Genome-wide association study of agronomic traits related to nitrogen use efficiency in Henan wheat.

BACKGROUND: Nitrogen use efficiency (NUE) is closely related to crop yield and nitrogen fertilizer application rate. Although NUE is susceptible to environments, quantitative trait nucleotides (QTNs) for NUE in wheat germplasm populations have been rarely reported in genome-wide associated study. RESULTS: In this study, 244 wheat accessions were phenotyped by three NUE-related traits in three environments and genotyped by 203,224 SNPs. All the phenotypes for each trait were used to associate with all the genotypes of these SNP markers for identifying QTNs and QTN-by-environment interactions via 3VmrMLM. Among 279 QTNs and one QTN-by-environment interaction for low nitrogen tolerance, 33 were stably identified, especially, one large QTN (r2 > 10%), qPHR3A.2, was newly identified for plant height ratio in one environment and multi-environment joint analysis. Among 52 genes around qPHR3A.2, four genes (TraesCS3A01G101900, TraesCS3A01G102200, TraesCS3A01G104100, and TraesCS3A01G105400) were found to be differentially expressed in low-nitrogen-tolerant wheat genotypes, while TaCLH2 (TraesCS3A01G101900) was putatively involved in porphyrin metabolism in KEGG enrichment analyses. CONCLUSIONS: This study identified valuable candidate gene for low-N-tolerant wheat breeding and provides new insights into the genetic basis of low N tolerance in wheat.

Identification of QTNs, QTN-by-environment interactions, and their candidate genes for salt tolerance related traits in soybean.

BACKGROUND: Salt stress significantly reduces soybean yield. To improve salt tolerance in soybean, it is important to mine the genes associated with salt tolerance traits. RESULTS: Salt tolerance traits of 286 soybean accessions were measured four times between 2009 and 2015. The results were associated with 740,754 single nucleotide polymorphisms (SNPs) to identify quantitative trait nucleotides (QTNs) and QTN-by-environment interactions (QEIs) using three-variance-component multi-locus random-SNP-effect mixed linear model (3VmrMLM). As a result, eight salt tolerance genes (GmCHX1, GsPRX9, Gm5PTase8, GmWRKY, GmCHX20a, GmNHX1, GmSK1, and GmLEA2-1) near 179 significant and 79 suggested QTNs and two salt tolerance genes (GmWRKY49 and GmSK1) near 45 significant and 14 suggested QEIs were associated with salt tolerance index traits in previous studies. Six candidate genes and three gene-by-environment interactions (GEIs) were predicted to be associated with these index traits. Analysis of four salt tolerance related traits under control and salt treatments revealed six genes associated with salt tolerance (GmHDA13, GmPHO1, GmERF5, GmNAC06, GmbZIP132, and GmHsp90s) around 166 QEIs were verified in previous studies. Five candidate GEIs were confirmed to be associated with salt stress by at least one haplotype analysis. The elite molecular modules of seven candidate genes with selection signs were extracted from wild soybean, and these genes could be applied to soybean molecular breeding. Two of these genes, Glyma06g04840 and Glyma07g18150, were confirmed by qRT-PCR and are expected to be key players in responding to salt stress. CONCLUSIONS: Around the QTNs and QEIs identified in this study, 16 known genes, 6 candidate genes, and 8 candidate GEIs were found to be associated with soybean salt tolerance, of which Glyma07g18150 was further confirmed by qRT-PCR.

Multi-omics analysis reveals novel loci and a candidate regulatory gene of unsaturated fatty acids in soybean (Glycine max (L.) Merr).

