Zhangxiong Liu

De novo assembly of soybean wild relatives for pan-genome analysis of diversity and agronomic traits

Wild relatives of crops are an important source of genetic diversity for agriculture, but their gene repertoire remains largely unexplored. We report the establishment and analysis of a pan-genome of Glycine soja, the wild relative of cultivated soybean Glycine max, by sequencing and de novo assembly of seven phylogenetically and geographically representative accessions. Intergenomic comparisons identified lineage-specific genes and genes with copy number variation or large-effect mutations, some of which show evidence of positive selection and may contribute to variation of agronomic traits such as biotic resistance, seed composition, flowering and maturity time, organ size and final biomass. Approximately 80% of the pan-genome was present in all seven accessions (core), whereas the rest was dispensable and exhibited greater variation than the core genome, perhaps reflecting a role in adaptation to diverse environments. This work will facilitate the harnessing of untapped genetic diversity from wild soybean for enhancement of elite cultivars.

Genetic structure and diversity of cultivated soybean ( Glycine max (L.) Merr.) landraces in China

The Chinese genebank contains 23,587 soybean landraces collected from 29 provinces. In this study, a representative collection of 1,863 landraces were assessed for genetic diversity and genetic differentiation in order to provide useful information for effective management and utilization. A total of 1,160 SSR alleles at 59 SSR loci were detected including 97 unique and 485 low-frequency alleles, which indicated great richness and uniqueness of genetic variation in this core collection. Seven clusters were inferred by STRUCTURE analysis, which is in good agreement with a neighbor-joining tree. The cluster subdivision was also supported by highly significant pairwise Fst values and was generally in accordance with differences in planting area and sowing season. The cluster HSuM, which contains accessions collected from the region between 32.0 and 40.5°N, 105.4 and 122.2°E along the central and downstream parts of the Yellow River, was the most genetically diverse of the seven clusters. This provides the first molecular evidence for the hypotheses that the origin of cultivated soybean is the Yellow River region. A high proportion (95.1%) of pairs of alleles from different loci was in LD in the complete dataset. This was mostly due to overall population structure, since the number of locus pairs in LD was reduced sharply within each of the clusters compared to the complete dataset. This shows that population structure needs to be accounted for in association studies conducted within this collection. The low value of LD within the clusters can be seen as evidence that much of the recombination events in the past have been maintained in soybean, fixed in homozygous self-fertilizing landraces.

Salinity tolerance in soybean is modulated by natural variation in GmSALT3

The identification of genes that improve the salt tolerance of crops is essential for the effective utilization of saline soils for agriculture. Here, we use fine mapping in a soybean (Glycine max (L.) Merr.) population derived from the commercial cultivars Tiefeng 8 and 85-140 to identify GmSALT3 (salt tolerance-associated gene on chromosome 3), a dominant gene associated with limiting the accumulation of sodium ions (Na+) in shoots and a substantial enhancement in salt tolerance in soybean. GmSALT3 encodes a protein from the cation/H+ exchanger family that we localized to the endoplasmic reticulum and which is preferentially expressed in the salt-tolerant parent Tiefeng 8 within root cells associated with phloem and xylem. We identified in the salt-sensitive parent, 85-140, a 3.78-kb copia retrotransposon insertion in exon 3 of Gmsalt3 that truncates the transcript. By sequencing 31 soybean landraces and 22 wild soybean (Glycine soja) a total of nine haplotypes including two salt-tolerant haplotypes and seven salt-sensitive haplotypes were identified. By analysing the distribution of haplotypes among 172 Chinese soybean landraces and 57 wild soybean we found that haplotype 1 (H1, found in Tiefeng 8) was strongly associated with salt tolerance and is likely to be the ancestral allele. Alleles H2-H6, H8 and H9, which do not confer salinity tolerance, were acquired more recently. H1, unlike other alleles, has a wide geographical range including saline areas, which indicates it is maintained when required but its potent stress tolerance can be lost during natural selection and domestication. GmSALT3 is a gene associated with salt tolerance with great potential for soybean improvement.

