The Myb73-GDPD2-GA2ox1 transcriptional regulatory module confers phosphate deficiency tolerance in soybean.

Phosphorus is indispensable in agricultural production. An increasing food supply requires more efficient use of phosphate due to limited phosphate resources. However, how crops regulate phosphate efficiency remains largely unknown. Here, we identified a major quantitative trait locus, qPE19, that controls 7 low-phosphate (LP)-related traits in soybean (Glycine max) through linkage mapping and genome-wide association studies. We identified the gene responsible for qPE19 as GLYCEROPHOSPHORYL DIESTER PHOSPHODIESTERASE2 (GmGDPD2), and haplotype 5 represents the optimal allele favoring LP tolerance. Overexpression of GmGDPD2 significantly affects hormone signaling and improves root architecture, phosphate efficiency and yield-related traits; conversely, CRISPR/Cas9-edited plants show decreases in these traits. GmMyb73 negatively regulates GmGDPD2 by directly binding to its promoter; thus, GmMyb73 negatively regulates LP tolerance. GmGDPD2 physically interacts with GA 2-oxidase 1 (GmGA2ox1) in the plasma membrane, and overexpressing GmGA2ox1 enhances LP-associated traits, similar to GmGDPD2 overexpression. Analysis of double mutants for GmGDPD2 and GmGA2ox1 demonstrated that GmGDPD2 regulates LP tolerance likely by influencing auxin and gibberellin dose-associated cell division in the root. These results reveal a regulatory module that plays a major role in regulating LP tolerance in soybeans and is expected to be utilized to develop phosphate-efficient varieties to enhance soybean production, particularly in phosphate-deficient soils.

Identification of soybean phosphorous efficiency QTLs and genes using chlorophyll fluorescence parameters through GWAS and RNA-seq.

MAIN CONCLUSION: Soybean phosphorous efficiency QTLs were identified and candidate genes were predicted using chlorophyll fluorescence parameters through GWAS and RNA-seq. Phosphorus (P) is an essential nutrient element for crop growth and development, lack of P uptake seriously affects yield in various crops. Photosynthesis is the basis of crop production, while it is very sensitive to P deficiency. It is of great importance to study the genetic relationship between photosynthesis and P efficiency to provide genetic insight for soybean improvement. In this study, a genome-wide association study (GWAS) was performed using 292,035 SNPs and the ratios of four main chlorophyll fluorescence parameters of 219 diverse soybean accessions under P deficiency and normal P across three experiments. In total, 52 SNPs in 12 genomic regions were detected in association with the four main chlorophyll fluorescence parameters under sufficient or deficient P levels. Combined it with RNA-seq analysis, we predicted three candidate genes for the significant genomic regions. For example, the expression level of the candidate gene (Glyma.18g092900) in P deficiency tolerant accession was three times higher than that of P deficiency sensitive one under phosphorous deficiency condition. This study provides insight into genetic links between photosynthetic and phosphorous efficiency and further functional analysis will provide valuable information for understanding the underlying genetic mechanism to facilitate marker-assisted breeding in soybean.

High-density QTL mapping of leaf-related traits and chlorophyll content in three soybean RIL populations.

BACKGROUND: Leaf size and shape, which affect light capture, and chlorophyll content are important factors affecting photosynthetic efficiency. Genetic variation of these components significantly affects yield potential and seed quality. Identification of the genetic basis for these traits and the relationship between them is of great practical significance for achieving ideal plant architecture and high photosynthetic efficiency for improved yield. RESULTS: Here, we undertook a large-scale linkage mapping study using three mapping populations to determine the genetic interplay between soybean leaf-related traits and chlorophyll content across two environments. Correlation analysis revealed a significant negative correlation between leaf size and shape, while both traits were positively correlated with chlorophyll content. This phenotypic relationship was verified across the three mapping populations as determined by principal component analysis, suggesting that these traits are under the control of complex and interrelated genetic components. The QTLs for leaf-related traits and chlorophyll are partly shared, which further supports the close genetic relationship between the two traits. The largest-effect major loci, q20, was stably identified across all population and environments and harbored the narrow leaflet gene Gm-JAG1 (Ln/ln), which is a key regulator of leaflet shape in soybean. CONCLUSION: Our results uncover several major QTLs (q4-1, q4-2, q11, q13, q18 and q20) and its candidate genes specific or common to leaf-related traits and chlorophyll, and also show a complex epistatic interaction between the two traits. The SNP markers closely linked to these valuable QTLs could be used for molecular design breeding with improved plant architecture, photosynthetic capacity and even yield.

