Phenotypic variation and genome-wide association studies of main culm panicle node number, maximum node production rate, and degree-days to heading in rice.

BACKGROUND: Grain yield is a complex trait that results from interaction between underlying phenotypic traits and climatic, edaphic, and biotic variables. In rice, main culm panicle node number (MCPNN; the node number on which the panicle is borne) and maximum node production rate (MNPR; the number of leaves that emerge per degree-day > 10°C) are primary phenotypic plant traits that have significant positive direct effects on yield-related traits. Degree-days to heading (DDTH), which has a significant positive effect on grain yield, is influenced by the interaction between MCPNN and MNPR. The objective of this research is to assess the phenotypic variation of MCPNN, MNPR, and DDTH in a panel of diverse rice accessions, determine regions in the rice genome associated with these traits using genome-wide association studies (GWAS), and identify putative candidate genes that control these traits. RESULTS: Considerable variation was observed for the three traits in a 220-genotype diverse rice population. MCPNN ranged from 8.1 to 20.9 nodes in 2018 and from 9.9 to 21.0 nodes in 2019. MNPR ranged from 0.0097 to 0.0214 nodes/degree day > 10°C in 2018 and from 0.0108 to 0.0193 nodes/degree-day > 10°C in 2019. DDTH ranged from 713 to 2,345 degree-days > 10°C in 2018 and from 778 to 2,404 degree-days > 10°C in 2019. Thirteen significant (P < 2.91 x 10-7) trait-single nucleotide polymorphism (SNP) associations were identified using the multilocus mixed linear model for GWAS. Significant associations between MCPNN and three SNPs in chromosome 2 (S02_12032235, S02_11971745, and S02_12030176) were detected with both the 2018 and best linear unbiased prediction (BLUP) datasets. Nine SNPs in chromosome 6 (S06_1970442, S06_2310856, S06_2550351, S06_1968653, S06_2296852, S06_1968680, S06_1968681, S06_1970597, and S06_1970602) were significantly associated with MNPR in the 2019 dataset. One SNP in chromosome 11 (S11_29358169) was significantly associated with the DDTH in the BLUP dataset. CONCLUSIONS: This study identifies SNP markers that are putatively associated with MCPNN, MNPR, and DDTH. Some of these SNPs were located within or near gene models, which identify possible candidate genes involved in these traits. Validation of the putative candidate genes through expression and gene editing analyses are necessary to confirm their roles in regulating MCPNN, MNPR, and DDTH. Identifying the underlying genetic basis for primary phenotypic traits MCPNN and MNPR could lead to the development of fast and efficient approaches for their estimation, such as marker-assisted selection and gene editing, which is essential in increasing breeding efficiency and enhancing grain yield in rice. On the other hand, DDTH is a resultant variable that is highly affected by nitrogen and water management, plant density, and several other factors.

GWASpro: a high-performance genome-wide association analysis server.

SUMMARY: We present GWASpro, a high-performance web server for the analyses of large-scale genome-wide association studies (GWAS). GWASpro was developed to provide data analyses for large-scale molecular genetic data, coupled with complex replicated experimental designs such as found in plant science investigations and to overcome the steep learning curves of existing GWAS software tools. GWASpro supports building complex design matrices, by which complex experimental designs that may include replications, treatments, locations and times, can be accounted for in the linear mixed model. GWASpro is optimized to handle GWAS data that may consist of up to 10 million markers and 10 000 samples from replicable lines or hybrids. GWASpro provides an interface that significantly reduces the learning curve for new GWAS investigators. AVAILABILITY AND IMPLEMENTATION: GWASpro is freely available at https://bioinfo.noble.org/GWASPRO. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

QTL mapping and confirmation for tolerance of anaerobic conditions during germination derived from the rice landrace Ma-Zhan Red.

