Nobuo Iwata

Tagging and combining bacterial blight resistance genes in rice using RAPD and RFLP markers

Four genes of rice,Oryza sativa L., conditioning resistance to the bacterial blight pathogenXanthomonas oryzae pv.oryzae (X. o. pv.oryzae), were tagged by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers. No recombinants were observed betweenxa-5 and RFLP marker lociRZ390, RG556 orRG207 on chromosome 5.Xa-3 andXa-4 were linked to RFLP locusXNpb181 at the top of chromosome 11, at distances of 2.3 cM and 1.7 cM, respectively. The nearest marker toXa-10, also located on chromosome 11, was the RAPD locusO072000 at a distance of 5.3 cM. From this study, the conventional map [19, 28] and two RFLP linkage maps of chromosome 11 [14, 26] were partially integrated. Using the RFLP and RAPD markers linked to the resistance genes, we selected rice lines homozygous for pairs of resistance genes,Xa-4 +xa-5 andXa-4 +Xa-10. Lines carryingXa-4 +xa-5 andXa-4 +Xa-10 were evaluated for reaction to eight strains of the bacterial blight pathogen, representing eight pathotypes and three genetic lineages. As expected, the lines carrying pairs of genes were resistant to more of the isolates than their single-gene parental lines. Lines carryingXa-4 +xa-5 were more resistant to isolates of race 4 than were either of the parental lines (‘quantitative complementation’). No such effects were seen forXa-4 +Xa-10. Thus, combinations of resistance genes provide broader spectra of resistance through both ordinary gene action expected and quantitative complementation.

Comparison of the genetic diversity of common wild rice (Oryza rufipogon Griff.) and cultivated rice (O. sativa L.) using RFLP markers

Abstract Forty fourth single-copy RFLP markers were used to evaluate the genetic diversity of 122 accessions of common wild rice (CWR, Oryza rufipogon Griff.) and 75 entries of cultivated rice (Oryza sativa L. ) from more than ten Asian countries. A comparison of the parameters showing genetic diversity, including the percentage of polymorphic loci (P), the average number of alleles per locus (A), the number of genotypes (Ng), the average heterozygosity (Ho) and the average genetic multiplicity (Hs) of CWR and indica and japonica subspecies of cultivated rice from different countries and regions, indicated that CWR from China possesses the highest genetic diversity, followed by CWR from South Asia and Southeast Asia. The genetic diversity of CWR from India is the second highest. Although the average gene diversity (Hs)of the South Asian CWR is higher than that of the Southeast Asian CWR, its percentage of polymorphic loci (P), number of alleles (Na) and number of genotypes (Ng) are all smaller. It was also found that the genetic diversity of cultivated rice is obviously lower than that of CWR. At the 44 loci investigated, the number of polymorphic loci of cultivated rice is only 3/4 that of CWR, while the number of alleles, 60%, and the number of genotypes is about 1/2 that of CWR. Of the two subspecies studied, the genetic diversity of indica is higher than that of japonica. The average heterozygosity of the Chinese CWR is the highest among all the entries studied. The average heterozygosity of CWR is about two-times that of cultivated rice. It is suggested that during the course of evolution from wild rice to cultivated rice, many alleles were lost through natural and human selection, leading to the lower heterozygosity and genetic diversity of the cultivated rice.

Mutants for rice storage proteins : 1. Screening of mutants for rice storage proteins of protein bodies in the starchy endosperm.

SummaryTo obtain genetic materials to breed qualitatively improved rice storage proteins, we screened about 3,000 mutant lines induced by the treatment of rice fertilized egg cell with N-methyl-N-nitrosourea (MNU). The screening was performed by comparing the profiles of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with that of the original variety, Kinmaze, especially focussing on the changes in polypeptides present in two kinds of protein bodies, PB-I and PB-II. We selected 17 mutant lines and classified them into 4 types on the basis of variations of the relative contents of the polypeptides. Determination of extracted protein in the starchy endosperm of the mutants revealed changes in the content of prolamin and glutelin but not globulin. In some mutants there was marked accumulation of 57 kDa polypeptide concomitant with the remarkable reduction of glutelin subunits. Treatment of the fertilized egg cell with MNU was found to be an effective method to induce mutations for storage proteins in protein bodies of rice.