A New Race (X12) of Soybean Cyst Nematode in China.

The soybean cyst nematode (SCN), Heterodera glycines, is a serious economic threat to soybean-producing regions worldwide. A new SCN population (called race X12) was detected in Shanxi province, China. Race X12 could reproduce on all the indicator lines of both race and Heterodera glycines (HG) type tests. The average number of females on Lee68 (susceptible control) was 171.40 with the lowest Female Index (FI) 61.31 on PI88788 and the highest FI 117.32 on Pickett in the race test. The average number of females on Lee68 was 323.17 with the lowest FI 44.18 on PI88788 and the highest FI 97.83 on PI548316 in the HG type test. ZDD2315 and ZDD24656 are elite resistant germplasms in China. ZDD2315 is highly resistant to race 4, the strongest infection race in the 16 races with FI 1.51 while being highly sensitive to race X12 with FI 64.32. ZDD24656, a variety derived from PI437654 and ZDD2315, is highly resistant to race 1 and race 2. ZDD24656 is highly sensitive to race X12 with FI 99.12. Morphological and molecular studies of J2 and cysts confirmed the population as the SCN H. glycines. This is a new SCN race with stronger virulence than that of race 4 and is a potential threat to soybean production in China.

Genome-Wide Association Studies Prioritize Genes Controlling Seed Size and Reproductive Period Length in Soybean.

Hundred-seed weight (HSW) and reproductive period length (RPL) are two major agronomic traits critical for soybean production and adaptation. However, both traits are quantitatively controlled by multiple genes that have yet to be comprehensively elucidated due to the lack of major genes; thereby, the genetic basis is largely unknown. In the present study, we conducted comprehensive genome-wide association analyses (GWAS) of HSW and RPL with multiple sets of accessions that were phenotyped across different environments. The large-scale analysis led to the identification of sixty-one and seventy-four significant QTLs for HSW and RPL, respectively. An ortholog-based search analysis prioritized the most promising candidate genes for the QTLs, including nine genes (TTG2, BZR1, BRI1, ANT, KLU, EOD1/BB, GPA1, ABA2, and ABI5) for HSW QTLs and nine genes (such as AGL8, AGL9, TOC1, and COL4) and six known soybean flowering time genes (E2, E3, E4, Tof11, Tof12, and FT2b) for RPL QTLs. We also demonstrated that some QTLs were targeted during domestication to drive the artificial selection of both traits towards human-favored traits. Local adaptation likely contributes to the increased genomic diversity of the QTLs underlying RPL. The results provide additional insight into the genetic basis of HSW and RPL and prioritize a valuable resource of candidate genes that merits further investigation to reveal the complex molecular mechanism and facilitate soybean improvement.

Identification of resistant sources from Glycine max against soybean cyst nematode.

Soybean cyst nematode (SCN, Heterodera glycines, HG) is one of the severe pests in plant-parasitic nematodes, which impairs root development and causes severe losses in soybean production worldwide. Breeding SCN-resistant cultivars is an important measure for securing harvests without affecting the environment, as can be done with pesticides. The majority of genetic resources for plant pest resistances are found in wild or closely related species which are often difficult to use in breeding due to crossing barriers or close linkage with unfavorable agronomic traits. In this study, 12 soybean cultivars were evaluated for their marker haplotype at the rhg1 and Rhg4 SCN resistance loci and their SCN resistance tested against multiple races in environmentally controlled bioassays. The results showed that all 12 cultivars displayed Peking-type resistance marker haplotypes and all of them proved to be resistant to multiple SCN races. These cultivars provide potential for improving H. glycines resistance of soybean as donor parent in breeding and can contribute to reduce SCN field populations as part of a sustainable agriculture management.

A genome-wide association study and genomic prediction for Phakopsora pachyrhizi resistance in soybean.