Wide adoption of direct-seeded rice practices has been hindered by poorly leveled fields, heavy rainfall and poor drainage, which cause accumulation of water in the fields shortly after sowing, leading to poor crop establishment. This is due to the inability of most rice varieties to germinate and reach the water surface under complete submergence. Hence, tolerance of anaerobic conditions during germination is an essential trait for direct-seeded rice cultivation in both rainfed and irrigated ecosystems. A QTL study was conducted to unravel the genetic basis of tolerance of anaerobic conditions during germination using a population derived from a cross between IR42, a susceptible variety, and Ma-Zhan Red, a tolerant landrace from China. Phenotypic data was collected based on the survival rates of the seedlings at 21 days after sowing of dry seeds under 10 cm of water. QTL analysis of the mapping population consisting of 175 F2:3 families genotyped with 118 SSR markers identified six significant QTLs on chromosomes 2, 5, 6, and 7, and in all cases the tolerant alleles were contributed by Ma-Zhan Red. The largest QTL on chromosome 7, having a LOD score of 14.5 and an R (2) of 31.7 %, was confirmed using a BC2F3 population. The QTLs detected in this study provide promising targets for further genetic characterization and for use in marker-assisted selection to rapidly develop varieties with improved tolerance to anaerobic condition during germination. Ultimately, this trait can be combined with other abiotic stress tolerance QTLs to provide resilient varieties for direct-seeded systems.

Genetic architecture of head rice and rice chalky grain percentages using genome-wide association studies.

High head rice and low chalky grain percentages are key grain quality traits selected in developing rice cultivars. The objectives of this research were to characterize the phenotypic variation of head rice and chalky grain percentages in a diverse collection of rice accessions, identify single nucleotide polymorphism (SNP) markers associated with each of these traits using genome-wide association studies (GWAS), and identify putative candidate genes linked to the SNPs identified by GWAS. Diverse rice varieties, landraces, and breeding lines were grown at the Texas A&M AgriLife Research Center in Beaumont. Head rice percentages (HRP) and chalky grain percentages (CGP) of 195 and 199 non-waxy accessions were estimated in 2018 and 2019, respectively. Phenotypic data were analyzed along with 854,832 SNPs using three statistical models: mixed linear model (MLM), multi-locus mixed model (MLMM), and fixed and random model circulating probability unification (FarmCPU). Significant variations in HRP and CGP were observed between rice accessions. Two significant marker-trait associations (MTAs) were detected on chromosomes 1 and 2, respectively, based on best linear unbiased prediction (BLUP) values in 2018, while in 2019, one SNP was significantly associated with HRP in each of chromosomes 6, 8, 9, and 11, and two in chromosome 7. CGP was significantly associated with five SNPs located in chromosomes 2, 4, 6, and 8 in the 2018 study and ten SNPs in chromosomes 1, 2, 3, 4, 7, 8, 11, and 12 in the 2019 study. The SNPs are located within or linked to putative candidate genes involved in HRP and CGP. This study reports five and ten novel MTAs for HRP and CGP, respectively, while three and five MTAs co-located with previously reported quantitative trait loci for HRP and CGP, respectively. The validation of candidate genes for their roles in determining HRP and CGP is necessary to design functional molecular markers that can be used to effectively develop rice cultivars with desirable grain quality.

Phenotypic and genome-wide association analyses for nitrogen use efficiency related traits in maize (Zea mays L.) exotic introgression lines.

Nitrogen (N) limits crop production, yet more than half of N fertilizer inputs are lost to the environment. Developing maize hybrids with improved N use efficiency can help minimize N losses and in turn reduce adverse ecological, economical, and health consequences. This study aimed to identify single nucleotide polymorphisms (SNPs) associated with agronomic traits (plant height, grain yield, and anthesis to silking interval) under high and low N conditions. A genome-wide association study (GWAS) was conducted using 181 doubled haploid (DH) lines derived from crosses between landraces from the Germplasm Enhancement of Maize (BGEM lines) project and two inbreds, PHB47 and PHZ51. These DH lines were genotyped using 62,077 SNP markers. The same lines from the per se trials were used as parental lines for the testcross field trials. Plant height, anthesis to silking interval, and grain yield were collected from high and low N conditions in three environments for both per se and testcross trials. We used three GWAS models, namely, general linear model (GLM), mixed linear model (MLM), and Fixed and Random model Circulating Probability Unification (FarmCPU) model. We observed significant genetic variation among the DH lines and their derived testcrosses. Interestingly, some testcrosses of exotic introgression lines were superior under high and low N conditions compared to the check hybrid, PHB47/PHZ51. We detected multiple SNPs associated with agronomic traits under high and low N, some of which co-localized with gene models associated with stress response and N metabolism. The BGEM panel is, thus, a promising source of allelic diversity for genes controlling agronomic traits under different N conditions.