Identification of a YAC clone carrying the Xa-1 allele, a bacterial blight resistance gene in rice

Map-based cloning methods have been applied for isolation of Xa-1, one of the bacterial blight resistance genes in rice.Xa-1 was previously mapped on chromosome 4 using molecular markers. For positional cloning of Xa-1, a high-resolution genetic map was made for theXa-1 region using an F2 population of 402 plants and additional molecular markers. Three restriction fragment length polymorphism (RFLP) markers, XNpb235, XNpb264 and C600 were found to be linked tightly to Xa-1, with no recombinants, and U08750 was mapped 1.5 cM from Xa-1. The screening of a yeast artificial chromosome (YAC) library using theseXa-1-linked RFLP markers resulted in the identification of ten contiguous YAC clones. Among these, one YAC clone, designated Y5212, with an insert of 340 kb, hybridized with all three tightly linked markers. This YAC was confirmed to possess the Xa-1 allele by mapping the Xa-1 gene between both end clones of this YAC (Y5212R and Y5212L).

Production of n + 1 plants and tetrasomics by means of anther culture of trisomic plants in rice (Oryza sativa L.)

SummaryEleven primary trisomics of rice, variety Nipponbare, were subjected to anther culture. The 12th trisomic did not produce normal anthers. A total of 3,734 plants were obtained, which were examined morphologically at the seedling stage in the greenhouse. A number of plants appeared in the progenies of ten trisomics which had unique morphological features. The frequency of these variant types differed among different progenies. Cytological observations revealed that 43 variant plants in the progenies of nine trisomics had 13 chromosomes (n + 1), and 56 were tetrasomics (2n = 26). The tetrasomic plants in the progeny of a trisomic were morphologically identical. Similarly, n + 1 plants in the progeny of a trisomic were also identical. Plants with 23, 25, 36, 39, and 73 chromosomes were also obtained. Results show that valuable aneuploids such as n + 1 and 2n + 2 can be obtained in the anther-culture-derived progenies of trisomics.

Integration of RFLP and conventional linkage maps in rice

To furnish significant information to genome study of rice, we have studied on integration of conventional linkage map in chromosomes 1, 2, 3 and 4 onto the RFLP linkage map. The orientation of both the linkage maps in the four chromosomes was completely understood and some RFLP markers were detected closely linked to conventional markers. In addition, the low degree of saturation of the RFLP linkage map on chromosomes 2 and 4 was found out. The RFLP map lacked the regions corresponding to d-29 — bl-1 and 1g — d-2 regions on chromosomes 2 and 4 in the conventional linkage map, respectively.

Tagging Genes for Bacterial Blight Resistance in Rice by RFLP and RAPD Markers

Molecular markers are being used to tag genes conferring resistance to bacterial blight of rice. Near-isogenic lines (NILs) for the resistance genes were compared with the susceptible recurrent parent, and polymorphic markers were used for linkage analysis on segregating populations. The genes Xa-1, Xa-2 and Xa-10 were located on restriction fragment length polymorphism (RFLP) linkage map. Xa-1and Xa-2, on chromosome 4, were linked to the RFLP marker XNpb264 at distances 1.97 cM and 2.04 cM, respectively. Xa-10 was located on chromosome 11, 15.7 cM from RFLP marker XNpb78. Random amplified polymorphic DNA (RAPD) markers flanking Xa-1 and Xa-10 were also identified using NILs. One marker was tightly linked to Xa-1. No recombinant was observed between Xa-1 and this marker in 84 F2 plants. Two markers were linked to Xa-10 at distances of 6.3 cM and 19.0 cM, respectively. IR-BB3 and IR-BB4, NILs with Xa-3 and Xa-4, respectively, were analyzed by using RFLP markers. In both lines, introgressed segments containing Xa-3 and Xa-4 were retained at the same end of the RFLP map of chromosome 11.

Rice Chromosome Mapping by Using Reciprocal Translocations and Tertiary Trisomics

The authors have attempted to construct the rice chromosome map. The three methods were adopted for this purpose; 1) cytological study for determining the position of interchange breakpoint on pachytene chromosome, 2) localization of interchange breakpoint on conventional linkage map by linkage analysis between breakpoints and conventional marker gene loci, 3) localization of interchange breakpoints on RFLP linkage map by RFLP gene dosage analysis for japonica-indica hybrid tertiary trisomics.