Soybean brown rust (SBR), caused by Phakopsora pachyrhizi, is a devastating fungal disease that threatens global soybean production. This study conducted a genome-wide association study (GWAS) with seven models on a panel of 3,082 soybean accessions to identify the markers associated with SBR resistance by 30,314 high quality single nucleotide polymorphism (SNPs). Then five genomic selection (GS) models, including Ridge regression best linear unbiased predictor (rrBLUP), Genomic best linear unbiased predictor (gBLUP), Bayesian least absolute shrinkage and selection operator (Bayesian LASSO), Random Forest (RF), and Support vector machines (SVM), were used to predict breeding values of SBR resistance using whole genome SNP sets and GWAS-based marker sets. Four SNPs, namely Gm18_57,223,391 (LOD = 2.69), Gm16_29,491,946 (LOD = 3.86), Gm06_45,035,185 (LOD = 4.74), and Gm18_51,994,200 (LOD = 3.60), were located near the reported P. pachyrhizi R genes, Rpp1, Rpp2, Rpp3, and Rpp4, respectively. Other significant SNPs, including Gm02_7,235,181 (LOD = 7.91), Gm02_7234594 (LOD = 7.61), Gm03_38,913,029 (LOD = 6.85), Gm04_46,003,059 (LOD = 6.03), Gm09_1,951,644 (LOD = 10.07), Gm10_39,142,024 (LOD = 7.12), Gm12_28,136,735 (LOD = 7.03), Gm13_16,350,701(LOD = 5.63), Gm14_6,185,611 (LOD = 5.51), and Gm19_44,734,953 (LOD = 6.02), were associated with abundant disease resistance genes, such as Glyma.02G084100, Glyma.03G175300, Glyma.04g189500, Glyma.09G023800, Glyma.12G160400, Glyma.13G064500, Glyma.14g073300, and Glyma.19G190200. The annotations of these genes included but not limited to: LRR class gene, cytochrome 450, cell wall structure, RCC1, NAC, ABC transporter, F-box domain, etc. The GWAS based markers showed more accuracies in genomic prediction than the whole genome SNPs, and Bayesian LASSO model was the ideal model in SBR resistance prediction with 44.5% ~ 60.4% accuracies. This study aids breeders in predicting selection accuracy of complex traits such as disease resistance and can shorten the soybean breeding cycle by the identified markers.

The Spatial Distribution and Genetic Diversity of the Soybean Cyst Nematode, Heterodera glycines, in China: It Is Time to Take Measures to Control Soybean Cyst Nematode.

The continuous evolution and spread of virulent forms of the soybean cyst nematode (SCN) driven by the environment and anthropogenic intervention is a serious threat to the soybean production worldwide, including China. Especially in China, the implemented measures to control SCN are insufficient for sustainable agricultural development yet. We summarized our knowledge about the spread and spatial distribution of SCN in China and the virulence diversity in the main soybean growing areas. To reveal the genetic relatedness and diversity of SCN populations, we re-sequenced 53 SCN genomes from the Huang-Huai Valleys, one of the two main soybean growing areas in China. We identified spreading patterns linked to the local agroecosystems and topographies. Moreover, we disclosed the first evidence for the selection of complex virulence in the field even under low selection pressure in an example from North Shanxi. SCN is present in all soybean growing areas in China but SCN susceptible cultivars are still largely grown indicating that SCN-related damage and financial loss have not received the attention they deserve yet. To prevent increasing yield losses and to improve the acceptance of resistant cultivars by the growers, we emphasized that it is time to accelerate SCN resistance breeding, planting resistant cultivars to a larger extent, and to support farmers to implement a wider crop rotation for sustainable development of the soybean production in China.

GmWRKY46, a WRKY transcription factor, negatively regulates phosphorus tolerance primarily through modifying root morphology in soybean.

Phosphorus (P) deficiency affects soybean growth and development, resulting in significant reduction of yields. However, the regulatory mechanism of P deficiency tolerance in soybean is still largely unclear. WRKY transcription factors are a family of regulators involved in a variety of abiotic stresses in plants while rarely reported in P deficiency. Here, we demonstrated that a soybean GmWRKY46 gene, belonging to group III of WRKY TF family, was involved in the regulation of P deficiency tolerance in soybean. The expression of GmWRKY46 in low P sensitive soybean varieties was significantly higher than that in tolerant soybean varieties. It was primarily expressed in roots and strongly induced by P deprivation. GmWRKY46 was localized in the nucleus. Compared with the control expressing the empty vector, overexpression of GmWRKY46 in soybean hairy roots exhibited more sensitive phenotypes to low P stress, while the RNA interfered GmWRKY46 significantly enhanced P deficiency tolerance by increasing the proliferation, elongation and P absorption efficiency of hairy roots. Expression patterns of a number of P-responsive genes (GmPht1;1, GmPht1;4, GmPTF1, GmACP1, GmPAP21 and GmExpansin-A7) were altered in both overexpression and gene silenced plants. The results provided a novel insight into how soybean responds to low P stress and new gene that may be used to improve soybean low P tolerance through gene editing approach.