Identifying novel QTLs for submergence tolerance in rice cultivars IR72 and Madabaru.

Short-term submergence is a recurring problem in many rice production areas. The SUB1 gene, derived from the tolerant variety FR13A, has been transferred to a number of widely grown varieties, allowing them to withstand complete submergence for up to 2 weeks. However, in areas where longer-term submergence occurs, improved varieties having higher tolerance levels are needed. To search for novel quantitative trait loci (QTLs) from other donors, an F(2:3) population between IR72 and Madabaru, both moderately tolerant varieties, was investigated. After a repeated phenotyping of 466 families under submergence stress, a subset of 80 families selected from the two extreme phenotypic tails was used for the QTL analysis. Phenotypic data showed transgressive segregation, with several families having an even higher survival rate than the FR13A-derived tolerant check (IR40931). Four QTLs were identified on chromosomes 1, 2, 9, and 12; the largest QTL on chromosome 1 had a LOD score of 11.2 and R (2) of 52.3%. A QTL mapping to the SUB1 region on chromosome 9, with a LOD score of 3.6 and R (2) of 18.6%, had the tolerant allele from Madabaru, while the other three QTLs had tolerant alleles from IR72. The identification of three non-SUB1 QTLs from IR72 suggests that an alternative pathway may be present in this variety that is independent of the ethylene-dependent pathway mediated by the SUB1A gene. These novel QTLs can be combined with SUB1 using marker assisted backcrossing in an effort to enhance the level of submergence tolerance for flood-prone areas.

Comparison of phenotypic versus marker-assisted background selection for the SUB1 QTL during backcrossing in rice.

Marker assisted backcrossing has been used effectively to transfer the submergence tolerance gene SUB1 into popular rice varieties, but the approach can be costly. The selection strategy comprising foreground marker and phenotypic selection was investigated as an alternative. The non-significant correlation coefficients between ranking of phenotypic selection and ranking of background marker selection in BC(2)F(1), BC(3)F(1) and BC(3)F(2) generations indicated inefficiency of phenotypic selection compared to marker-assisted background selection with respect to recovery of the recipient genome. In addition, the introgression size of the chromosome fragment containing SUB1 was approximately 17 Mb, showing the effects of linkage drag. The significant correlation coefficient between rankings of phenotypic selection with the percentage of recipient alleles in the BC(1)F(1) generation suggested that background selection could be avoided in this generation to minimize the genotyping cost. The phenotypically selected best plant of the BC(3)F(1) generation was selfed and backcross recombinant lines were selected in the resulting BC(3)F(4) generation. The selection strategy could be appropriate for the introgression of SUB1 QTL in countries that lack access to high-throughput genotyping facilities.

Development of submergence-tolerant rice cultivars: the Sub1 locus and beyond.

BACKGROUND AND AIMS: Submergence is a recurring problem in the rice-producing rainfed lowlands of south and south-east Asia. Developing rice cultivars with tolerance of submergence and with agronomic and quality traits acceptable to farmers is a feasible approach to address this problem. The objectives of this study were to (a) develop mega varieties with Sub1 introgression that are submergence tolerant, (b) assess the performance of Sub1 in different genetic backgrounds, (c) determine the roles of the Sub1A and Sub1C genes in conferring tolerance, and (d) assess the level of tolerance in F(1) hybrids heterozygous for the Sub1A-1-tolerant allele. METHODS: Tolerant varieties were developed by marker-assisted backcrossing through two or three backcrosses, and their performance was evaluated to determine the effect of Sub1 in different genetic backgrounds. The roles of Sub1A and Sub1C in conferring the tolerant phenotype were further investigated using recombinants identified within the Sub1 gene cluster based on survival and gene expression data. KEY RESULTS: All mega varieties with Sub1 introgression had a significantly higher survival rate than the original parents. An intolerant Sub1C allele combined with the tolerant Sub1A-1 allele did not significantly reduce the level of tolerance, and the Sub1C-1 expression appeared to be independent of the Sub1A allele; however, even when Sub1C-1 expression is completely turned off in the presence of Sub1A-2, plants remained intolerant. Survival rates and Sub1A expression were significantly lower in heterozygotes compared with the homozygous tolerant parent. CONCLUSIONS: Sub1 provided a substantial enhancement in the level of tolerance of all the sensitive mega varieties. Sub1A is confirmed as the primary contributor to tolerance, while Sub1C alleles do not seem important. Lack of dominance of Sub1 suggests that the Sub1A-1 allele should be carried by both parents for developing tolerant rice hybrids.

