An efficient method for screening rice breeding lines against races of Magnaporthe oryzae.

Rice blast, caused by Magnaporthe oryzae, is the most destructive rice disease worldwide. The disease symptoms are usually expressed on the leaf and panicle. The leaf disease intensity in controlled environmental conditions is frequently quantified using a 0-5 scale, where 0 represents the absence of symptoms and 5 represents large eyespot lesions. However, this scale restricts the qualitative classification of the varieties into intermediate resistant and susceptible categories. Here we develop a 0-6 scale for blast disease that allows proper assignment of rice breeding lines and varieties into six resistance levels (highly resistant, resistant, moderate resistant, moderate susceptible, susceptible, and highly susceptible). We evaluated 41 common rice varieties against four major blast races (IB1, IB17, IB49, and IE1-K). Varieties carrying the Pi-ta gene were either highly resistant, resistant, or moderate resistant to IB17. The IE1-K race was able to break Pi-ta-mediate resistance of the rice varieties. The Pi-z gene conferred resistance to the IB17 and IE1-K races. The varieties M201, Cheniere, and Frontier were highly susceptible (score 6; 100% disease) to the race IE1-K. Moreover, varieties that were resistant or susceptible to all four blast races also showed similar levels of resistance/susceptibility to blast disease in the field. Taken together, our data proved that the 0-6 blast scale can efficiently determine the resistance levels of rice varieties against major blast races. This robust method will assist rice breeding programs to incorporate durable resistance against major and emerging blast races.

Construction of a single nucleotide polymorphism linkage map and identification of quantitative trait loci controlling heat tolerance in cowpea, Vigna unguiculata (L.) Walp.

Plant tolerance to heat or high temperature is crucial to crop production, especially in the situation of elevated temperature resulting from global climate change. Cowpea, Vigna unguiculata (L.) Walp., is an internationally important legume food crop and an excellent pool of genes for numerous traits resilient to environmental extremes, particularly heat and drought. Here, we report a single nucleotide polymorphism (SNP) genetic map for cowpea and identification of the loci controlling the heat tolerance in the species. The SNP map consists of 531 bins containing 4,154 SNPs grouped into 11 linkage groups, and collectively spans 1,084.7 cM, thus having a density of one SNP in 0.26 cM or 149 kb. The 11 linkage groups of the map were aligned to the 11 cowpea chromosomes. Quantitative trait locus (QTL) mapping identified nine QTLs responsible for the cowpea heat tolerance on seven of the 11 chromosomes, with each QTL explaining 6.5-21.8% of heat tolerance phenotypic variation. Moreover, we aligned these nine QTLs to the cowpea genome. Each of the QTLs was positioned in a genomic region ranging from 209,000 bp to 12,590,450 bp, and the QTL with the largest effect (21.8%) on heat tolerance, qHT4-1, was located within an interval of only 234,195 bp. These results provide SNP markers useful for marker-assisted selection for heat tolerance and lay a foundation for cloning, characterization, and applications of the genes controlling the cowpea heat tolerance for heat tolerance genetic improvement in cowpea and related crops.

Identification and mapping of a novel resistance gene to the rice pathogen, Cercospora janseana.

KEY MESSAGE: The genetic architecture of resistance to Cercospora janseana was examined, and a single resistance locus was identified. A SNP marker was identified and validated for utilization in U.S. breeding germplasm Cercospora janseana (Racib.) is a fungal pathogen that causes narrow brown leaf spot (NBLS) in rice. Although NBLS is a major disease in the southern United States and variation in resistance among U.S. rice germplasm exists, little is known about the genetic architecture underlying the trait. In this study, a recombinant inbred line population was evaluated for NBLS resistance under natural disease infestation in the field across three years. A single, large-effect QTL, CRSP-2.1, was identified that explained 81.4% of the phenotypic variation. The QTL was defined to a 532 kb physical interval and 13 single nucleotide polymorphisms (SNPs) were identified across the region to characterize the haplotype diversity present in U.S. rice germplasm. A panel of 387 U.S. rice germplasm was genotyped with the 13 haplotype SNPs and phenotyped over two years for NBLS resistance. Fourteen haplotypes were identified, with six haplotypes accounting for 94% of the panel. The susceptible haplotype from the RIL population was the only susceptible haplotype observed in the U.S. germplasm. A single SNP was identified that distinguished the susceptible haplotype from all resistant haplotypes, explaining 52.7% of the phenotypic variation for NBLS resistance. Pedigree analysis and haplotype characterization of historical germplasm demonstrated that the susceptible haplotype was introduced into Southern U.S. germplasm through the California line L-202 into the Louisiana variety Cypress. Cypress was extensively used as a parent over the last 25 years, resulting in the susceptible CRSP-2.1 allele increasing in frequency from zero to 44% in the modern U.S. germplasm panel.