BACKGROUND: Soybean is a major oil crop; the nutritional components of soybean oil are mainly controlled by unsaturated fatty acids (FA). Unsaturated FAs mainly include oleic acid (OA, 18:1), linoleic acid (LLA, 18:2), and linolenic acid (LNA, 18:3). The genetic architecture of unsaturated FAs in soybean seeds has not been fully elucidated, although many independent studies have been conducted. A 3 V multi-locus random single nucleotide polymorphism (SNP)-effect mixed linear model (3VmrMLM) was established to identify quantitative trait loci (QTLs) and QTL-by-environment interactions (QEIs) for complex traits. RESULTS: In this study, 194 soybean accessions with 36,981 SNPs were calculated using the 3VmrMLM model. As a result, 94 quantitative trait nucleotides (QTNs) and 19 QEIs were detected using single-environment (QTN) and multi-environment (QEI) methods. Three significant QEIs, namely rs4633292, rs39216169, and rs14264702, overlapped with a significant single-environment QTN. CONCLUSIONS: For QTNs and QEIs, further haplotype analysis of candidate genes revealed that the Glyma.03G040400 and Glyma.17G236700 genes were beneficial haplotypes that may be associated with unsaturated FAs. This result provides ideas for the identification of soybean lipid-related genes and provides insights for breeding high oil soybean varieties in the future.

Identification of QTL, QTL-by-environment interactions, and their candidate genes for resistance HG Type 0 and HG Type 1.2.3.5.7 in soybean using 3VmrMLM.

Introduction: Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is an important disease affecting soybean yield in the world. Potential SCN-related QTLs and QTL-by-environment interactions (QEIs) have been used in SCN-resistant breeding. Methods: In this study, a compressed variance component mixed model, 3VmrMLM, in genome-wide association studies was used to detect QTLs and QEIs for resistance to SCN HG Type 0 and HG Type 1.2.3.5.7 in 156 different soybean cultivars materials. Results and discussion: The results showed that 53 QTLs were detected in single environment analysis; 36 QTLs and 9 QEIs were detected in multi-environment analysis. Based on the statistical screening of the obtained QTLs, we obtained 10 novel QTLs and one QEI which were different from the previous studies. Based on previous studies, we identified 101 known genes around the significant/suggested QTLs and QEIs. Furthermore, used the transcriptome data of SCN-resistant (Dongnong L-10) and SCN-susceptible (Suinong 14) cultivars, 10 candidate genes related to SCN resistance were identified and verified by Quantitative real time polymerase chain reaction (qRT-PCR) analysis. Haplotype difference analysis showed that Glyma.03G005600 was associated with SCN HG Type 0 and HG Type 1.2.3.5.7 resistance and had a haplotype beneficial to multi-SCN-race resistance. These results provide a new idea for accelerating SCN disease resistance breeding.

Compressed variance component mixed model reveals epistasis associated with flowering in Arabidopsis.

Introduction: Epistasis is currently a topic of great interest in molecular and quantitative genetics. Arabidopsis thaliana, as a model organism, plays a crucial role in studying the fundamental biology of diverse plant species. However, there have been limited reports about identification of epistasis related to flowering in genome-wide association studies (GWAS). Therefore, it is of utmost importance to conduct epistasis in Arabidopsis. Method: In this study, we employed Levene's test and compressed variance component mixed model in GWAS to detect quantitative trait nucleotides (QTNs) and QTN-by-QTN interactions (QQIs) for 11 flowering-related traits of 199 Arabidopsis accessions with 216,130 markers. Results: Our analysis detected 89 QTNs and 130 pairs of QQIs. Around these loci, 34 known genes previously reported in Arabidopsis were confirmed to be associated with flowering-related traits, such as SPA4, which is involved in regulating photoperiodic flowering, and interacts with PAP1 and PAP2, affecting growth of Arabidopsis under light conditions. Then, we observed significant and differential expression of 35 genes in response to variations in temperature, photoperiod, and vernalization treatments out of unreported genes. Functional enrichment analysis revealed that 26 of these genes were associated with various biological processes. Finally, the haplotype and phenotypic difference analysis revealed 20 candidate genes exhibiting significant phenotypic variations across gene haplotypes, of which the candidate genes AT1G12990 and AT1G09950 around QQIs might have interaction effect to flowering time regulation in Arabidopsis. Discussion: These findings may offer valuable insights for the identification and exploration of genes and gene-by-gene interactions associated with flowering-related traits in Arabidopsis, that may even provide valuable reference and guidance for the research of epistasis in other species.