Genome-wide association studies of doubled haploid exotic introgression lines for root system architecture traits in maize (Zea mays L.).

Root system architecture (RSA) is becoming recognized as important for water and nutrient acquisition in plants. This study focuses on finding single nucleotide polymorphisms (SNPs) associated with seedling RSA traits from 300 doubled haploid (DH) lines derived from crosses between Germplasm Enhancement of Maize (GEM) accessions and inbred lines PHB47 and PHZ51. These DH lines were genotyped using 62,077 SNP markers, while root and shoot phenotype data were collected from 14-day old seedlings. Genome-wide association studies (GWAS) were conducted using three models to offset false positives/negatives. Multiple SNPs associated with seedling root traits were detected, some of which were within or linked to gene models that showed expression in seedling roots. Significant trait associations involving the SNP S5_152926936 on Chromosome 5 were detected in all three models, particularly the trait network area. The SNP is within the gene model GRMZM2G021110, which is expressed in roots at seedling stage. SNPs that were significantly associated with seedling root traits, and closely linked to gene models that encode proteins associated with root development were also detected. This study shows that the GEM-DH panel may be a source of allelic diversity for genes controlling seedling root development.

Brassinosteroid and gibberellin control of seedling traits in maize (Zea mays L.).

In this study, we established two doubled haploid (DH) libraries with a total of 207 DH lines. We applied BR and GA inhibitors to all DH lines at seedling stage and measured seedling BR and GA inhibitor responses. Moreover, we evaluated field traits for each DH line (untreated). We conducted genome-wide association studies (GWAS) with 62,049 genome wide SNPs to explore the genetic control of seedling traits by BR and GA. In addition, we correlate seedling stage hormone inhibitor response with field traits. Large variation for BR and GA inhibitor response and field traits was observed across these DH lines. Seedling stage BR and GA inhibitor response was significantly correlate with yield and flowering time. Using three different GWAS approaches to balance false positive/negatives, multiple SNPs were discovered to be significantly associated with BR/GA inhibitor responses with some localized within gene models. SNPs from gene model GRMZM2G013391 were associated with GA inhibitor response across all three GWAS models. This gene is expressed in roots and shoots and was shown to regulate GA signaling. These results show that BRs and GAs have a great impact for controlling seedling growth. Gene models from GWAS results could be targets for seeding traits improvement.

Gibberellins Promote Brassinosteroids Action and Both Increase Heterosis for Plant Height in Maize (Zea mays L.).

Brassinosteroids (BRs) and Gibberellins (GAs) are two classes of plant hormones affecting plant height (PHT). Thus, manipulation of BR and GA levels or signaling enables optimization of crop grain and biomass yields. We established backcross (BC) families, selected for increased PHT, in two elite maize inbred backgrounds. Various exotic accessions used in the germplasm enhancement in maize project served as donors. BC1-derived doubled haploid lines in the same two elite maize inbred backgrounds established without selection for plant height were included for comparison. We conducted genome-wide association studies to explore the genetic control of PHT by BR and GA. In addition, we used BR and GA inhibitors to compare the relationship between PHT, BR, and GA in inbred lines and heterozygotes from a physiological and biological perspective. A total of 73 genomic loci were discovered to be associated with PHT, with seven co-localized with GA, and two co-localized with BR candidate genes. PHT determined in field trials was significantly correlated with seedling stage BR and GA inhibitor responses. However, this observation was only true for maize heterozygotes, not for inbred lines. Path analysis results suggest that heterozygosity increases GA levels, which in turn promote BR levels. Thus, at least part of heterosis for PHT in maize can be explained by increased GA and BR levels, and seedling stage hormone inhibitor response is promising to predict heterosis for PHT.