Quantitative trait loci influencing days to flowering and plant height in cowpea, Vigna unguiculata (L.) Walp.

Cowpea (Vigna unguiculate (L.) Walp.) is a worldwide important multifunctional legume crop for food grain, vegetable, fodder, and cover crop. Nevertheless, only limited research has been conducted on agronomic traits. Here, we report quantitative trait locus (QTL) analysis of the days to flowering (DTF) and plant height (PH) using a dense SNP linkage map recently developed from a recombinant inbred line (RIL) population derived from a cross between Golden Eye Cream and IT98K-476-8. The population was phenotyped for DTF and PH through field and greenhouse trials under two environments. The QTLs controlling these traits were mapped using multiple-environment combined and individual trial phenotypic data. The combined data analysis identified one major QTL (qDTF9.1) for DTF, and one major QTL (qPH9.1) and a minor QTL (qPH4.1) for PH. qDTF9.1 and qPH9.1 were adjacent to each other on Chromosome 9 and each explained 29.3% and 29.5% of the phenotypic variation (PVE), respectively. The individual trial data analysis identified a minor QTL (qDTF2.1) on Chromosome 2 for DTF and two minor QTLs (qPH4.1 and qPH4.2) on Chromosome 4 for PH, while the major QTLs, qDTF9.1 and qPH9.1, were consistently identified in all trials conducted. Epistasis analysis revealed that qDTF9.1 interacted with one locus on Chromosome 4, contributed 50% of the PVE, and qPH9.1 interacted with one locus on each of Chromosomes 4 and 6, contributing 30% and 23% of the PVE, respectively, suggesting that epistasis plays an important role in the trait performance. These results, therefore, provide a deeper understanding of the genetic architecture of plant DTF and PH, and molecular tools necessary for cloning the genes and for enhanced cowpea breeding.

Genetic Characterization of the Partial Disease Resistance of Rice to Bacterial Panicle Blight and Sheath Blight by Combined QTL Linkage and QTL-seq Analyses.

Bacterial panicle blight (BPB) and sheath blight (SB) are major diseases of rice and few cultivars have shown a high level of resistance to these diseases. A recombinant inbred line (RIL) population developed from the U.S. cultivars Jupiter (moderately resistant) and Trenasse (susceptible) was investigated to identify loci associated with the partial disease resistance to BPB and SB. Disease phenotypes in BPB and SB, as well as the days-to-heading (DTH) trait, were evaluated in the field. DTH was correlated to BPB and SB diseases, while BPB was positively correlated to SB in the field trials with this RIL population. Genotyping was performed using Kompetitive Allele Specific PCR (KASP) assays and whole-genome sequence (WGS) analyses. Quantitative trait locus (QTL) mapping and bulk segregant analysis using a set of WGS data (QTL-seq) detected a major QTL on the upper arm of chromosome 3 for BPB, SB, and DTH traits within the 1.0-1.9 Mb position. Additional QTLs associated with BPB and SB were also identified from other chromosomes by the QTL-seq analysis. The QTLs identified in this study contain at least nine candidate genes that are predicted to have biological functions in defense or flowering. These findings provide an insight into the complex nature of the quantitative resistance to BPB and SB, which may also be closely linked to the flowering trait.

Discovery and Validation of Grain Shape Loci in U.S. Rice Germplasm Through Haplotype Characterization.

Rice grain shape is a major determinant of rice market value and the end-use. We mapped quantitative trait loci (QTL) for grain shape traits in a bi-parental recombinant inbred line population (Trenasse/Jupiter) and discovered two major grain length QTLs-qGL3.1 and qGL7.1. Previously, a major grain shape gene GS3 was reported in the qGL3.1 region and grain length gene GL7 was reported to be encompassing qGL7.1 locus. The re-sequencing SNP data on the International Rice Research Institute (IRRI) 3K Rice Genome Project (RGP) panel were obtained from the IRRI SNP-Seek database for both genes and haplotype diversity was characterized for each gene in this diverse panel. United States rice germplasm was not well represented in the IRRI 3K RGP database. Therefore, a minimum SNP set was identified for each gene that could differentiate all the characterized haplotypes. These haplotypes in the 3K RGP panel were screened across 323 elite U.S. genotypes using the minimum SNP set. The screening of haplotypes and phenotype association confirmed the role of GS3 under qGL3.1. However, screening of the GL7 haplotypes in the U.S. germplasm panel showed that GL7 did not play a role in qGL7.1, and in addition, GL7.1 did not segregate in the Trenasse/Jupiter RIL population. This concluded that qGL7.1 is a novel QTL discovered on chr7 for grain shape in the Trenasse/Jupiter RIL population. A high-throughput KASP-based SNP marker for each locus (GS3 and qGL7.1) was identified and validated in elite U.S. rice germplasm to be used in an applied rice breeding program.

Development and validation of an optimized marker set for genomic selection in southern U.S. rice breeding programs.