Detection of epistasis interaction loci for fiber quality-related trait via 3VmrMLM in upland cotton.

Cotton fiber quality-related traits, such as fiber length, fiber strength, and fiber elongation, are affected by complex mechanisms controlled by multiple genes. Determining the QTN-by-QTN interactions (QQIs) associated with fiber quality-related traits is therefore essential for accelerating the genetic enhancement of cotton breeding. In this study, a natural population of 1,245 upland cotton varieties with 1,122,352 SNPs was used for detecting the main-effect QTNs and QQIs using the 3V multi-locus random-SNP-effect mixed linear model (3VmrMLM) method. A total of 171 significant main-effect QTNs and 42 QQIs were detected, of which 22 were both main-effect QTNs and QQIs. Of the detected 42 QQIs, a total of 13 significant loci and 5 candidate genes were reported in previous studies. Among the three interaction types, the AD interaction type has a preference for the trait of FE. Additionally, the QQIs have a substantial impact on the enhancement predictability for fiber quality-related traits. The study of QQIs is crucial for elucidating the genetic mechanism of cotton fiber quality and enhancing breeding efficiency.

Identification of QTNs, QTN-by-environment interactions, and their candidate genes for grain size traits in main crop and ratoon rice.

Although grain size is an important quantitative trait affecting rice yield and quality, there are few studies on gene-by-environment interactions (GEIs) in genome-wide association studies, especially, in main crop (MC) and ratoon rice (RR). To address these issues, the phenotypes for grain width (GW), grain length (GL), and thousand grain weight (TGW) of 159 accessions of MC and RR in two environments were used to associate with 2,017,495 SNPs for detecting quantitative trait nucleotides (QTNs) and QTN-by-environment interactions (QEIs) using 3VmrMLM. As a result, 64, 71, 67, 72, 63, and 56 QTNs, and 0, 1, 2, 2, 2, and 1 QEIs were found to be significantly associated with GW in MC (GW-MC), GL-MC, TGW-MC, GW-RR, GL-RR, and TGW-RR, respectively. 3, 4, 7, 2, 2, and 4 genes were found to be truly associated with the above traits, respectively, while 2 genes around the above QEIs were found to be truly associated with GL-RR, and one of the two known genes was differentially expressed under two soil moisture conditions. 10, 7, 1, 8, 4, and 3 candidate genes were found by differential expression and GO annotation analysis to be around the QTNs for the above traits, respectively, in which 6, 3, 1, 2, 0, and 2 candidate genes were found to be significant in haplotype analysis. The gene Os03g0737000 around one QEI for GL-MC was annotated as salt stress related gene and found to be differentially expressed in two cultivars with different grain sizes. Among all the candidate genes around the QTNs in this study, four were key, in which two were reported to be truly associated with seed development, and two (Os02g0626100 for GL-MC and Os02g0538000 for GW-MC) were new. Moreover, 1, 2, and 1 known genes, along with 8 additional candidate genes and 2 candidate GEIs, were found to be around QTNs and QEIs for GW, GL, and TGW, respectively in MC and RR joint analysis, in which 3 additional candidate genes were key and new. Our results provided a solid foundation for genetic improvement and molecular breeding in MC and RR.

Identification of QTN-by-environment interactions for yield related traits in maize under multiple abiotic stresses.