Activation of a rice endogenous retrotransposon Tos17 in tissue culture is accompanied by cytosine demethylation and causes heritable alteration in methylation pattern of flanking genomic regions

Tos17 is a copia-like, cryptic retrotransposon of rice, but can be activated by tissue culture. To study possible epigenetic mechanism controlling activity of Tos17, we subjected three rice lines (the parental line cv. Matsumae and two introgression lines, RZ2 and RZ35) that harbor different copies of the element to tissue culture. For each line, we investigated transcription and transposition of Tos17 in seed plants, calli and regenerated plants, cytosine-methylation status at CG and CNG positions within Tos17, effect of 5-azacytidine on methylation status and activity of Tos17, and cytosine-methylation states in genomic regions flanking original and some newly transposed copies of Tos17 in calli and regenerated plants. We found that only in introgression line RZ35 wasTos17 transcriptionally activated and temporarily mobilized by tissue culture, which was followed by repression before or upon plant regeneration. The activity and inactivity of Tos17 in calli and regenerated plants of RZ35 are accompanied by hypo- and hyper-CG methylation and hemi- and full CNG methylation, respectively, within the element, whereas immobilization of the element in the other two lines is concomitant with near-constant, full hypermethylation. Treatment with 5-azacytidine induced both CG and CNG partial hypomethylation of Tos17 in two lines (Matsumae and RZ35), which, however, was not accompanied by activation of Tos17 in any line. Heritable alteration in cytosine-methylation patterns occurred in three of seven genomic regions flanking Tos17 in calli and regenerated plants of RZ35, but in none of the five regions flanking dormant Tos17 in the other two lines.

Establishment of Chinese soybean (Glycine max) core collections with agronomic traits and SSR markers

It is very important to efficiently study and use genetic diversity resources in crop breeding and sustainable agriculture. In this study, different sampling methods and sample sizes were compared in order to optimize the strategies for building a rationally sized core collection of Chinese soybean (Glycine max). The diversity in the core collection captured more than 70% of that in the pre-core collection, no matter what sampling methods were used, at a sampling proportion of 1%. Core collections established with both simple sequence repeat (SSR) marker data and agronomic traits were more representative than those chosen on an independent basis. An optimal sampling method for a soybean core collection was determined, in which strategy ‘S’ (allocating accessions to clusters according to the proportion of square root of the original sample size within each ecotype) was used based on SSR and agronomic data. Curve estimation was used to estimate the allelic richness of the entire Chinese soybean germplasm and a minimum sample size for a core collection, on which a sampling proportion of about 2% was determined to be optimal for a core collection. Further analysis on the core collection with fourteen agronomic traits and allelic constitution at 60 SSR loci suggested that it highly represented the entire collections both on genetic structure and diversity distribution. This core collection would provide an effective platform in proper exploitation of soybean germplasm resources for the study of complex traits and discovering important novel traits for crop genetic development.

Production of a highly asymmetric somatic hybrid between rice and Zizania latifolia (Griseb): evidence for inter-genomic exchange

Abstract A highly asymmetric and fertile somatic hybrid plant was obtained via protoplast fusion in an intergenric combination. Gamma-ray-irradiated Zizania latifolia (Griseb). Turcz. ex Stapf mesophyll protoplasts were electrofused with idoacetamide-inactivated rice protoplasts derived from a 2-month-old suspension cell culture. Two of the six putative hybrid calli regenerated plants. Cytological observation showed that the somatic chromosome numbers of both plants were the same as the rice parent (2n=24). Nevertheless, the hybrid nature and inter-genomic exchange events of one of the plants, i.e. SH6 (SH for somatic hybrid), were confirmed by Southern analysis using both total genomic DNA and moderate-copy, Z. latifolia-abundant DNA sequences as probes; in both cases, parental specific and/or new intergenomic recombinant hybridization fragments were detected. In both plant and seed morphology, the hybrid (SH6) was distinct from its rice parental cultivar, as well as from the wild donor species, Z. latifolia.

Genetic diversity comparison between Chinese and Japanese soybeans ( Glycine max (L.) Merr.) revealed by nuclear SSRs

Soybean (Glycine max (L.) Merr.) has a long planting history in both China and Japan. In order to investigate the genetic relationship between Chinese and Japanese soybeans, 205 Chinese soybean accessions, that represent the seven different soybean ecotypes, and 39 Japanese soybean accessions from various regions were analyzed by using 46 SSR loci. In total 745 alleles were detected with an average allele number of 16.2 per locus. The allelic frequencies varied from 0.002 to 0.554 with an average of 0.06. Cluster analysis with UPGMA separated the Chinese accessions from Japanese accessions, suggesting that soybean in these two countries form different gene pools. When comparing the Japanese soybean with that from seven different Chinese soybean ecotypes, 164–200 alleles were specific to the Chinese accessions and 64–112 specific to the Japanese accessions. The comparison of SSRs diversity revealed that accessions from China exhibited more genetic diversity than those from Japan. The data were analyzed to resolve the genetic structure and to interpret the evolutionary relationships between groups. Three distinct groups were identified, corresponding to Japanese soybean, Northern China soybean, Southern China soybean and a mixed group in which most accessions were from central China. The results indicate that accessions from Japan are distinct from Chinese ones, and Japanese accessions had more close relationship with Chinese northeast spring and southern spring ecotypes. We further analyzed five agronomic trait-related SSR loci and found that the preponderant alleles are different in Chinese and Japanese soybean. Our study provides important insights into further utilization of Japanese soybean in Chinese soybean breeding.