The potential of genomic selection (GS) to increase the efficiency of breeding programs has been clearly demonstrated; however, the implementation of GS in rice (Oryza sativa L.) breeding programs has been limited. In recent years, efforts have begun to work toward implementing GS into the Louisiana State University (LSU) Agricultural Center rice breeding program. One of the first steps for successful GS implementation is to establish a suitable marker set for the target germplasm and a reliable, cost-effective genotyping platform capable of providing informative marker data with an adequate turnaround time. The objective of this study was to develop a marker set for routine GS and demonstrate its effectiveness in southern U.S. rice germplasm. The utility of the resulting marker set, the LSU500, for GS applications was demonstrated using four years of breeding data across 7,607 experimental lines and four elite biparental populations. The predictive ability of GS ranged from 0.13 to 0.78 for key traits across different market classes and yield trials. Comparisons between phenotypic selection and GS within biparental populations demonstrates similar performance of GS compared with phenotypic selection in predicting future performance. The prediction accuracies obtained with the LSU500 marker set demonstrates the utility of this marker set for cost-effective GS applications in southern U.S. rice breeding programs. The LSU500 marker set has been established through the genotyping service provider Agriplex Genomics, and in the future, it will undergo improvements to reduce the cost and increase the accuracy of GS.

A SNP marker set was developed for genomic selection in southern U.S. rice breeding programs. Predictive ability across target germplasm was shown with 3 yr of data (4,078 lines). Within-population predictive ability was shown across four biparental populations. Genomic and phenotypic selection ability to predict future performance was compared.

Characterization of Haplotype Diversity in the BADH2 Aroma Gene and Development of a KASP SNP Assay for Predicting Aroma in U.S. Rice.

BACKGROUND: Aroma is an important grain quality trait in rice, controlled by mutations within the BADH2 gene. The trait is simply inherited, and its importance in variety development makes it a practical target for marker-assisted selection in applied breeding programs. The predominant functional mutation within BADH2, an 8-bp indel, can be reliably detected using a PCR-based assay, but the available assays and associated genotyping platforms are insufficient for large-scale applied molecular breeding applications and are not compatible with outsourcing genotyping services. RESULTS: We first characterized SNP diversity across the BADH2 gene in a collection of 2932 rice varieties to determine the number of gene haplotypes in O. sativa. Using 297 gene-based SNPs, 11 haplotype groups were detected, and subsequently identified a minimal set of nine informative SNPs that uniquely identified the BADH2 haplotypes. These nine SNPs were developed into KASP assays and used to examine a panel of 369 U.S. rice accessions. The panel represented modern breeding germplasm and included all known aroma pedigree sources in U.S. rice. Six haplotypes were detected within the U.S. panel, of which two were found in majority (85%) of varieties. A representative set of 39 varieties from all haplotype groups was evaluated phenotypically to distinguish aromatic from non-aromatic lines. CONCLUSION: One haplotype (Hap 6) was found to be perfectly associated with the aromatic phenotype. A single KASP SNP unique to Hap 6 was demonstrated to reliably differentiate aromatic from non-aromatic rice varieties across U.S. germplasm.

Haplotype Characterization of the sd1 Semidwarf Gene in United States Rice.

CORE IDEAS: Genomic data from diverse germplasm used for application in targeted breeding germplasm. Six SNPs identified that can characterize all haplotypes present at SD1 locus in diverse rice. Three alleles of the SD1 gene identified in US rice germplasm including two semidwarf alleles. Two SNPs identified and validated that differentiate the SD1 allele present in US germplasm. KASP assays designed for both SNPs for use in high-throughput breeding applications. Plant height is an important target in US rice (Oryza sativa L.) breeding programs and the large effect of the sd1 semidwarf gene makes it a suitable target for marker-assisted selection. Although the deletion underlying the semidwarf allele is known and a gel-based DNA marker is available, this marker is not ideal for applied breeding because of throughput and cost constraints. The objectives of this study were to characterize the haplotype diversity at the SD1 locus within US rice germplasm and develop a single nucleotide polymorphism (SNP) assay for breeding applications. The International Rice Research Institute (IRRI) SNP-Seek database was used to characterize the haplotype diversity present at the SD1 locus across a set of rice accessions and seven haplotypes were identified. The US rice germplasm was not well represented in the IRRI database, so a set of six SNPs was identified that could differentiate all detected haplotypes. These SNPs were designed into Kompetitive allele specific polymerase chain reaction (KASP) assays and screened across 359 elite US genotypes. Of the seven haplotypes, two were present within the US germplasm, one of which was the semidwarf deletion allele. A third haplotype was observed within the US medium-grain germplasm and demonstrated to be a semidwarf allele derived from the induced mutation in the 'Calrose76'. Two SNPs were identified that distinguish the three SD1 haplotypes present in the US germplasm. These SNPs were validated across the US germplasm and two biparental populations.