Re-sequencing Resources to Improve Starch and Grain Quality in Rice.

Next-generation sequencing can identify differences in the rice genome that explain the genetic basis of grain quality variation. Differences in rice grain quality are mainly associated with differences in the major component of the grain, starch. Association of rice quality variation with rice genome variation can be conducted at the gene or whole-genome level. Re-sequencing of specific genes or whole genomes can be used depending on the extent to which candidate genes for the traits of interest are known. Amplicon sequencing of genes involved in starch metabolism can help in targeted discovery of the molecular genetic basis of differences in starch related quality attributes. Whole-genome re-sequencing can complement this, when the genetic basis of the trait is expected to be outside the coding region of starch metabolism genes. These approaches have been used successfully to understand the rice genome at specific loci and over the whole genome.

Application of large-scale sequencing to marker discovery in plants.

Advances in DNA sequencing provide tools for efficient large-scale discovery of markers for use in plants. Discovery options include large-scale amplicon sequencing, transcriptome sequencing, gene-enriched genome sequencing and whole genome sequencing. Examples of each of these approaches and their potential to generate molecular markers for specific applications have been described. Sequencing the whole genome of parents identifies all the polymorphisms available for analysis in their progeny. Sequencing PCR amplicons of sets of candidate genes from DNA bulks can be used to define the available variation in these genes that might be exploited in a population or germplasm collection. Sequencing of the transcriptomes of genotypes varying for the trait of interest may identify genes with patterns of expression that could explain the phenotypic variation. Sequencing genomic DNA enriched for genes by hybridization with probes for all or some of the known genes simplifies sequencing and analysis of differences in gene sequences between large numbers of genotypes and genes especially when working with complex genomes. Examples of application of the above-mentioned techniques have been described.

A high-throughput assay for rapid and simultaneous analysis of perfect markers for important quality and agronomic traits in rice using multiplexed MALDI-TOF mass spectrometry.

The application of single nucleotide polymorphisms (SNPs) in plant breeding involves the analysis of a large number of samples, and therefore requires rapid, inexpensive and highly automated multiplex methods to genotype the sequence variants. We have optimized a high-throughput multiplexed SNP assay for eight polymorphisms which explain two agronomic and three grain quality traits in rice. Gene fragments coding for the agronomic traits plant height (semi-dwarf, sd-1) and blast disease resistance (Pi-ta) and the quality traits amylose content (waxy), gelatinization temperature (alk) and fragrance (fgr) were amplified in a multiplex polymerase chain reaction. A single base extension reaction carried out at the polymorphism responsible for each of these phenotypes within these genes generated extension products which were quantified by a matrix-assisted laser desorption ionization-time of flight system. The assay detects both SNPs and indels and is co-dominant, simultaneously detecting both homozygous and heterozygous samples in a multiplex system. This assay analyses eight functional polymorphisms in one 5 microL reaction, demonstrating the high-throughput and cost-effective capability of this system. At this conservative level of multiplexing, 3072 assays can be performed in a single 384-well microtitre plate, allowing the rapid production of valuable information for selection in rice breeding.