Arnold Parco

Fine mapping and DNA marker-assisted pyramiding of the three major genes for blast resistance in rice

Abstract Three major genes (Pi1, Piz-5 and Pita) for blast resistance on chromosomes 11, 6 and 12, respectively, were fine-mapped and closely linked RFLP markers identified. New markers for Pi1 and Pita were found that were flanking the genes. The three genes were pyramided using RFLP markers. A PCR-based SAP (sequence amplified polymorphism) marker was used to identify Piz-5 in the segregating population. The plants carrying the two- and three-gene combinations that were tested for resistance to leaf blast in the Philippines and India indicated that combinations including Piz-5 have enhanced resistance than when it is present alone. The genes from the pyramided lines are at present being deployed into agronomically superior ricevarieties by marker-aided selection (MAS).

Polymorphism, distribution, and segregation of AFLP markers in a doubled haploid rice population

We exploited the newly developed amplified fragment length polymorphism (AFLP) technique to study the polymorphism, distribution and inheritance of AFLP markers with a doubled haploid rice population derived from ‘IR64’/‘Azucena’. Using only 20 pairs of primer combinations, we detected 945 AFLP bands of which 208 were polymorphic. All 208 AFLP markers were mapped and distributed over all 12 chromosomes. When these were compared with RFLP markers already mapped in the population, we found the AFLP markers to be highly polymorphic in rice and to follow Mendelian segregation. As linkage map of rice can be generated rapidly with AFLP markers they will be very useful for marker-assisted backcrossing.

RFLP mapping of isozymes, RAPD and QTLs for grain shape, brown planthopper resistance in a doubled haploid rice population

We have developed an RFLP framework map with 146 RFLP markers based on a doubled haploid population derived from a cross between an indica variety IR64 and a japonica variety Azucena. The population carries 50.2% of IR64 loci and 49.8% of Azucena loci, indicating an equal amount of genetic materials from each parent has been transmitted to the progenies through anther culture. However, some markers show segregation distortion. These distorted marker loci are located on 10 chromosomal segments. Using this map we were able to place 8 isozymes, 14 RAPDs, 12 cloned genes, 1 gene for brown planthopper (BPH) resistance, and 12 QTLs for grain length, grain width and length/width ratio onto rice chromosomes. The major gene for BPH resistance was mapped on chromosome 12 near RG463 and isozyme Sdh-1. Most of the QTLs identified for the three grain characters were closely linked on chromosomes 1, 2, 3 and 10. We concluded that the RFLP framework map presented here will be useful for mapping other genes segregating in this doubled haploid population. Thus rapid generation of doubled haploid lines and their unbiased segregation make it very attractive for gene mapping.

Mapping of Seed Shattering Loci Provides Insights into Origin of Weedy Rice and Rice Domestication

Seed shattering is an important trait that distinguishes crop cultivars from the wild and weedy species. The genetics of seed shattering was investigated in this study to provide insights into rice domestication and the evolution of weedy rice. Quantitative trait locus (QTL) analysis, conducted in 2 recombinant inbred populations involving 2 rice cultivars and a weedy rice accession of the southern United States, revealed 3-5 QTLs that controlled seed shattering with 38-45% of the total phenotypic variation. Two QTLs on chromosomes 4 and 10 were consistent in both populations. Both cultivar and weedy rice contributed alleles for increased seed shattering. Genetic backgrounds affected both QTL number and the magnitude of QTL effects. The major QTL qSH4 and a minor QTL qSH3 were validated in near-isogenic lines, with the former conferring a significantly higher degree of seed shattering than the latter. Although the major QTL qSH4 overlapped with the sh4, the presence of the nonshattering single nucleotide polymorphism allele in the weedy rice accession suggested involvement of a linked locus or an alternative molecular genetic mechanism. Overlapping of several QTLs with those from earlier studies indicated that weedy rice may have been derived from the wild species Oryza rufipogon. Natural hybridization of rice cultivars with the highly variable O. rufipogon present in different geographic regions might be responsible for the evolution of a wide range of phenotypic and genotypic variabilities seen in weedy rice populations worldwide.

A Chromosome Segment Substitution Library of Weedy Rice for Genetic Dissection of Complex Agronomic and Domestication Traits

Chromosome segment substitution lines (CSSLs) are a powerful alternative for locating quantitative trait loci (QTL), analyzing gene interactions, and providing starting materials for map-based cloning projects. We report the development and characterization of a CSSL library of a U.S. weedy rice accession ‘PSRR-1’ with genome-wide coverage in an adapted rice cultivar ‘Bengal’ background. The majority of the CSSLs carried a single defined weedy rice segment with an average introgression segment of 2.8 % of the donor genome. QTL mapping results for several agronomic and domestication traits from the CSSL population were compared with those obtained from two recombinant inbred line (RIL) populations involving the same weedy rice accession. There was congruence of major effect QTLs between both types of populations, but new and additional QTLs were detected in the CSSL population. Although, three major effect QTLs for plant height were detected on chromosomes 1, 4, and 8 in the CSSL population, the latter two escaped detection in both RIL populations. Since this was observed for many traits, epistasis may play a major role for the phenotypic variation observed in weedy rice. High levels of shattering and seed dormancy in weedy rice might result from an accumulation of many small effect QTLs. Several CSSLs with desirable agronomic traits (e.g. longer panicles, longer grains, and higher seed weight) identified in this study could be useful for rice breeding. Since weedy rice is a reservoir of genes for many weedy and agronomic attributes, the CSSL library will serve as a valuable resource to discover latent genetic diversity for improving crop productivity and understanding the plant domestication process through cloning and characterization of the underlying genes.

Identification of cold-responsive genes in energycane for their use in genetic diversity analysis and future functional marker development.

Breeding for cold tolerance in sugarcane will allow its cultivation as a dedicated biomass crop in cold environments. Development of functional markers to facilitate marker-assisted breeding requires identification of cold stress tolerance genes. Using suppression subtractive hybridization, 465 cold-responsive genes were isolated from the cold-tolerant energycane Ho02-144. Predicted gene interactions network indicated several associated pathways that may coordinately regulate cold tolerance responses in energycane. Expression analysis of a select set of genes, representing signaling and transcription factors, genes involved in polyamine and antioxidant biosynthesis, protein degradation and in the repair of damaged proteins in the cytosol, showed their time-dependent regulation under cold-stress. Comparative expression profiles of these genes between Ho02-144 and a cold-sensitive clone (L79-1002) showed that almost all genes were induced immediately upon imposition of cold stress and maintained their expression in Ho02-144 whereas they were either downregulated or their upregulation was very low in L79-1002. Simple sequence repeat markers derived from 260 cold-responsive genes showed allelic diversity among the cold-sensitive commercial hybrids that were distinct from the Saccharum spontaneum clones. Future efforts will target sequence polymorphism information of these genes in our ongoing QTL and association mapping studies to identify functional markers associated with cold tolerance in sugar/energycane.