Exploitation And Application Of Intron Length Polymorphism Markers In Rice (Oryza Sativa L.)

Polymorphisms in DNA sequences can be exploited as molecular markers, which are very useful tools for genetic research (e.g. construction of genetic maps, mapping of genes or quantitative trait loci) and breeding (e.g. marker-assisted selection). Introns are non-coding sequences in a gene that have little functional significance; therefore, introns are more variable than coding sequences. There could be various polymorphisms in introns, but intron length polymorphism is the most easily recognizable type. The present research have performed a genome-wide search of ILPs between two subspecies (indica and japonica) in rice using the draft genomic sequences of cultivars 93-11 (indica) and Nipponbare (japonica) and 32127 full length cDNA sequences of Nipponbare obtained from public databases. We have developed 5811 candidate ILP markers and further applied ILP to construction of linkage map, development of single segment substitution lines, genetic diversity evaluation and rice evolution. The main results are as follows:1 13308 putative ILP markers were identified and 5811 candidate ILP markers were developed via e-PCR with primers designed in flanking exons. A database for depositing the information of the 5811 ILP markers have been established.2 Out of 215 candidate ILP markers tested on 93-11, Nipponbare and their hybrid, we successfully exploited 173 (80.47%) codominant ILP markers and 6 dominant ILP markers.3 Analyses on 10 rice accessions showed that these ILP markers were widely applicable and most (71.1%) exhibited subspecies specificity. This feature suggests that ILPs would be useful for the studies of genome evolution and inter-subspecies heterosis and for cross-subspecies marker-assisted selection in rice. In addition, by testing 51 pairs of the ILP primers on five Gramineae plants and three dicot plants, we found another desirable characteristic of rice ILP markers that they have high transferability to other plants.4 A linkage map comprising 172 ILP markers and 13 SSR markers was constructed based on the Nipponbare/93-11// Nipponbare BC₁F₁ population consisting of 102 plants. The map covers 1905.70cM in total length with an average distance of 10.30cM between adjacent markers. Among these ILP and SSR markers, 30 markers (16.22%) showed the genetic segregation distortion (P<0.05). There are 28 (93.33%) distorted markers deviated toward donor parent 93-11, only 2 markers deviated toward Nipponbare.5 138 overlapping single segment substitution lines with the Nipponbare genetic background have been developed, which include 54 SSILs distributed on chromosomes 1-11 excluding chromosome 12. The length of the 54 single substituted segments ranges from 3～121.2cM, covering 2046.5cM in total length with an average length 37.90cM. All the SSILs provide coverage of 61.58% of the rice genome.6 71 accessions were divided into four major clusters, indica, japonica and two medial groups according to the UPGMA cluster diagram based on 115 alleles produced by 53 ILP primer pairs, this results indicated that ILP markers could be used for the estimation of genetic diversity and the identification of rice cultivars.7 Cluster analysis shows that the O. sativa and O. rufipogon accessions can be unambiguously classified into two groups, with one group consisting of all the indica rice accessions and all the wild rice accessions from tropical (South and Southeast) Asia, and the other group consisting of all the japonica rice accessions and all the wild rice accessions from southern China. This results strongly suggested that the O. rufipogon from tropical Asia might be the progenitor of indica rice, while that from southern China might be the progenitor of japonica rice. Therefore, we conclude that the two subspecies of Asian cultivated rice might be domesticated independently from different origins.