Microsatellite analysis of genetic variation and population genetic differentiation in autotetraploid and diploid rice.

Genetic diversity and population genetic structure of autotetraploid and diploid populations of rice collected from Chengdu Institute of Biology, Chinese Academy of Sciences, were studied based on 36 microsatellite loci. Among 50 varieties, a moderate to high level of genetic diversity was observed at the population level, with the number of alleles per locus (Ae) ranging from 2 to 6 (mean 3.028) and polymorphism information content ranging from 0.04 to 0.76 (mean 0.366). The expected heterozygosity (He) varied from 0.04 to 0.76 (mean 0.370) and Shannon's index (I) from 0.098 to 1.613 (mean 0.649). The autotetraploid populations showed slightly higher levels of Ae, He, and I than the diploid populations. Rare alleles were observed at most of the simple sequence repeat loci in one or more of the 50 accessions, and a core fingerprint database of the autotetraploid and diploid rice was constructed. The F-statistics showed genetic variability mainly among autotetraploid populations rather than diploid populations (Fst = 0.066). Cluster analysis of the 50 accessions showed four major groups. Group I contained all of the autotetraploid and diploid indica maintainer lines and an autotetraploid and its original diploid indica male sterile lines. Group II contained only the original IR accessions. Group III was more diverse than either Group II or Group IV, comprising both autotetraploid and diploid indica restoring lines. Group IV included a japonica cluster of the autotetraploid and diploid rices. Furthermore, genetic differences at the single-locus and two-locus levels, as well as components due to allelic and gametic differentiation, were revealed between autotetraploid and diploid varieties. This analysis indicated that the gene pools of diploid and autotetraploid rice were somewhat dissimilar, as variation exists that distinguishes autotetraploid from diploid rices. Using this variation, we can breed new autotetraploid varieties with some important agricultural characters that were not found in the original diploid rice varieties.

[Wx sequence analysis of a autotetraploid indica mutant rice and establishment of molecular marker for identifying].

The amylose content of the mutant of autotetraploid indica rice D4063-1 is 5.23% anout, which was half of its origin diploid rice Minghui 63. The whole sequence of Waxy gene of D4063-1 was amplified and sequenced. A base was absent on the Wx of D4063-1 in exon sequence, which resulted in frameshift mutation and terminating codon occurred ahead in the 9 exon. The mutation of Wx also led to the change of some mutation in the 9 exon and terminating codon occurred early. The change of Wx also led to changes of the sites of common restriction endonuclease. The results showed that D4063-1 added two sph sites compared to indica and japonica rice; Compared to japonica rice, D4063-1decreased six Acc sites, and added 4 Xba, a Pst and a Sal restriction sites. Phylogenic analysis showed that the DNA sequence of Waxy gene of D4063-1 was closer to indica rice. We supposed that the Waxy gene of D4063-1 originated from genotype of Wxa. According to the differences of Wx in D4063-1, we deduced the absent base led to RNA splicing obstacle, which was the main cause of low amylose content and it might be related to the soft rice phenotype. Based on analysis of Wx of D4063-1, indica and japonica and according to the special sites of the three species, primers as markers-AUT4063-I were designed to distinguish D4063-1 from other rice. Combining with primer pair F5, dominant and codominant ways were established for discriminating them, and rapid and correct identification of D4063-1 from other rice could be done.

[Cytogenetical comparison of restorers TP-4 and D minghui63 and maintainer D46B of autotetraploid rice].

Cytogenetical comparison was made between high seed set restorers TP-4 and D minghui63 and eminent maintainer line D46B of autotetraploid rice. The meiosis observation demonstrated the genomes of our autotetraploid materials were all 2n = 48, the same as those in mitosis observation. Low percentages of univalent and trivalent in metaphase I (MI) of restorers TP-4 and D minghui63 and in metaphase I (MI) of maintainer line D46B of autotetraploid rice were observed. And the percentages of chromosome pairing were all over 99%, showing eminent cytological character. The frequency of TP-4 and D minghui63 in metaphase I (MI) was 2.00/PMC and 2.26/PMC, respectively. However the frequency of D46B was 6.00/PMC, significantly higher than those of TP-4 and D minghui63. It indicated that the maintainer D46B has better chromosome pairing capability in metaphase I (MI). While, the frequency of lagging chromosomes of the maintainer D46B in anaphase I (AI) was 10.62%, significantly lower than that of TP-4 (19.44%) or D minghui63 (23.14%), and it was close to the level of diploid control (7.30%). In telophase I (TI), maintainer D46B exhibited a lower frequency of microkernel, and in telophase II (TII) the frequency of normal quartered microspore of maintainer D46B was not only higher than that of TP-4 or D minghui63 but also than that of diploid control. The percentage of the cell observed chromosome lagging in A1 and the percentage of abnormal cell in TI showed a greatly significant positive correlation. That may demonstrate chromo some separation in anaphase I (AI) and microkernel formation in telophase I (TI) are controlled by the same dominant single gene or the major gene of QTL.