The Usage Of Fluorescent In Situ Hybridization And Molecular Markers On The Research Of Rice And Wheat Germplasm Resources

The most important foodstuff crops in the world concentrate in the grass family (Gramineae), such as wheat, corn and rice. These crops have been cultivated for a long time and only few cultivated varieties have spreaded to great area. The genetic resources of these crops has become more and more limited. Damage from diseases and insects and abiotic stress has become more and more serious. To resolve the problem of global population expansion and great demand for food, fine genie resources from wild species have been continually explored, and cloned orthologous genes have been used widely in other species by transformed methods. To understand genetics characteristics of the two halotolerance genes (RHL and P5CS) of cereal, and the genetic characteristics of hybrids between wheat/rice and its wild species, to utilize the two halotolerance genes and useful genetic resources from wild species of wheat and rice, and to explore the application of fine resources, a serials of genetic research were developed by fluorescent in situ hybridization (FISH) and other genetic methods.1. Through fluorescence in situ hybridization, RHL gene cDNA was mapped on rice, corn and barley chromosomes. RHL gene probe was detected on chromosomes of three species: RHL gene was localized at the interstitial region on the long arm of rice chromosome 9; it has two sites respectively localized at the distal end on the long arm of maize chromosome 2 and near centromere on the long arm of maize chromosome 7; it was also localized at the distal end on the long arm of barley chromosome 5. The result was compared with the grass genomic consensus map published on Science magazine (Gale and Devos, Science, 1998), and the consistency that they all contained homologous region of rice chromosome 6 was confirmed. Further, Touchdown PCR with gene-specific primer also proved the existence of RHL gene with same length in a same region in corn and barley genome. Using a biotin-labeled fluorescent in situ hybridization (FISH) technique, P5CS cDNA clone from mothbean (Vigna aconitifolia) as a probe was approximately located onto the long arm of rice chromosome 9. The percentage distance (from the hybridization sites to centromere) was 51.79 + 1.24%. The prospect of comparative plant biology for crop improvement is discussed.2. Intergenomic hybrid plants between Oryza sativa (AA, 2n=2X=24) and O. minuta (BBCC, 2n=4X=48) and between Oryza sativa and O. meyeriana (GG, 2n=2X=24)were provided by Dr. Chen Baotang, which were produced by embryo rescue after sexual cross. Observation on morphology of hybrid between cultivar rice and O. minuta, chromosome counts confirmed their hybrid status (ABC, 2n=3X=36). Genomic in situ hybridization (GISH) was further applied to establish the parentage of the chromosomes of Fl hybrids, and O. minuta and O. saliva chromosomes were distinguishable in the hybrids by different colors. RAPD profiles unequivocally revealed their hybrids with double parent patterns. The results of blast tests showed that hybrids acquired resistant character from O. minuta, and had a level of susceptibility intermediate between that of the parents; Observation on morphology of hybrid between rice and O. meyeriana, the counts of chromosomes in the hybrids varied in two manners. One is the number of chromosomes was 24. The other is the number of chromosomes was various from 28 to 47, which majoraty of hybrids belong to the latter. Chromosome duplication and subsequent partial separation of the parental genomes during mitosis might explain the results observed in the hybrids between rice and O. meyeriana. The process was similar with the first division of meiosis. The result of GISH revealed the parent chromosomes in the hybrids could be identificated by two colors, but the most of lost chromosomes belonged to the cultivated rice. RAPD profiles suggested the patterns of these hybrids were obviously different. The result of two RAPD patterns accord with the counts of chromosomes and the result of GISH. Combined with RAPD and G...