Introduction: Quantitative trait nucleotide (QTN)-by-environment interactions (QEIs) play an increasingly essential role in the genetic dissection of complex traits in crops as global climate change accelerates. The abiotic stresses, such as drought and heat, are the major constraints on maize yields. Multi-environment joint analysis can improve statistical power in QTN and QEI detection, and further help us to understand the genetic basis and provide implications for maize improvement. Methods: In this study, 3VmrMLM was applied to identify QTNs and QEIs for three yield-related traits (grain yield, anthesis date, and anthesis-silking interval) of 300 tropical and subtropical maize inbred lines with 332,641 SNPs under well-watered and drought and heat stresses. Results: Among the total 321 genes around 76 QTNs and 73 QEIs identified in this study, 34 known genes were reported in previous maize studies to be truly associated with these traits, such as ereb53 (GRMZM2G141638) and thx12 (GRMZM2G016649) associated with drought stress tolerance, and hsftf27 (GRMZM2G025685) and myb60 (GRMZM2G312419) associated with heat stress. In addition, among 127 homologs in Arabidopsis out of 287 unreported genes, 46 and 47 were found to be significantly and differentially expressed under drought vs well-watered treatments, and high vs. normal temperature treatments, respectively. Using functional enrichment analysis, 37 of these differentially expressed genes were involved in various biological processes. Tissue-specific expression and haplotype difference analysis further revealed 24 candidate genes with significantly phenotypic differences across gene haplotypes under different environments, of which the candidate genes GRMZM2G064159, GRMZM2G146192, and GRMZM2G114789 around QEIs may have gene-by-environment interactions for maize yield. Discussion: All these findings may provide new insights for breeding in maize for yield-related traits adapted to abiotic stresses.

Genome-Wide Association Studies Using 3VmrMLM Model Provide New Insights into Branched-Chain Amino Acid Contents in Rice Grains.

Rice (Oryza sativa L.) is a globally important food source providing carbohydrates, amino acids, and dietary fiber for humans and livestock. The branched-chain amino acid (BCAA) level is a complex trait related to the nutrient quality of rice. However, the genetic mechanism underlying the BCAA (valine, leucine, and isoleucine) accumulation in rice grains remains largely unclear. In this study, the grain BCAA contents and 239,055 SNPs of a diverse panel containing 422 rice accessions were adopted to perform a genome-wide association study (GWAS) using a recently proposed 3VmrMLM model. A total of 357 BCAA-content-associated main-effect quantitative trait nucleotides (QTNs) were identified from 15 datasets (12 BCAA content datasets and 3 BLUP datasets of BCAA). Furthermore, the allelic variation of two novel candidate genes, LOC_Os01g52530 and LOC_Os06g15420, responsible for the isoleucine (Ile) content alteration were identified. To reveal the genetic basis of the potential interactions between the gene and environmental factor, 53 QTN-by-environment interactions (QEIs) were detected using the 3VmrMLM model. The LOC_Os03g24460, LOC_Os01g55590, and LOC_Os12g31820 were considered as the candidate genes potentially contributing to the valine (Val), leucine (Leu), and isoleucine (Ile) accumulations, respectively. Additionally, 10 QTN-by-QTN interactions (QQIs) were detected using the 3VmrMLM model, which were putative gene-by-gene interactions related to the Leu and Ile contents. Taken together, these findings suggest that the implementation of the 3VmrMLM model in a GWAS may provide new insights into the deeper understanding of BCAA accumulation in rice grains. The identified QTNs/QEIs/QQIs serve as potential targets for the genetic improvement of rice with high BCAA levels.

Genome-wide association study of coleoptile length with Shanxi wheat.

