack and generated targeted mutants of E1 gene controlling soybean flowering. We obtained two novel types of mutations, 11 bp and 40 bp deletion at E1 coding region, respectively, and frameshift mutations produced premature translation termination codons and truncated E1 proteins, causing obvious early flowering under long day condition. In addition, no off-target effects were observed by predicting and analyzing the potential off-target sites of E1 targets. Significant decreased E1 gene expression of two novel mutants showed that the truncated E1 protein disinhibited GmFT2a/5a and increasing GmFT2a/5a gene expressions resulted obvious early flowering. Homozygous trans-clean mutants without T-DNA elements were also obtained and showed early flowering under long day condition. The photo-insensitive soybean transformation receptor we created laid a foundation for breeding excellent transgenic receptors suitable for high latitudes.

Glyphosate is a widely used non-selective herbicide with broad spectrum of weed control around the world. At present, most of the commercial glyphosate tolerant soybeans utilize glyphosate tolerant gene CP4-EPSPS or glyphosate acetyltransferase gene GAT separately. In this study, both glyphosate tolerant gene G2-EPSPS and glyphosate degraded gene GAT were co-transferred into soybean and transgenic plants showed high tolerance to glyphosate. Molecular analysis including PCR, Sothern blot, qRT-PCR, and Western blot revealed that target genes have been integrated into genome and expressed effectively at both mRNA and protein levels. Furthermore, the glyphosate tolerance analysis showed that no typical symptom was observed when compared with a glyphosate tolerant line HJ06-698 derived from GR1 transgenic soybean even at fourfold labeled rate of Roundup. Chlorophyll and shikimic acid content analysis of transgenic plant also revealed that these two indexes were not significantly altered after glyphosate application. These results indicated that co-expression of G2-EPSPS and GAT conferred high tolerance to the herbicide glyphosate in soybean. Therefore, combination of tolerant and degraded genes provides a new strategy for developing glyphosate tolerant transgenic crops.