Molecular Marker-Assisted Rice Improvement, 2011

 

Project Leader

Thomas Tai, research geneticist USDA-ARS, Crops Pathology and Genetics Research Unit, Department of Plant Sciences, UC Davis

 

The overall objective of this ongoing project is to integrate advanced technologies in molecular ge-netics with conventional breeding methods to develop improved germplasm for the California rice industry.

Primary emphasis is on the development and application of DNA markers to predict the presence or absence of traits of interest, such as disease resistance, cold tolerance, and grain quality. Use of these markers is intended to accelerate the selection process and to streamline the breeding of im-proved varieties.

Basic genetic studies have resulted in the identification of DNA markers for many important traits. Genes underlying traits for yield, fertility, grain size, grain quality, and others have been identified. The major objectives of 2011 research were to initiate marker development and gene characterization with next-generation sequencing technology and to develop genetic mapping and mutant populations of rice to facilitate trait and gene discovery.

New DNA marker technology

Until the last few years, DNA markers were identified using indirect methods that have limitations with regard to distinguishing between very closely related individuals such as breeding lines. Directly sequencing DNA of individuals is the most powerful way to detect differences between them (i.e. markers). New methods and instruments for sequencing—next-generation sequencing (NGS)—have greatly increased affordability. More and more research and breeding programs are taking advantage of NGS to complement or replace conventional DNA marker methods for genetic studies and variety development.

Restriction Enzyme Site Comparative Analysis (RESCAN) is a DNA marker detection and finger-printing method developed by Professor Luca Comai and colleagues at UC Davis. RESCAN and similar methods isolate DNA samples from individuals that contain a small but representative fraction of their genomes (i.e. their chromosomes). The same fraction is prepared from each individual and then compared using NGS to search for differences. In this way, thousands of markers in hundreds of individuals or hundreds of markers in thousands of individuals can be detected.

As a first step toward using NGS for marker detection and fingerprinting, researchers initiated the analysis of 45 California rice varieties using the RESCAN method. Two versions of RESCAN—standard fractionation and LabChip fractionation—are being evaluated. Sequencing of the varieties using the standard fractionation was completed and the data from all the varieties are being analyzed.

Preliminary data analysis of four of the 45 varieties (M-206, M-204, M-203, and S-301) confirmed the ability of RESCAN to detect thousands of DNA markers among these four varieties. The highly detailed comparison of M-206, M-204, and S-301 identified more than 9,000 DNA markers and revealed that M-206 appears to contain DNA not derived from S-301 or M-204 (although these are the parents of M-206). This could reflect differences in seed sources from when the variety was bred and the source used for DNA analysis. A similar comparison of M-203 and M-206 identified more than 11,000 markers. Markers distinguishing M-203 and M-206 are of interest because of work on a mapping population to investigate milling yield and stability, among other traits.

The ability to distinguish differences in the narrow germplasm base in these varieties, and the relative ease of generating thousands of markers, is indicative of the power of this approach for gene mapping and isolation. Analysis of the remaining sequenced varieties is continuing.

Evaluation of the LabChip fractionation version will be completed in 2012. This version of RESCAN may allow more individuals to be sequenced at the same time, thus further reducing the cost of analysis.

Geneticists plan to use the RESCAN method to analyze hundreds of varieties, breeding lines, and crosses in 2012. Among the high-value targets are high-yielding lines derived from the stem-rot resistant 87Y550 and genetic mapping populations for the analysis of head-rice yield, cold toler-ance, and stem-rot resistance.

New rice populations

Work also continued on the development of genetic mapping populations derived from California rice varieties. Use of the RESCAN method will facilitate use of these populations to map genes im-portant for traits such as milling yield and stability. Plans are to grow out these lines in 2012 to perform preliminary characterization of heading date, yield (total and milling), seed shape and size, and other agronomic traits.

California rice mapping populations under development in 2011
Population Origin (2008) Original lines 2011
MS2041

M-204/s-301
F1 plants (seed from Dr. V. Andaya)

7 plants ranging from 700 to 1000 F2 seeds per plant Generation advance to F4 (485 lines)
SM3014 S-301/M-204
F1 plants (seed from Dr. V. Andaya)
8 plants ranging from 800 to 1000 F2 seeds per plant Generation advance to F4 (487)
SM3016 S-301/M-206
F2 plants (seeds from Dr. V. Andaya)
290 F2 plants, harvested single panicles of F3 seeds Generation advance to F5 (286 lines)
M2036 M-203/M-206
F2 plants (seeds from Dr. V. Andaya)
294 F2 plants, harvested single panicles for F3 seeds Generations advance to F5 (285 lines) and F6 (234 lines)

In previous years, small mutant populations had been developed using California varieties such as S-102 and Terso. In 2011 a new effort was initiated to produce a larger, more diverse mutant population. M-204, which is in the pedigree of the mapping populations, has been selected to produce these populations. M-204 should increase the chance of useful mutants being integrated into the RES breeding projects and may also complement trait and gene discovery in the rice mapping populations.

Using both gamma radiation and the DNA-modifying chemical sodium azide, seeds of M-204 were treated to produce mutant populations. Characterization of these populations is ongoing. In 2012 they will be evaluated to identify useful variants and will undergo further development to produce fixed mutant lines.

Cold Tolerance

Several varieties from the U.S. collection were tested for reproductive cold tolerance in the RES cold-blanking greenhouse. Severe blanking was observed. Conditions for assessment of cold-blanking tolerance will be refined for 2012.