Identification of Candidate Genes Controlling Soybean Cyst Nematode Resistance in "Handou 10" Based on Genome and Transcriptome Analyzes.

Soybean cyst nematode (SCN; Heterodera glycines Ichinohe) is a highly destructive pathogen for soybean production worldwide. The use of resistant varieties is the most effective way of preventing yield loss. Handou 10 is a commercial soybean variety with desirable agronomic traits and SCN resistance, however genes underlying the SCN resistance in the variety are unknown. An F2:8 recombinant inbred line (RIL) population derived from a cross between Zheng 9525 (susceptible) and Handou 10 was developed and its resistance to SCN HG type 2.5.7 (race 1) and 1.2.5.7 (race 2) was identified. We identified seven quantitative trait loci (QTLs) with additive effects. Among these, three QTLs on Chromosomes 7, 8, and 18 were resistant to both races. These QTLs could explain 1.91-7.73% of the phenotypic variation of SCN's female index. The QTLs on chromosomes 8 and 18 have already been reported and were most likely overlapped with rhg1 and Rhg4 loci, respectively. However, the QTL on chromosome 7 was novel. Candidate genes for the three QTLs were predicted through genes functional analysis and transcriptome analysis of infected roots of Handou 10 vs. Zheng 9525. Transcriptome analysis performed also indicated that the plant-pathogen interaction played an important role in the SCN resistance for Handou 10. The information will facilitate SCN-resistant gene cloning, and the novel resistant gene will be a source for improving soybeans' resistance to SCN.

Immersive virtual reality as analgesia for women during hysterosalpingography: study protocol for a randomized controlled trial.

BACKGROUND: Hysterosalpingography (HSG) is an accepted radiologic diagnostic modality for initial infertility workup, and is generally considered uncomfortable and painful. However, the management of pain related to HSG remains inefficient. As an emerging nonpharmacologic and noninvasive pain control strategy, virtual reality (VR) distraction has been successfully used in areas such as burns, blunt force trauma, hospital-based needle procedures, dental/periodontal procedures, and urological endoscopy patients. This study aims to evaluate the analgesic effect of VR during HSG. METHODS/DESIGN: A single-center, parallel-group, randomized controlled trial will be carried out in the Radiology Department of Yinchuan Women and Children Healthcare Hospital, Yinchuan. A total of 200 participants who are scheduled for HSG will be enrolled in this study. The participants will be randomized (1:1) into two groups: a VR group and a blank control group. The VR group will receive routine care plus immersive VR intervention and the blank control group will receive routine care. Outcomes will be monitored at baseline, immediately after HSG and 15 min after HSG for each group. The primary outcome is the worst pain score during HSG by a visual analog scale (VAS). The secondary outcomes include: affective pain, cognitive pain, and anxiety during the HSG procedure; worst pain within 15 min after HSG; patient satisfaction and acceptance with pain management; physiological parameters; adverse effects; HSG results; and immersion perception score of the VR system (for the VR condition only). DISCUSSION: This study will focus on exploring a simply operated, noninvasive and low-cost analgesia during the HSG procedure. The results of this trial will provide data on the feasibility and safety of VR distraction therapy during HSG. TRIAL REGISTRATION: Chinese Clinical Trial Register, ChiCTR1900021342. Registered on 16 February 2019.

Chromosome-level reference genome of X12, a highly virulent race of the soybean cyst nematode Heterodera glycines.