In arid and semi-arid regions, coleoptile length is a vital agronomic trait for wheat breeding. The coleoptile length determines the maximum depth that seeds can be sown, and it is critical for establishment of the crop. Therefore, identifying loci associated with coleoptile length in wheat is essential. In the present study, 282 accessions from Shanxi Province representing wheat breeding for the Loess Plateau were grown under three experimental conditions to study coleoptile length. The results of phenotypic variation indicated that drought stress and light stress could lead to shortening of coleoptile length. Under drought stress the growth rate of environmentally sensitive cultivars decreased more than insensitive cultivars. The broad-sense heritability (H 2) of BLUP (best linear unbiased prediction) under various conditions showed G × E interaction for coleoptile length but was mainly influenced by heredity. Correlation analysis showed that correlation between plant height-related traits and coleoptile length was significant in modern cultivars whereas it was not significant in landraces. A total of 45 significant marker-trait associations (MTAs) for coleoptile length in the three conditions were identified using the 3VmrMLM (3 Variance-component multi-locus random-SNP-effect Mixed Linear Model) and MLM (mixed linear model). In total, nine stable genetic loci were identified via 3VmrMLM under the three conditions, explaining 2.94-7.79% of phenotypic variation. Five loci on chromosome 2B, 3A, 3B, and 5B have not been reported previously. Six loci had additive effects toward increasing coleoptile length, three of which are novel. Molecular markers for the loci with additive effects on coleoptile length can be used to breed cultivars with long coleoptiles.

Genome-wide association studies reveal novel QTLs, QTL-by-environment interactions and their candidate genes for tocopherol content in soybean seed.

Genome-wide association studies (GWAS) is an efficient method to detect quantitative trait locus (QTL), and has dissected many complex traits in soybean [Glycine max (L.) Merr.]. Although these results have undoubtedly played a far-reaching role in the study of soybean biology, environmental interactions for complex traits in traditional GWAS models are frequently overlooked. Recently, a new GWAS model, 3VmrMLM, was established to identify QTLs and QTL-by-environment interactions (QEIs) for complex traits. In this study, the GLM, MLM, CMLM, FarmCPU, BLINK, and 3VmrMLM models were used to identify QTLs and QEIs for tocopherol (Toc) content in soybean seed, including δ-Tocotrienol (δ-Toc) content, γ-Tocotrienol (γ-Toc) content, α-Tocopherol (α-Toc) content, and total Tocopherol (T-Toc) content. As a result, 101 QTLs were detected by the above methods in single-environment analysis, and 57 QTLs and 13 QEIs were detected by 3VmrMLM in multi-environment analysis. Among these QTLs, some QTLs (Group I) were repeatedly detected three times or by at least two models, and some QTLs (Group II) were repeatedly detected only by 3VmrMLM. In the two Groups, 3VmrMLM was able to correctly detect all known QTLs in group I, while good results were achieved in Group II, for example, 8 novel QTLs were detected in Group II. In addition, comparative genomic analysis revealed that the proportion of Glyma_max specific genes near QEIs was higher, in other words, these QEIs nearby genes are more susceptible to environmental influences. Finally, around the 8 novel QTLs, 11 important candidate genes were identified using haplotype, and validated by RNA-Seq data and qRT-PCR analysis. In summary, we used phenotypic data of Toc content in soybean, and tested the accuracy and reliability of 3VmrMLM, and then revealed novel QTLs, QEIs and candidate genes for these traits. Hence, the 3VmrMLM model has broad prospects and potential for analyzing the genetic structure of complex quantitative traits in soybean.

Identification of QTNs, QTN-by-environment interactions and genes for yield-related traits in rice using 3VmrMLM.

Rice, which supports more than half the population worldwide, is one of the most important food crops. Thus, potential yield-related quantitative trait nucleotides (QTNs) and QTN-by-environment interactions (QEIs) have been used to develop efficient rice breeding strategies. In this study, a compressed variance component mixed model, 3VmrMLM, in genome-wide association studies was used to detect QTNs for eight yield-related traits of 413 rice accessions with 44,000 single nucleotide polymorphisms. These traits include florets per panicle, panicle fertility, panicle length, panicle number per plant, plant height, primary panicle branch number, seed number per panicle, and flowering time. Meanwhile, QTNs and QEIs were identified for flowering times in three different environments and five subpopulations. In the detections, a total of 7~23 QTNs were detected for each trait, including the three single-environment flowering time traits. In the detection of QEIs for flowering time in the three environments, 21 QTNs and 13 QEIs were identified. In the five subpopulation analyses, 3~9 QTNs and 2~4 QEIs were detected for each subpopulation. Based on previous studies, we identified 87 known genes around the significant/suggested QTNs and QEIs, such as LOC_Os06g06750 (OsMADS5) and LOC_Os07g47330 (FZP). Further differential expression analysis and functional enrichment analysis identified 30 candidate genes. Of these candidate genes, 27 genes had high expression in specific tissues, and 19 of these 27 genes were homologous to known genes in Arabidopsis. Haplotype difference analysis revealed that LOC_Os04g53210 and LOC_Os07g42440 are possibly associated with yield, and LOC_Os04g53210 may be useful around a QEI for flowering time. These results provide insights for future breeding for high quality and yield in rice.

