Robert G. Fjellstrom

Analysis of rice blast resistance gene Pi-z in rice germplasm using pathogenicity assays and DNA markers

The Pi-z gene in rice confers resistance to a wide range of races of the rice blast fungus, Magnaporthe oryzae. The objective of this study was to characterize Pi-z in 111 rice germplasm accessions using DNA markers and pathogenicity assays. The existence of Pi-z in rice germplasm was detected by using four simple sequence repeat (SSR) markers (RM527, AP4791, AP5659-1, AP5659-5) closely linked to Pi-z, and was verified using pathogenicity assays with an avirulent strain (IE1k) and two virulent races (IB33 and IB49). Among 111 germplasm accessions evaluated, 73 were found to contain the Pi-z gene using both SSR markers and pathogenicity assays. The remaining 38 germplasm accessions were found to be inconsistent in their responses to the blast races IB33, IEIk and IB49 with expected SSR marker alleles, suggesting the presence of unexpected SSR alleles and additional R gene(s). These characterized germplasm can be used for genetic studies and marker-assisted breeding for improving blast resistance in rice.

RFLP marker analysis supports tetrasonic inheritance in Lotus corniculatus L.

Abstract Lotus corniculatus is a tetraploid (2n=4x=24) perennial forage legume and has been reported to have tetrasomic inheritance for several traits, although it has also been reported to show disomic inheritance. Molecular markers were used to clarify whether tetrasomic inheritance, disomic inheritance, or a combination of both, was found within an F2 population arising from a cross between two diverse L. corniculatus accessions. The inheritance of ”tetra-allelic” RFLP markers (markers with four segregating bands) indicated that disomic inheritance could not account for the phenotypic F2 classes observed, and that only tetrasomic inheritance would explain the observed results. Goodness of fit tests for ”tetra-allelic” and ”tri-allelic” (three segregating bands) RFLP marker data suggested support for chromosomal-type tetrasomic inheritance. RFLP genotypes interpreted from autoradiographic signal intensity provided additional support for tetrasomic inheritance and the occurrence of preferential pairing between parental chromosomes. Bivalent pairing was predominant in the two parental lines and their F1 hybrid in cytological analyses. L. corniculatus has been classified as both an autotetraploid and an allotetraploid species. RFLP evidence of tetrasomic inheritance gives support for L. corniculatus being classified as an autotetraploid species. Even though bivalent pairing occurs, as seen in other autotetraploid species, pairing between any of the four homologous chromosomes is possible. Preferential pairing in the F1 hybrid suggests that genome differentiation appears to be minimal between homologs within an accession, while genome differentiation is greater between homologs from different accessions of this genetically diverse species.

Phylogenetic analysis and evolution of the genusJuglans (Juglandaceae) as determined from nuclear genome RFLPs

RFLPs were studied in 13Juglans species to determine phylogenetic relationships inJuglans. Allele frequency data were used to generate genetic distance matrices and fragment data were used to generate genetic distances based upon shared-fragments and to perform parsimony analysis. Although similar cluster analyses result from analysing allelic and shared-fragment distance, the two types of distance values displayed variable correspondence with each other. Parsimony analysis produced a tree similar to distance data trees, but with additional phylogenetic resolution agreeing with previous systematic studies. All analyses indicate an ancient origin ofJ. regia, previously considered a recently derived species.

Alternative ecotilling protocol for rapid, cost-effective single-nucleotide polymorphism discovery and genotyping in rice (Oryza sativa L.)

