Comparative Physical Mapping of Bph3 with BAC-FISH in Oryza officinalis and O. sativa

A FISH procedure was adopted to physical mapping rice RFLP marker RZ69 and the BAC clone 38J9 screened by RZ69 which is linked to gene Bph3 in Oryza saliva L. and O. officinalis Well ex: Watt. The FISH results showed that both 38J9 and RZ69 were located in the middle of 4S in O. officinalis and the centromere area of 4S in O. sativa. In O. officinalis the percentage distances from the centromere to the hybridization sites were 20.00 +/- 5.40 and 22.12 +/- 3.44, the detection rates were 50.00% and 6.14%, but in O. sativa they were 0 and 0, 56.10% and 6.29% correspondingly. The results obtained from the BAC and RFLP clone were almost the same in the cultivated rice and O. officinalis. It was suggested that die marker RZ69 of the cultivated rice and its homologous sequence in O. officinalis were in the same BAC clone, the homologous sequence of Bph3 in O. officinalis should be located at the sites hybridized by both RZ69 and 38J9. Many signals on different chromosomes of O. officinalis were observed under no blocking with Cot-1 DNA, showing that the repetitive sequences were also homologous between O. sativa and O. officinalis. The identification of chromosome 4 of O. officinalis is based on comparative map with RG214 and BAC clone screened by RG214. The feasibility of comparative physical mapping performed between O. sativa and O. officinalis with rice BAC clones was discussed.

Cytological Behavior of Hybridization Barriers Between Oryza sativa and Oryza officinalis

Oryza officinalis is one of the important wild species in the tertiary gene pool of Oryza sativa. It has a number of elite genes for rice breeding in resistance or tolerance. However, breeding barriers are so serious that the gene transfer is much difficult by sexual cross method between O. sativa and O. officinalis. Characteristics of the breeding barriers were systemically studied in this paper. When both the diploid （AA, 2n=2x=24） and autotetraploid （AAAA, 2n=4x=48） cultivated rice were crossed as maternal parents with O. officinalis （CC, 2n=2x=24）, none F1 hybrid seeds were obtained. The young hybrid ovaries aborted at 13-16 d after pollinations （DAP）. By rescuing hybrid embryos, in vitro F1 plantlets were obtained in 2x×2x combinations with the crossabilities lower than 0.5%. Lower rates of double-fertilization and abnormal development of hybrid embryo and endosperm were mainly observed in both combinations of 2x×2x and 4x×2x. Free endosperm nuclei in hybrid degenerated early at 1 DAP in a large scale. Almost no normal endosperm cells formed at 3 DAP. Development of a lot of embryos ceased at globularor pear-shaped stage as well as some degenerated gradually. The hybrid plantlets were both male and female sterility. Due to the abnormal development, a diversity of abnormal embryo sacs formed in hybrids, and hybrid pollen grains were typically abortive. It showed that conflicts of genome A and C in hybrid induced abnormal meioses of meiocytes.

Comparative Embryological Studies on Infertility of Interspecific Hybridizations Between Oryza sativa with Different Ploidy Levels and O. officinalis

As maternal parents, diploid （L202-2x） and autotetraploid （L202-4x） of Oryza sativa cv. L2O2 were crossed with O. officinalis. Embryo development and fertilization in these two crosses were comparatively studied. There were no mature hybrid seeds obtained because all the hybridized spikelets died 30 days after pollination. The main reasons for no seed set were abnormal fertilization and development of the embryos and endosperms in the interspecific hybrids. There were doublefertilization, egg cell single-fertilization and non-fertilization in these crosses. Although 59.45% and 54.87% of hybrid embryos produced in the crosses of L202-2x/O. officinalis and L202-4x/O. officinalis, respectively, hybrid embryos ceased to develop or degenerated and plenty of free endosperm nuclei were in disaggregating state without developing cellular endosperms three days after pollination. Besides, some embryological differences in these two crosses were found, that is, the rate of double-fertilization and total rate of doubleand single-fertilization in L202-2x/O. officinalis were higher than those in L202-4x/O. officinalis. The embryo and endosperm of hybrids developed more slowly, and embryos and free endosperm nuclei were more severely degenerated in L202-4x/O. officinalis than in L202-2x/O. officinalis. Five days after pollination, a few of embryos in L202-2x/O. officinalis developed into pear-shaped ones, however, embryos in L202-4x/O. officinalis were all degenerated. Therefore, it is more difficult to obtain interspecific hybrids by wide crosses between autotetraploid of O. sativa and O. officinalis.

