SNP-based analysis of genetic diversity in anther-derived rice by whole genome sequencing.

BACKGROUND: Anther culture has advantage to obtain a homozygous progeny by induced doubling of haploid chromosomes and to improve selection efficiency for invaluable agronomical traits. Therefore, anther culturing is widely utilized to breed new varieties and to induce genetic variations in several crops including rice. Genome sequencing technologies allow the detection of a massive number of DNA polymorphism such as SNPs and Indels between closely related cultivars. These DNA polymorphisms permit the rapid identification of genetic diversity among cultivars and genomic locations of heritable traits. To estimate sequence diversity derived from anther culturing, we performed whole-genome resequencing of five Korean rice accessions, including three anther culture lines (BLB, HY-04 and HY-08), their progenitor cultivar (Hwayeong), and an additional japonica cultivar (Dongjin). RESULTS: A total of 1,165 × 106 raw reads were generated with over 58× coverage that detected 1,154,063 DNA polymorphisms between the Korean rice accessions and Nipponbare. We observed that in Hwayeong and its progenies, 0.64 SNP was found per one kb of Nipponbare genome, while Dongjin, bred by a conventional breeding method, had a lower number of SNPs (0.45 SNP/kb). Among 1,154,063 DNA polymorphisms, 29,269 non-synonymous SNPs located on 30,013 genes and these genes were functionally classified based on gene ontology (GO). We also analyzed line-specific SNPs which were estimated 1 ~ 3% of the total SNPs. The frequency of non-synonymous SNPs in each accession ranged from 26 SNPs in Hwayeong to 214 SNPs in HY-04. CONCLUSIONS: The genetic difference we detected between the progenies derived from anther culture and their mother cultivar is due to somaclonal variation during tissue culture process, such as karyotype change, chromosome rearrangement, gene amplification and deletion, transposable element, and DNA methylation. Detection of genome-wide DNA polymorphisms by high-throughput sequencer enabled to identify sequence diversity derived from anther culturing and genomic locations of heritable traits. Furthermore, it will provide an invaluable resource to identify molecular markers and genes associated with diverse traits of agronomical importance.

Inactivation of the UGPase1 gene causes genic male sterility and endosperm chalkiness in rice (Oryza sativa L.).

A rice genic male-sterility gene ms-h is recessive and has a pleiotropic effect on the chalky endosperm. After fine mapping, nucleotide sequencing analysis of the ms-h gene revealed a single nucleotide substitution at the 3'-splice junction of the 14th intron of the UDP-glucose pyrophosphorylase 1 (UGPase1; EC2.7.7.9) gene, which causes the expression of two mature transcripts with abnormal sizes caused by the aberrant splicing. An in vitro functional assay showed that both proteins encoded by the two abnormal transcripts have no UGPase activity. The suppression of UGPase by the introduction of a UGPase1-RNAi construct in wild-type plants nearly eliminated seed set because of the male defect, with developmental retardation similar to the ms-h mutant phenotype, whereas overexpression of UGPase1 in ms-h mutant plants restored male fertility and the transformants produced T(1) seeds that segregated into normal and chalky endosperms. In addition, both phenotypes were co-segregated with the UGPase1 transgene in segregating T(1) plants, which demonstrates that UGPase1 has functional roles in both male sterility and the development of a chalky endosperm. Our results suggest that UGPase1 plays a key role in pollen development as well as seed carbohydrate metabolism.

A Ds-insertion mutant of OSH6 (Oryza sativa Homeobox 6) exhibits outgrowth of vestigial leaf-like structures, bracts, in rice.

OSH6 (Oryza sativa Homeobox6) is an ortholog of lg3 (Liguleless3) in maize. We generated a novel allele, termed OSH6-Ds, by inserting a defective Ds element into the third exon of OSH6, which resulted in a truncated OSH6 mRNA. The truncated mRNA was expressed ectopically in leaf tissues and encoded the N-terminal region of OSH6, which includes the KNOX1 and partial KNOX2 subdomains. This recessive mutant showed outgrowth of bracts or produced leaves at the basal node of the panicle. These phenotypes distinguished it from the OSH6 transgene whose ectopic expression led to a "blade to sheath transformation" phenotype at the midrib region of leaves, similar to that seen in dominant Lg3 mutants. Expression of a similar truncated OSH6 cDNA from the 35S promoter (35S::DeltaOSH6) confirmed that the ectopic expression of this product was responsible for the aberrant bract development. These data suggest that OSH6-Ds interferes with a developmental mechanism involved in bract differentiation, especially at the basal nodes of panicles.

Analysis of gene-trap Ds rice populations in Korea.

Insertional mutagen-mediated gene tagging populations have been essential resources for analyzing the function of plant genes. In rice, maize transposable elements have been successfully utilized to produce transposant populations. However, many generations and substantial field space are required to obtain a sufficiently sized transposant population. In rice, the japonica and indica subspecies are phenotypically and genetically divergent. Here, callus cultures with seeds carrying Ac and Ds were used to produce 89,700 lines of Dongjin, a japonica cultivar, and 6,200 lines of MGRI079, whose genome is composed of a mixture of the genetic backgrounds of japonica and indica. Of the more than 3,000 lines examined, 67% had Ds elements. Among the Ds-carrying lines, 81% of Dongjin and 63% of MGRI079 contained transposed Ds, with an average of around 2.0 copies. By examining more than 15,000 lines, it was found that 12% expressed the reporter gene GUS during the early-seedling stage. GUS was expressed in root hairs and crown root initials at estimated frequencies of 0.78% and 0.34%, respectively. The 5,271 analyzed Ds loci were found to be randomly distributed over all of the rice chromosomes.

Characterization and mapping of a shattering mutant in rice that corresponds to a block of domestication genes.

Easy shattering reduces yield due to grain loss during harvest in cereals. Shattering is also a hindrance in breeding programs that use wild accessions because the shattering habit is often linked to desirable traits. We characterized a shattering mutant line of rice, Hsh, which was derived from a nonshattering japonica variety, Hwacheong, by N-methyl-N-nitrosourea (MNU) treatment. The breaking tensile strength (BTS) of the grain pedicel was measured using a digital force gauge to evaluate the degree of shattering of rice varieties at 5, 10, 15, 20, 25, 30, 35, and 40 days after heading (DAH). The BTS of Hwacheong did not decrease with increasing DAH, maintaining a level of 180-240 gf, while that of Hsh decreased greatly during 10-20 DAH and finally stabilized at 50 gf. Optical microscopy revealed that Hsh had a well-developed abscission layer similar to the wild rice Oryza nivara (accession IRGC105706), while Hwacheong did not produce an abscission layer, indicating that the shattering of Hsh was caused by differentiation of the abscission layer. On the basis of the BTS value and morphology of the abscission layer of F(1) plants and segregation data in F(2) populations, it was concluded that the easy shattering of Hsh was controlled by the single recessive gene sh-h. The gene sh-h was determined to be located on rice chromosome 7 by bulked segregant analysis. Using 14 SSR markers on rice chromosome 7, the gene sh-h was mapped between the flanking markers RM8262 and RM7161 at distances of 1.6 and 2.0 cM, respectively. An SSR marker Rc17 cosegregated with the gene sh-h. The locus sh-h for shattering was tightly linked to the Rc locus conferring red pericarp, as well as a QTL qSD(s)-7-1 for seed dormancy, implying that this region might represent a domestication block in the evolutionary pathway of rice.