Ecotilling is a high-throughput method of discovery and analysis of single-nucleotide polymorphism (SNP) variations in natural populations, but it requires a substantial investment in sophisticated equipment, costly reagents, and specialized software programs and implementation of several time-consuming steps that limit its use in laboratories with modest financial resources. Moreover, labeling efficiency of PCR primers with fluorescent dyes during Ecotilling can be reduced by unwanted exonuclease activity of single strand-specific nucleases. A new alternative protocol involving a simplified gel system, unlabeled primers, DNA staining after single strand-specific nuclease digestion, and standard gel data analysis was optimized to address these constraints. Using this alternative protocol, we successfully identified four new SNPs verified by sequencing in a collection of 57 diverse rice accessions along with 2 previously reported SNPs in a 922-bp DNA region from thealk gene. An SNP cluster containing a deletion within a 472-bp fragment of thewaxy gene was also characterized. In addition, 4 previously reported SNPs in thealk andwaxy genes were faithfully genotyped among the 57 accessions based on comparisons with sequencing results. Associations between the genotyped SNPs and amylose class and starch gelatinization temperature were as anticipated. These results, along with detailed time and cost comparisons between the 2 methods, suggest that alternative Ecotilling is a simple and reproducible method for SNP discovery and genotyping in rice that leads to substantial savings in equipment, reagents, software, and time compared with the standard Ecotilling procedure.

Identification of the Rice Blast Resistance Gene Pib in the National Small Grains Collection

The Pib gene in rice confers resistance to a wide range of races of the rice blast pathogen, Magnaporthe oryzae, including race IE1k that overcomes Pita, another broad-spectrum resistance gene. In this study, the presence of Pib was determined in 164 rice germplasm accessions from a core subset of the National Small Grains Collection utilizing DNA markers and pathogenicity assays. The presence of Pib was evaluated with two simple sequence repeat (SSR) markers and a dominant marker (Pib-dom) derived from the Pib gene sequence. Pathogenicity assays using two avirulent races (IE1k and IB1) and a virulent race (IB54) were performed to verify the resistance responses of accessions. Of the 164 accessions evaluated, 109 contained the Pib gene as determined using both SSR markers and pathogenicity assays, albeit different haplotypes were detected. The remaining 52 germplasm accessions were different in their responses to the blast races IB54, IE1k, and IB1, thus indicating the presence of R gene(s) other than Pib. The accessions characterized in this study could be used for marker-assisted breeding to improve blast resistance in indica and japonica cultivars worldwide.

Sheath-blight resistance QTLS in japonica rice germplasm

Sheath blight (SB), caused by Rhizoctonia solani, is a serious disease of cultivated rice (Oryza sativa L.) for which genetic resistance is in demand by breeders. With the goal of resistance (SBR)-QTL discovery in U. S. japonica breeding material, 197 doubled-haploid lines from a cross between MCR10277 (resistant) and Cocodrie (susceptible) were evaluated in field and greenhouse assays with U. S. and Colombian pathogen isolates and genotyped at 111 microsatellite marker loci. Four SBR QTLs from MCR10277 were identified, together accounting for 47% of field genetic variation. In all trials the strongest effect was provided by a chromosome-9 QTL, qsbr_9.1, but some QTLs differed for U. S. and Colombian R. solani isolates. SBR QTLs coincided with only two of several height or heading-time QTLs, suggesting that the relationship between these developmental traits and SBR is not simple. For the U. S. isolates, a microchamber greenhouse assay revealed the same QTLs as did field inoculation.

Genetic Variation and Association Mapping of Protein Concentration in Brown Rice Using a Diverse Rice Germplasm Collection

ABSTRACT Protein is the second most abundant constituent in the rice grain next to starch. Association analysis for protein concentration in brown rice was performed using a “mini-core” collection, which represents the germplasm diversity found in the USDA rice world collection. Protein concentration was determined in replicated trials conducted in two southern U.S. locations, and association mapping was performed by using 157 genomewide DNA markers. Protein concentration ranged from 5.4 to 11.9% among the 202 accessions. Protein variation owing to accession and accession × location interaction were highly significant. Ample variation was seen within each subpopulation by ancestry, as well as within the 14 geographic regions where the accessions originated. Accessions from Eastern Europe had the highest level of protein. Ten markers on eight chromosomes were significantly associated with protein concentration. Five of these markers occurred near known protein precursor genes or quantitative trait loci, an...