Single-seeded InDel fingerprints in rice:An effective tool for indica-japonica rice classification and evolutionary studies

Asian cultivated rice(Oryza sativa L.),an important cereal crop worldwide,was domesticated from its wild ancestor 8000 years ago.During its long-term cultivation and evolution under diverse agroecological conditions, Asian cultivated rice has differentiated into indica and japonica subspecies.An effective method is required to identify rice germplasm for its indica and japonica features,which is essential in rice genetic improvements.We developed a protocol that combined DNA extraction from a single rice seed and the insertion/deletion(InDel) molecular fingerprint to determine the indica and japonica features of rice germplasm.We analyzed a set of rice germplasm,including 166 Asian rice varieties,two African rice varieties,30 accessions of wild rice species,and 42 weedy rice accessions,using the single-seeded InDel fingerprints(SSIF).The results show that the SSIF method can efficiently determine the indica and japonica features of the rice germplasm.Further analyses revealed significant indica and japonica differentiation in most Asian rice varieties and weedy rice accessions.In contrast,African rice varieties and nearly all the wild rice accessions did not exhibit such differentiation.The pattern of cultivated and wild rice samples illustrated by the SSIF supports our previous hypothesis that indica and japonica differentiation occurred after rice domestication under different agroecological conditions.In addition,the divergent pattern of rice cultivars and weedy rice accessions suggests the possibility of an endoferal origin(from crop)of the weedy rice included in the present study.     

Genetic Signature of Rice Domestication Shown by a Variety of Genes

Cultivated rice was domesticated from common wild rice. However, little is known about genetic adaptation under domestication. We investigated the nucleotide variation of both cultivated rice and its wild progenitors at 22 R-gene and 10 non–R-gene loci. A significant regression was observed between wild rice and rice cultivars in their polymorphic levels, particularly in their nonsynonymous substitutions (θ _a ). Our data also showed that a similar proportion (approximately 60%) of nucleotide variation in wild rice was retained in cultivated rice in both R-genes and non–R-genes. Interestingly, the slope always was >1 and the intercept always >0 in linear regressions when a cultivar’s polymorphism was x-axis. The slope and intercept values can provide a basis by which to estimate the founder effect and the strength of artificial direct selection. A larger founder effect than previously reported and a strong direct-selection effect were shown in rice genes. In addition, two-directional selection was commonly found in differentiated genes between indica and japonica rice subspecies. This kind of selection may explain the mosaic origins of indica and japonica rice subspecies. Furthermore, in most R-genes, no significant differentiation between cultivated and wild rice was detected. We found evidence for genetic introgression from wild rice, which may have played an important role during the domestication of rice R-genes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Yuanli Zhang and Jiao Wang contributed equally to this work.     

Large-scale DNA polymorphism study of <Emphasis Type="Italic">Oryza sativa</Emphasis> and <Emphasis Type="Italic">O. rufipogon</Emphasis> reveals the origin and divergence of Asian rice

Polymorphism over ∼26 kb of DNA sequence spanning 22 loci and one region distributed on chromosomes 1, 2, 3 and 4 was studied in 30 accessions of cultivated rice, Oryza sativa, and its wild relatives. Phylogenetic analysis using all the DNA sequences suggested that O. sativa ssp. indica and ssp. japonica were independently domesticated from a wild species O. rufipogon. O. sativa ssp. indica contained substantial genetic diversity (π = 0.0024), whereas ssp. japonica exhibited extremely low nucleotide diversity (π = 0.0001) suggesting the origin of the latter from a small number of founders. O. sativa ssp. japonica contained a larger number of derived and fixed non-synonymous substitutions as compared to ssp. indica. Nucleotide diversity and genealogical history substantially varied across the 22 loci. A locus, RLD15 on chromosome 2, showed a distinct genealogy with ssp. japonica sequences distantly separated from those of O. rufipogon and O. sativa ssp. indica. Linkage disequilibrium (LD) was analyzed in two different regions. LD in O. rufipogon decays within 5 kb, whereas it extends to ∼50 kb in O. sativa ssp. indica. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Analysis of indica- and japonica-specific markers of Oryza sativa and their applications

Asian rice, Oryza sativa L., is one of the most important crop species. Genetic analysis has established that rice consists of several genetically differentiated variety groups, with two main groups, namely, O. sativa ssp. japonica kata and ssp. indica kata. To determine the genetic diversity of indica and japonica rice, 45 rice varieties, including domesticated rice and Asia common wild rice (O. rufipogon Griff.), were analyzed using sequence-related amplified polymorphism, target region amplified polymorphism, simple sequence repeat, and intersimple sequence repeat marker systems. A total of 90 indica- and japonica-specific bands between typical indica and japonica subspecies were identified, which greatly helped in determining whether domesticated rice is of the indica or japonica type, and in analyzing the consanguinity of hybrid rice with japonica, which were bred from indica and japonica crossed offspring. These specific bands were both located in the coding and non-encoding region, and usually connected with quantitative trait loci. Utilizing the indica-japonica-specific markers, japonica consanguinity was detected in sterile hybrid rice lines. Many indica-japonica-specific bands were found in O. rufipogon. This result supports the multiple-origin model for domesticated rice. Javanica exhibited a greater number of indica-japonica-specific bands, which indicates that it is a subspecies of O. sativa L.     