Genome-wide association studies for soybean epicotyl length in two environments using 3VmrMLM.

Germination of soybean seed is the imminent vital process after sowing. The status of plumular axis and radicle determine whether soybean seed can emerge normally. Epicotyl, an organ between cotyledons and first functional leaves, is essential for soybean seed germination, seedling growth and early morphogenesis. Epicotyl length (EL) is a quantitative trait controlled by multiple genes/QTLs. Here, the present study analyzes the phenotypic diversity and genetic basis of EL using 951 soybean improved cultivars and landraces from Asia, America, Europe and Africa. 3VmrMLM was used to analyze the associations between EL in 2016 and 2020 and 1,639,846 SNPs for the identification of QTNs and QTN-by-environment interactions (QEIs)".A total of 180 QTNs and QEIs associated with EL were detected. Among them, 74 QTNs (ELS_Q) and 16 QEIs (ELS_QE) were identified to be associated with ELS (epicotyl length of single plant emergence), and 60 QTNs (ELT_Q) and 30 QEIs (ELT_QE) were identified to be associated with ELT (epicotyl length of three seedlings). Based on transcript abundance analysis, GO (Gene Ontology) enrichment and haplotype analysis, ten candidate genes were predicted within nine genic SNPs located in introns, upstream or downstream, which were supposed to be directly or indirectly involved in the process of seed germination and seedling development., Of 10 candidate genes, two of them (Glyma.04G122400 and Glyma.18G183600) could possibly affect epicotyl length elongation. These results indicate the genetic basis of EL and provides a valuable basis for specific functional studies of epicotyl traits.

Identification of QTN-by-environment interactions and their candidate genes for soybean seed oil-related traits using 3VmrMLM.

Introduction: Although seed oil content and its fatty acid compositions in soybean were affected by environment, QTN-by-environment (QEIs) and gene-by-environment interactions (GEIs) were rarely reported in genome-wide association studies. Methods: The 3VmrMLM method was used to associate the trait phenotypes, measured in five to seven environments, of 286 soybean accessions with 106,013 SNPs for detecting QTNs and QEIs. Results: Seven oil metabolism genes (GmSACPD-A, GmSACPD-B, GmbZIP123, GmSWEET39, GmFATB1A, GmDGAT2D, and GmDGAT1B) around 598 QTNs and one oil metabolism gene GmFATB2B around 54 QEIs were verified in previous studies; 76 candidate genes and 66 candidate GEIs were predicted to be associated with these traits, in which 5 genes around QEIs were verified in other species to participate in oil metabolism, and had differential expression across environments. These genes were found to be related to soybean seed oil content in haplotype analysis. In addition, most candidate GEIs were co-expressed with drought response genes in co-expression network, and three KEGG pathways which respond to drought were enriched under drought stress rather than control condition; six candidate genes were hub genes in the co-expression networks under drought stress. Discussion: The above results indicated that GEIs, together with drought response genes in co-expression network, may respond to drought, and play important roles in regulating seed oil-related traits together with oil metabolism genes. These results provide important information for genetic basis, molecular mechanisms, and soybean breeding for seed oil-related traits.