Screening And Identification Of The Centromeic BAC Clones For Different Genomes In Genus Oryza And Its Application In The Research Of Interspecific Hybrids

Rice (Oryza sativa) is the most important staple crop, feeding more than half of the world's population. Since China is the largest producer and consumer of rice worldwide, Chinese government has always attached great importance to the application of advanced technologies, such as hybrid rice and genetic engineering, in selecting and breeding new rice varieties and expected, by means of these technologies, to culture the rice varieties with high and stable yield potentials, ideal quality and enhanced stress resistance. However, due to the multiplication of population, the ill ecological balance, the deteriorating rice growth habitat and other adverse factors, rice production has been severely damaged by pests, the unrestrained use of insecticides and chemical fertilizers, and the scarcity of water and energy. To address the mentioned issues, more and more rice scientists, in the modern rice breeding program, have paid their attention to wild rice species which promise an important gene pool with potential elite genes.The rice genus, Oryza, comprises approximately 24 species distributed in a wide range of tropical and subtropical area of Asia, Africa, Australia, and Central and South America. Based on interspecific crossing and subsequent cytogenetic analyses and genomic DNA hybridization , 10 genome sets have been recognized for 24 Oryza species including A, B, C, D, E, F, G, H,J, and K. Besides two cultivated species, O. sativa and O. glaberrima, the genus of Oryza consists of approximately 22 wild species, representing 6 diploid genomes (AA, BB, CC, EE, FF and GG) and 4 tetraploid ones (BBCC, CCDD,HHJJ and HHKK). Obviously in the near future it is one of the most important approaches to apply rice breeding program to transfer the elite genes of wild species into cultivated rice. In order to utilize the elite genes of wild species to best advantage, it is necessary to conduct systematic research into wild rice germplasm. In the present study, we have constructed six genomic BAC libraries of Oryza glaberrima (AA genome,W0025), O.punctata (BB genome,W1577), O.officinalis (CC genome,W1263), O.australiensis (EE genome,W0008), O.brachyantha(FF genome,W1057)and O.granulata (GG genome,102118), respectively. Together with the technique of colony blot hybridiation and FISH, different centromeric BAC clones have been screened and identified from their corresponding BAC libraries.A few cases, which transferred useful traits from wild species into cultivated rice, were reported due to distant genetic relationship and diversity of DNA sequence, especially rapid evolutive centromeric DNA sequence. In the most interspecific hybrids between O. sativa and its wild species, highly divergent DNA sequence was usually one of the main reasons resulted in poor chromosome paring and synapsis, which given rise to many reproduction barriers such as hybrid sterility, hybrid weakness and abortion of hybrid embryos. The barriers caused some problem to introgress useful genes from wild species to cultivated rice through interspecific hybridization. With the aid of embryo rescue, we have obtained some interspecific hybrids which have been confirmed by Genomic in situ hybridization (GISH) and FISH probed by each genomic DNA and the above-mentioned identified BAC clones. These developed interspecific hybrids lays a platform for directly making use of elite genes of wild species in the near future.The major results of the present study are as follows:Six BAC libraries of diploid wild rice species were constructed. The species possessed respective different genome constitution, including AA, BB, CC, EE, FF and GG genome which covered the majority of genomes of genus Oryza. By analyzed the results of CHEF (contour-clamped homogeneous electric field) gels, the average insert size of the six libraries ranges from 60.5 to 92 Kb. The six libraries represent, in conjunction with their respective containing the number of clones and the size of haploid genome, approximately 3.7×(AA genome), 1.2×(BB), 2.5×(CC), 1.5×(EE), 7.5×(FF) and 2.6×(GG), respectively.By screening of the six BAC libraries probed by respective genomic DNA, we obtained some clones which contained rich repeat sequence. Subsequently the centromere-related clones were selected and identified by FISH analysis. Meanwhile, co-hybridization was detected between these centromere-related clones and the six genomes. The results from this study demonstrated that: (1) there were centromere-specific satellite repeat in O. officinalis, O. brachyantha, O.granulata, respectively, and centromere-specific CRR-related sequence was found in O. brachyantha and O.granulata ; (2) homology sequences of CentO and CRR of O. sativa were detected on all centromeres of O. glaberrima, O. punctata and O. australiensis; and (3) the two somatic chromosomes of O. officinalis comprised of homology sequences of CentO satellites as revealed FISH analysis probed with RCS2. Homology sequences of CRR of O. sativa were also detected on all centromeres of O. officinalis.Coupled with the technique of subclone and FISH, centromere-specific satellites were isolated and identified from O. punctata and O.brachyantha, respectively. The results demonstrated that (1) 165 bp CentO repeat is the major centromeric elements in O. punctata, which have high homology with the CentO satellites in O.sativa. (2) according to its sequence length and structure, CentO monomers in O.punctata can be classified into three groups: 155 bp, 165 bp and incomplete CentO subfamilies which had high homology among their sequences. (3) based on the homology of DNA sequence,154 bp CentO-F satellites have also three types (CentO-Fa, CentO-Fb and CentO-Fc) in O.brachyantha, and low homology was found among their sequences. (4) the distribution of the three kinds of CentO-F satellites in different centromeres is variable. It indicates that centromeric elements of different centomeres are highly diverged in O.brachyantha.In this study, some interspecific hybrids from crosses between O.sativa and its wild rice were obtained by means of sexual hybridization and embryo rescue. GISH and BAC-FISH were used to distinguish the chromosomes of O. sativa from wild species in their interspecific hybrid and the chromosome pairing at metaphaseⅠin meiosis of the hybrid was also investigated. The results indicated that (1) the seed setting percentage of intergenomic hybrids depended on the crossing combination and was relation with genotype of both wild rice species and the used varieties of cultivated rice. (2) in the cells of these hybrids at metaphaseⅠin meiosis, poor chromosome pairing was identified and most of the chromosome were existed as univalents. (3) different genomes could be identified by using blocking DNA to eliminate the cohybridization in GISH analysis of their interspedific hybrids.