Soybean cyst nematode (SCN, Heterodera glycines) is a major pest of soybean that is spreading across major soybean production regions worldwide. Increased SCN virulence has recently been observed in both the United States and China. However, no study has reported a genome assembly for H. glycines at the chromosome scale. Herein, the first chromosome-level reference genome of X12, an unusual SCN race with high infection ability, is presented. Using whole-genome shotgun (WGS) sequencing, Pacific Biosciences (PacBio) sequencing, Illumina paired-end sequencing, 10X Genomics linked reads and high-throughput chromatin conformation capture (Hi-C) genome scaffolding techniques, a 141.01-megabase (Mb) assembled genome was obtained with scaffold and contig N50 sizes of 16.27 Mb and 330.54 kilobases (kb), respectively. The assembly showed high integrity and quality, with over 90% of Illumina reads mapped to the genome. The assembly quality was evaluated using Core Eukaryotic Genes Mapping Approach and Benchmarking Universal Single-Copy Orthologs. A total of 11,882 genes were predicted using de novo, homolog and RNAseq data generated from eggs, second-stage juveniles (J2), third-stage juveniles (J3) and fourth-stage juveniles (J4) of X12, and 79.0% of homologous sequences were annotated in the genome. These high-quality X12 genome data will provide valuable resources for research in a broad range of areas, including fundamental nematode biology, SCN-plant interactions and co-evolution, and also contribute to the development of technology for overall SCN management.

High-Density Genetic Mapping Identifies New Major Loci for Tolerance to Low-Phosphorus Stress in Soybean.

Phosphorus (P) is essential for all living cells and organisms, and low-P stress represents a major constraint on plant growth and yield worldwide. Soybean is an important economical resource of protein and oil for human and animals, and soybean is also a high-P demand species that is sensitive to low-P stress, which is considered a major constraint on soybean production. However, P efficiency is an important complex quantitative trait involving multiple genes, and the mechanisms underlying soybean P efficiency are largely unknown. Here, we reported the construction of a high-density genetic map using a specific-locus amplified fragment sequencing (SLAF-seq) strategy in soybean. This map, spanning 3020.59 cM in length, contained 6159 markers on 20 chromosomes, with an average distance of 0.49 cM between adjacent markers. Based on this map, 20 loci, including eight novel loci, associated with P efficiency-related traits were identified across multiple years and treatments. The confidence intervals of almost all QTLs were refined significantly, and the accuracy of this map was evidenced by coincident detections of the previously identified P efficiency-related genes GmACP1 and GmPT1. Notably, a highly significant novel QTL located on chromosome 4, q4-2, was identified across traits, years and treatments. Several candidate genes, such as a pectin methylesterase-encoding gene (Glyma.04G214000) and a protein kinase gene (Glyma.13G161900), with significantly differential expression upon low-P stress were considered as promising candidates involved in regulating soybean P efficiency. Markers that tightly associated with P efficiency could be used for marker-assisted selection in a soybean P efficient breeding program. Further, dissection of these QTLs will facilitate gene cloning underlying P efficiency in soybean, and increase our understanding of efficient use of P in enhancing crop yield.

Genome-Wide Association Study of Resistance to Soybean Cyst Nematode (Heterodera glycines) HG Type 2.5.7 in Wild Soybean (Glycine soja).

Soybean cyst nematode (SCN) is the most destructive soybean pest worldwide. Host plant resistance is the most environmentally friendly and cost-effective way of mitigating SCN damage to soybeans. However, overuse of the resistant soybean [Glycine max (L.) Merr.] cultivars from limited genetic resources has resulted in SCN race shifts in many soybean-growing areas. Thus, exploration of novel sources of SCN resistance and dissection of the genetic basis are urgently needed. In this study, we screened 235 wild soybean (Glycine soja Sieb. & Zucc.) accessions to identify genotypes resistant to SCN HG Type 2.5.7 (race 5), a less investigated type but is prevalent in the southeastern US. We also dissected the genetic basis of SCN resistance using a genome-wide association study with SNPs genotyped by SoySNP50k iSelect BeadChip. In total, 43 resistant accessions (female index < 30) were identified, with 10 SNPs being significantly associated with SCN HG 2.5.7 resistance in this wild species. Furthermore, four significant SNPs were localized to linked regions of the known quantitative trait locus (QTL) rhg1 on chromosome 18. The other four SNPs on chromosome 18 and two SNPs on chromosome 19 are novel. Genes encoding disease resistance-related proteins with a leucine-rich region, a mitogen-activated protein kinase (MAPK) on chromosome 18, and a MYB transcription factor on chromosome 19 were identified as promising candidate genes. The identified SNPs and candidate genes will benefit future marker-assisted breeding and dissection of the molecular mechanisms underlying the soybean-SCN interaction.