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.

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.

Genetic Dissection of Seedling Stage Salinity Tolerance in Rice Using Introgression Lines of a Salt Tolerant Landrace Nona Bokra.

Salinity is an important abiotic stress affecting rice production worldwide. Development of salt tolerant varieties is the most feasible approach for improving rice productivity in salt affected soils. In rice, seedling stage salinity tolerance is crucial for better crop establishment. Quantitative trait loci (QTL) mapping using introgression lines (ILs) is useful for identification and simultaneous transfer of desirable alleles into elite genetic background. In the present study, 138 ILs derived from the cross between a high yielding elite salt susceptible japonica rice cultivar Jupiter and a salt tolerant indica landrace Nona Bokra were evaluated for salt tolerance at seedling stage in a hydroponics experiment and were genotyped using 126 simple sequence repeat markers. A total of 33 additive QTLs were detected by composite interval mapping for 8 morphophysiological traits. The phenotypic responses, genomic composition, and QTLs identified from the study indicated that Na/K ratio is the key factor for salinity tolerance. The mechanisms of tolerance might be due to homeostasis between Na+ and K+ or Na+ compartmentation. Gene ontology (GO) analysis revealed that significant GO terms in the selected QTL regions were associated with the genes/pathways involved in signaling, enzyme inhibition, and ion transport. Because majority of QTLs are with small effects, marker-assisted recurrent selection is proposed to accumulate favorable alleles for improving salt tolerance using the tolerant ILs identified in this study. The tolerant ILs also provide an opportunity for functional genomics studies to provide molecular insights into salt tolerance mechanisms in Nona Bokra.

Identification and validation of QTLs for seedling salinity tolerance in introgression lines of a salt tolerant rice landrace 'Pokkali'.

Salinity is a major threat to rice production worldwide. Several studies have been conducted to elucidate the molecular basis of salinity tolerance in rice. However, the genetic information such as quantitative trait loci (QTLs) and molecular markers, emanating from these studies, were rarely exploited for marker-assisted breeding. To better understand salinity tolerance and to validate previously reported QTLs at seedling stage, a set of introgression lines (ILs) of a salt tolerant donor line 'Pokkali' developed in a susceptible high yielding rice cultivar 'Bengal' background was evaluated for several morphological and physiological traits under salt stress. Both SSR and genotyping-by-sequencing (GBS) derived SNP markers were utilized to characterize the ILs and identify QTLs for traits related to salinity tolerance. A total of eighteen and thirty-two QTLs were detected using SSR and SNP markers, respectively. At least fourteen QTLs detected in the RIL population developed from the same cross were validated in IL population. Analysis of phenotypic responses, genomic composition, and QTLs present in the tolerant ILs suggested that the mechanisms of tolerance could be Na+ dilution in leaves, vacuolar Na+ compartmentation, and possibly synthesis of compatible solutes. Our results emphasize the use of salt injury score (SIS) QTLs in marker-assisted breeding to improve salinity tolerance. The tolerant lines identified in this study will serve as improved breeding materials for transferring salinity tolerance without the undesirable traits of Pokkali. Additionally, the lines will be useful for fine mapping and map-based cloning of genes responsible for salinity tolerance.

Molecular Dissection of Seedling Salinity Tolerance in Rice (Oryza sativa L.) Using a High-Density GBS-Based SNP Linkage Map.

BACKGROUND: Salinity is one of the many abiotic stresses limiting rice production worldwide. Several studies were conducted to identify quantitative trait loci (QTLs) for traits associated to salinity tolerance. However, due to large confidence interval for the position of QTLs, utility of reported QTLs and the associated markers has been limited in rice breeding programs. The main objective of this study is to construct a high-density rice genetic map for identification QTLs and candidate genes for salinity tolerance at seedling stage. RESULTS: We evaluated a population of 187 recombinant inbred lines (RILs) developed from a cross between Bengal and Pokkali for nine traits related to salinity tolerance. A total of 9303 SNP markers generated by genotyping-by-sequencing (GBS) were mapped to 2817 recombination points. The genetic map had a total map length of 1650 cM with an average resolution of 0.59 cM between markers. For nine traits, a total of 85 additive QTLs were identified, of which, 16 were large-effect QTLs and the rest were small-effect QTLs. The average interval size of QTL was about 132 kilo base pairs (Kb). Eleven of the 85 additive QTLs validated 14 reported QTLs for shoot potassium concentration, sodium-potassium ratio, salt injury score, plant height, and shoot dry weight. Epistatic QTL mapping identified several pairs of QTLs that significantly contributed to the variation of traits. The QTL for high shoot K+ concentration was mapped near the qSKC1 region. However, candidate genes within the QTL interval were a CC-NBS-LRR protein, three uncharacterized genes, and transposable elements. Additionally, many QTLs flanked small chromosomal intervals containing few candidate genes. Annotation of the genes located within QTL intervals indicated that ion transporters, osmotic regulators, transcription factors, and protein kinases may play essential role in various salt tolerance mechanisms. CONCLUSION: The saturation of SNP markers in our linkage map increased the resolution of QTL mapping. Our study offers new insights on salinity tolerance and presents useful candidate genes that will help in marker-assisted gene pyramiding to develop salt tolerant rice varieties.

Genetic variation in Southern USA rice genotypes for seedling salinity tolerance.

The success of a rice breeding program in developing salt tolerant varieties depends on genetic variation and the salt stress response of adapted and donor rice germplasm. In this study, we used a combination of morphological and physiological traits in multivariate analyses to elucidate the phenotypic and genetic variation in salinity tolerance of 30 Southern USA rice genotypes, along with 19 donor genotypes with varying degree of tolerance. Significant genotypic variation and correlations were found among the salt injury score (SIS), ion leakage, chlorophyll reduction, shoot length reduction, shoot K(+) concentration, and shoot Na(+)/K(+) ratio. Using these parameters, the combined methods of cluster analysis and discriminant analysis validated the salinity response of known genotypes and classified most of the USA varieties into sensitive groups, except for three and seven varieties placed in the tolerant and moderately tolerant groups, respectively. Discriminant function and MANOVA delineated the differences in tolerance and suggested no differences between sensitive and highly sensitive (HS) groups. DNA profiling using simple sequence repeat markers showed narrow genetic diversity among USA genotypes. However, the overall genetic clustering was mostly due to subspecies and grain type differentiation and not by varietal grouping based on salinity tolerance. Among the donor genotypes, Nona Bokra, Pokkali, and its derived breeding lines remained the donors of choice for improving salinity tolerance during the seedling stage. However, due to undesirable agronomic attributes and photosensitivity of these donors, alternative genotypes such as TCCP266, Geumgangbyeo, and R609 are recommended as useful and novel sources of salinity tolerance for USA rice breeding programs.