Identification of Quantitative Trait Loci Underlying Plant Height and Seed Weight in Soybean

For clarifying the genetic base of the variation of yield‐related traits in soybean [Glycine max (L.) Merr.], we mapped quantitative trait loci (QTLs) for plant height and seed weight using a recombinant inbred line population derived from a cross between Chinese elite line Zhongpin03‐5373 and cultivar Zhonghuang13. We detected 11 QTLs for plant height and 18 QTLs for seed weight across six diverse environments. These included three pairs of plant height‐ vs. seed weight‐related QTLs located in close proximity to each other, with two pairs, qPH‐7 vs. qSW‐7‐2 and qPH‐19‐2 vs. qSW‐19, sharing the same direction of additive effects. Individual QTLs explained 2.02 to 47.60% of the variation in plant height and 2.13 to 14.35% in seed weight. Two and five of the major QTLs discovered for plant height and seed weight, respectively, that were stable across environments in our study have been reported previously. Among them, four QTLs, qPH‐13, qSW‐11, qSW‐12‐2, and qSW‐18, were not involved in digenic epistatic interaction in our biparental population, indicating that these QTLs will be useful for marker‐assisted selection and should be targeted for the future identification of candidate genes. Moreover, eight QTLs for both plant height and seed weight were newly identified in our population.

The worldwide utilization of the Chinese soybean germplasm collection

This article focuses on advances in both basic and applied research on soybean germplasm resources collected from China and dispersed to the world. Many landraces developed over the course of the 4500 years since its domestication in the Huangdi period. Systematic germplasm collection was begun in the early 20th century by Professor Shou Wang, and since then over 170,000 accessions have been conserved worldwide. Evaluation with respect to key morphological characteristics, pest resistance, abiotic stress tolerance and nutritional quality attributes has been widely carried out. In addition, genetic diversity has been assessed at the DNA level, and used to establish core collections for both cultivated ( Glycine max ) and wild ( Glycine soja ) soybean. Some core sets have targeted the most used parental lines, and others have focused on specific traits, such as resistance to the soybean cyst nematode or to soybean mosaic virus, or enhanced phosphorus use efficiency. The recent acquisition of the soybean genome sequence should accelerate the utilization of not only the Chinese soybean germplasm collection, but also those maintained elsewhere in the world.

GmSALT3, which confers improved soybean salt tolerance in the field, increases leaf Cl⁻ exclusion prior to Na⁺ exclusion but does not improve early vigor under salinity

Soil salinity reduces soybean growth and yield. The recently identified GmSALT3 (Glycine max salt Tolerance-associated gene on chromosome 3) has the potential to improve soybean yields in salinized conditions. Here we evaluate the impact of GmSALT3 on soybean performance under saline or non-saline conditions. Three sets of near isogenic lines (NILs), with genetic similarity of 95.6–99.3% between each pair of NIL-T and NIL-S, were generated from a cross between two varieties 85–140 (salt-sensitive, S) and Tiefeng 8 (salt-tolerant, T) by using marker-assisted selection. Each NIL-T; 782-T, 820-T and 860-T, contained a common ~1000 kb fragment on chromosome 3 where GmSALT3 was located. We show that GmSALT3 does not contribute to an improvement in seedling emergence rate or early vigor under salt stress. However, when 12-day-old seedlings were exposed to NaCl stress, the NIL-T lines accumulated significantly less leaf Na+ compared with their corresponding NIL-S, while no significant difference of K+ concentration was observed between NIL-T and NIL-S; the magnitude of Na+ accumulation within each NIL-T set was influenced by the different genetic backgrounds. In addition, NIL-T lines accumulated less Cl- in the leaf and more in the root prior to any difference in Na+; in the field they accumulated less pod wall Cl- than the corresponding NIL-S lines. Under non-saline field conditions, no significant differences were observed for yield related traits within each pair of NIL-T and NIL-S lines, indicating there was no yield penalty for having the GmSALT3 gene. In contrast, under saline field conditions the NIL-T lines had significantly greater plant seed weight and 100-seed weight than the corresponding NIL-S lines, meaning GmSALT3 conferred a yield advantage to soybean plants in salinized fields. Our results indicated that GmSALT3 mediated regulation of both Na+ and Cl- accumulation in soybean, and contributes to improved soybean yield through maintaining a higher seed weight under saline stress.