Isozyme analysis on progenes of somatic hybrids of Oryza sativa L.and O.officinalis

Some wild species of Oryza possess agronomi-cally useful traits such as disease/insect resis-tance and salt tolerance.However,the use ofwild Oryza species for rice improvement hasbeen hampered by the sexual incompatibility orsterility of hybrids obtained by conventionalmethods.We have used protoplast fusion as amethod to incorporate useful trait of wild O-ryza species into cultivated rice and~ obtainedsomatic hybrid plants between O.sativa and

Genetic Analysis of Monosomic Alien Addition Line MAAL8 of O. officinalis(CC)-O. sativa(AA)

[Objective] By carrying out the anther culture on the monosomic alien addition line MAAL8 of O.officinalis-O.sativa and the back crossing with O.sativa H1493,the genetic characteristics of monosomic alien addition line were studied.[Method] The phenotype analysis was used to study the separation proportion of progeny.Moreover,SSR and the methylation analysis were used to study the transmission behavior of nonhomologous chromosome.[Result] 78 plants of 145 backcross progenies preserved the rolled leaf mark trait of MAAL8.In 32 anther culture plants,five plants had the normal rolled leaves,and two plants had the extremely rolled leaves.The rest had the flat leaves.14 couples of SSR markers were used to analyze,and it indicated that all the rolled-leaf plants could obtain the characteristic band type of O.officinalis,but the flat-leaf plants showed none of them.11 polymorphic RFLP markers were used to carry out Methylation-Sensitive Southern analysis.It showed that the methylation variation manners of eight markers between AA and CC genomes were different.[Conclusion] The nonhomologous chromosome of MAAL8 could pass to the progenies independently and integrally via the meiosis,and the phenotype characteristics didn＇t change.Moreover,the methylation manner of O.officinalis could inherit stably in the hybrid progeny as the addition of single chromosome.The stability of methylation might have the certain effect on the relatively independent inheritance of nonhomologous chromosome in the genome environment of O.sativa.

Ortholog Alleles at Xa3/Xa26 Locus Confer Conserved Race-Specific Resistance against Xanthomonas oryzae in Rice

The rice disease resistance （R） gene Xa3/Xa26 （having also been named Xa3 and Xa26） against Xanthomonas oryzae pv. oryzae （Xoo）, which causes bacterial blight disease, belongs to a multiple gene family clustered in chromosome 11 and is from an AA genome rice cultivar （Oryza sativa L.）. This family encodes leucine-rich repeat （LRR） receptor kinase- type proteins, Here, we show that the orthologs （alleles） of Xa3/Xa26, Xa3/Xa26-2, and Xa3/Xa26-3, from wild Oryza spe- cies O. officinalis （CC genome） and O. minuta （BBCC genome）, respectively, were also R genes against Xoo. Xa3/Xa26-2 and Xa3/Xa26-3 conferred resistance to 16 of the 18 Xoo strains examined. Comparative sequence analysis of the Xa3/Xa26 families in the two wild Oryza species showed that Xa3/Xa26-3 appeared to have originated from the CC genome of O. minuta. The predicted proteins encoded by Xa3/Xa26, Xa3/Xa26-2, and Xa3/Xa26-3 share 91-99% sequence identity and 94-99% sequence similarity. Transgenic plants carrying a single copy of Xa3/Xa26, Xa3/Xa26-2, or Xa3PXa26-3, in the same genetic background, showed a similar resistance spectrum to a set of Xoo strains, although plants carrying Xa3/Xa26-2 or Xa3/Xa26-3 showed lower resistance levels than the plants carrying Xa3/Xa26. These results suggest that the Xa3/Xa26 locus predates the speciation of A and C genome, which is approximately 7.5 million years ago. Thus, the resistance spec- ificity of this locus has been conserved for a long time.

Analysis of collinear regions of Oryza AA and CC genomes

Comparative analyses of genome structure and sequence of closely related species have yielded insights into the evolution and function of plant genomes. A total of 103,844 BAC end sequences delegated -73.8 Mb of O. officinalis that belongs to the CC genome type of the rice genus Oryza were obtained and compared with the genome sequences office cultivar, O. sativa ssp.japonica cv. Nipponbare. We found that more than 45% of O. officinalis genome consists of repeat sequences, which is higher than that of Nipponbare cultivar. To further investigate the evolutionary divergence of AA and CC genomes, two BAC-contigs of O. officinalis were compared with the collinear genomic regions of Nipponbare. Of 57 genes predicted in the AA genome orthologous regions, 39 had orthologs in the regions of the CC genome. Alignment of the orthologous regions indicated that the CC genome has undergone expansion in both genic and intergenic regions through primarily retroelement insertion. Particularly, the density of RNA transposable elements was 17.95% and 1.78% in O. officinalis and O. sativa, respectively. This explains why the orthologous region is about 100 kb longer in the CC genome in comparison to the AA genome.