Development And Application Of PCR-based RGA Markers In Rice

Resistant gene analogs (RGA) is an unpopular marker system during the decades due to RGA is a probe of Restriction Fragment Length Polymorphism (RFLP) which is difficulty to dectect. From the application view of Quantitative Trait Loci (QTL) and Marker Assistant Selection (MAS), the specified marker (e. g. RGA) is much more useful. In order to develop the specified RGA marker based on PCR, Wang et al (2005) have developed 402 co-dominant rice RGA candidate markers based on the whole genomic sequence messages of japonica (Nipponbare) and indica (93-11) using the bioinformatics methods. All the candidate RGA markers exhibited one single band and co-dominant detected by the electronic PCR (e-PCR). We randomly chose 40 primer pairs to investigate the feasibility of the RGA markers, such as polymorphism, commonality and subspecies specificity. The results of the experiment are as follows:(1) The 40 primers were selected randomly to test by PCR and the results showed that 35 primers exhibited polymorphisms between 93-11 and Nipponbare. 31 markers (77.5%) could obtain clear bands in two parents and exhibited polymorphisms, which was consistent with the e-PCR results. Thus, 31 markers could be considered as co-dominant markers. However, RGA29, RGA35, RGA28 and RGA33 were used as dominant markers. 3 primer pairs (RGA8,RGA 17 and RGA 18) had clear bands in the two parents but without polymorphisms. In addition, 2 primers (RGA 19 and RGA25) failed to obtain the band. In conclusion, 35 (87.5%) of the 40 markers had polymorphisms and was considered as specified markers.(2)We detected 65 clear bands with an average of 2.10 bands for each marker when 31 co-dominant markers were used to test commonality on 24 rice subspecies. The successfully amplication rate of the 31 markers in 24 rice varieties ranged from 75% to 100%, with an average 94.79%. 19 markers had stable bands in 24 rice varieties and 12 makers could obtain bands among 18 to 23 varieties. This result showed that RGA markers had good commonality. In addition, 6 markers presented new bands among 1 to 4 varieties due to the R genes revolution. We detected 5 and 3heterozygous loci in Diwani and Lemont, respectively. This may be caused by the purification of Diwani and Lemont. 25 markers were detected 2 bands and the band size are consistent with the 93-11 or Nipponbare. This suggested that the RGA wasconserved in the rice evolution.(3) The PIC value of the 35 markers varied 0.16 (RGA40) to 0.58 (RGA14) with an average 0.46. Therefore, the results showed that RGA markers had high polymorphisms.(4) We also performed cluster analysis for 24 varieties using 19 RGA amplified PCR products. When the similarity coefficient was setting at 0.47, the 24 varieties were clearly grouped into two classes. The larger clade included 14 japonica varieties and 2 indica varieties and the other clade composed of 8 indica. varieties. The results implied that RGA markers are conserved in japonica, whereas relative high variation in subspecies, japonica and indica, and distinguished well between the subspecies.