Elucidation of genomic organizations of transgenic soybean plants through de novo genome assembly with short paired-end reads.

Elucidation of the genomic organizations of transgene insertion sites is essential for the genetic studies of transgenic plants. Herein, we establish an analysis pipeline that identifies the transgene insertion sites as well as the presence of vector backbones, through de novo genome assembly with high-throughput sequencing data in two transgenic soybean lines, AtYUCCA6-#5 and 35S-UGT72E3/2-#7. Sequencing data of approximately 28× and 29× genome coverages for each line generated by high-throughput sequencing were de novo assembled. The databases generated from the de novo assembled sequences were used to search contigs that contained putative insertion sites and their flanking sequences (integration sites) of transgene fragments using transgenic vector sequences as queries. The predicted integration site sequences, which are located at three annotated genes that might regulate plant development or confer disease resistance, were then confirmed by local alignment against the soybean reference genome and PCR amplification. As results, we revealed the precise transgene-flanking sequences and sequence rearrangements at insertion sites in both the transgenic lines, as well as the aberrant insertion of a transgene fragment. Consequently, relative to experimental or enrichment technologies, our approach is straightforward and time-effective, providing an alternative method for the identification of insertion sites in transgenic plants.

Chromosomal features revealed by comparison of genetic maps of Glycine max and Glycine soja.

Recombination is a crucial component of evolution and breeding. New combinations of variation on chromosomes are shaped by recombination. Recombination is also involved in chromosomal rearrangements. However, recombination rates vary tremendously among chromosome segments. Genome-wide genetic maps are one of the best tools to study variation of recombination. Here, we describe high density genetic maps of Glycine max and Glycine soja constructed from four segregating populations. The maps were used to identify chromosomal rearrangements and find the highly predictable pattern of cross-overs on the broad scale in soybean. Markers on these genetic maps were used to evaluate assembly quality of the current soybean reference genome sequence. We find a strong inversion candidate larger than 3 Mb based on patterns of cross-overs. We also identify quantitative trait loci (QTL) that control number of cross-overs. This study provides fundamental insights relevant to practical strategy for breeding programs and for pan-genome researches.

Genetic analysis of the Lf1 gene that controls leaflet number in soybean.

KEY MESSAGE: A high-resolution genetic map that was constructed for the Lf1 -residing region will provide valuable information for map-based cloning and genetic improvement efforts in soybean. Changes in leaf architecture as photosynthesis factories remain a major challenge for the improvement of crop productivity. Unlike most soybeans, which have compound leaves comprising three leaflets, the soybean Lf1 mutant has a high frequency of compound leaves with five leaflets in a partially dominant manner. Here, we generated a fine genetic map to determine the genetic basis of this multifoliolate leaf trait. A five-leaflet variant Dusam was found in a recently collected landrace cultivar. Phenotypic data were collected from the F2 population of a cross between the Dusam and three-leaflet cultivar V94-5152. The mapping results generated using public markers indicated that the five-leaflet determining gene in Dusam is an allele of the previously studied Lf1 gene on chromosome 8. A high-resolution map delimited the genomic region controlling the leaflet number trait to a sequence length of 49 kb. AP2 domain-containing Glyma.08g281900 annotated in this 49 kb region appeared to be a strong candidate for the Lf1-encoding gene, as members of the AP2-type transcription factor family regulate lateral organ development. Dusam additionally exhibits visually distinct phenotypes for shattering and seed-coat cracking traits. However, the two traits were clearly unlinked to the Lf1 gene in our mapping population. Interestingly, the mapping results suggest that the Lf1 gene most likely exerts a pleiotropic effect on the number of seeds per pod. Thus, our results provide a strong foundation towards the cloning of this compound leaf development gene and marker-assisted selection of the seeds per pod trait.

Identification of haplotypes at the Rsv4 genomic region in soybean associated with durable resistance to soybean mosaic virus.

KEY MESSAGE: Discovery of new germplasm sources and identification of haplotypes for the durable Soybean mosaic virus resistance gene, Rsv 4, provide novel resources for map-based cloning and genetic improvement efforts in soybean. The Soybean mosaic virus (SMV) resistance locus Rsv4 is of interest because it provides a durable type of resistance in soybean [Glycine max (L.) Merr.]. To better understand its molecular basis, we used a population of 309 BC3F2 individuals to fine-map Rsv4 to a ~120 kb interval and leveraged this genetic information in a second study to identify accessions 'Haman' and 'Ilpumgeomjeong' as new sources of Rsv4. These two accessions along with three other Rsv4 and 14 rsv4 accessions were used to examine the patterns of nucleotide diversity at the Rsv4 region based on high-depth resequencing data. Through a targeted association analysis of these 19 accessions within the ~120 kb interval, a cluster of four intergenic single-nucleotide polymorphisms (SNPs) was found to perfectly associate with SMV resistance. Interestingly, this ~120 kb interval did not contain any genes similar to previously characterized dominant disease resistance genes. Therefore, a haplotype analysis was used to further resolve the association signal to a ~94 kb region, which also resulted in the identification of at least two Rsv4 haplotypes. A haplotype phylogenetic analysis of this region suggests that the Rsv4 locus in G. max is recently introgressed from G. soja. This integrated study provides a strong foundation for efforts focused on the cloning of this durable virus resistance gene and marker-assisted selection of Rsv4-mediated SMV resistance in soybean breeding programs.

The patterns of deleterious mutations during the domestication of soybean.

Globally, soybean is a major protein and oil crop. Enhancing our understanding of the soybean domestication and improvement process helps boost genomics-assisted breeding efforts. Here we present a genome-wide variation map of 10.6 million single-nucleotide polymorphisms and 1.4 million indels for 781 soybean individuals which includes 418 domesticated (Glycine max), 345 wild (Glycine soja), and 18 natural hybrid (G. max/G. soja) accessions. We describe the enhanced detection of 183 domestication-selective sweeps and the patterns of putative deleterious mutations during domestication and improvement. This predominantly selfing species shows 7.1% reduction of overall deleterious mutations in domesticated soybean relative to wild soybean and a further 1.4% reduction from landrace to improved accessions. The detected domestication-selective sweeps also show reduced levels of deleterious alleles. Importantly, genotype imputation with this resource increases the mapping resolution of genome-wide association studies for seed protein and oil traits in a soybean diversity panel.