Biased distribution of microsatellite motifs in the rice genome

Microsatellites are useful tools to study the extent of divergence between two taxonomic groups that show high sequence similarity. We have compared microsatellite distribution to illustrate genetic variation between the two rice genomes, Oryza sativa L. ssp. indica and Oryza sativa L. ssp. japonica. Microsatellite distribution proved to be non random as certain regions of very high microsatellite density have been identified. Microsatellite density in the subspecies japonica was computed marginally higher than in the subspecies indica in the genomic regions compared between the two subspecies. Unexpectedly high microsatellite densities were observed in 5′-untranslated regions of genes. These regions also displayed a clear motif bias. Some of the longest microsatellite repeats were found in intron sequences. Frequency, as well as motif bias was also noted with respect to the association of microsatellites with transposable elements. Microsatellite mutability values were exemplarily estimated for 90 loci by aligning the microsatellite containing regions between the two genomes. Poor rates of finding an orthologue corresponded with high microsatellite mutability in rice. These insights are likely to play a significant role in selecting microsatellite loci to be used in molecular breeding and studying evolutionary dynamics of the two subspecies.     

Genetic diversity of six isozyme loci in cultivated barley of Tibet

Summary A random sample of 463 accessions of cultivated barley from the Tibet Hordeum germplasm collection was assayed electorphoretically for genetic diversity at six isozyme loci. Two loci (Acp-1 and Got-1) were found to be monomorphic and extensive variation was detected at the remaining four loci (Est-1, Est-2, Est-3 and Est-4). The allelic composition of Tibetan barley appeared to be distinct as compared to the results of previous studies of barleys from other parts of the world. Partitioning of genetic diversity showed that approximately 96% of the total variation was maintained at the within-subregion level and only about 4% was accounted for by differentiation among the eight subregions. Analysis of multilocus genotypes revealed non-random association of the alleles at the four loci, both in the entire sample and in all the subregions, although the four major multilocus genotypes did not show significant departure from the expectation based on complete random association. The possible causes for the establishment of these multilocus associations were discussed.     

Genetic characterization of weedy rice (Oryza sativa L.) based on morpho-physiology, isozymes and RAPD markers

 Weedy rice (Oryza sativa L.) is an important resource for breeding and for studying the evolution of rice. The present study was carried out to identify the genetic basis of the weedy rices distributed in various countries of the world. One hundred and fifty two strains of weedy rice collected from Bangladesh, Brazil, Bhutan, China, India, Japan, Korea, Nepal, Thailand and the USA were tested for variations in six morpho-physiological characteristics and in 14 isozyme loci. Twenty six weedy strains selected from the above materials were assayed for the Est-10 locus, six RAPD loci of the nuclear genome, and one chloroplast locus. From the results of multivariate analysis based on the morpho-physiological characteristics and the isozymes, weedy rice strains were classified into indica and japonica types, and each type was further divided into forms resembling cultivated and wild rice. Thus, four groups designated as I, II, III and IV were identified. Weedy strains of group I (indica-type similar to cultivars) were distributed mostly in temperate countries, group II (indica-type similar to wild rice) in tropical countries, group III (japonica-type similar to cultivars) in Bhutan and Korea, group IV ( japonica-type similar to wild rice) in China and Korea. In group I, classified as indica, several strains showed japonica-specific RAPD markers, while some others had japonica cytoplasm with indica-specific RAPD markers in a heterozygous state at several loci. One weedy strain belonging to group II showed a wild rice-specific allele at the Est-10 locus. However, in groups III and IV, no variation was ound either for the markers on Est-10 or for the RAPD loci tested. Judging from this study, weedy rice of group I might have originated at least partly from gene flow between indica and japonica, whereas that of group II most probably originated from gene flow between wild and cultivated indica rice. Weedy rice of group III is thought to have originated from old rice cultivars which had reverted to a weedy form, and that of group IV from gene flow between japonica cultivars and wild rice having japonica backgrounds. Received: 2 May 1996 / Accepted: 30 August 1996     

Genetic structure and differentiation of <Emphasis Type="Italic">Oryza sativa</Emphasis> L. in China revealed by microsatellites

China is one of the largest centers of genetic diversity of Oryza sativa L. in the world. Using a genetically representative primary core collection of 3,024 rice landraces in China, we analyzed the genetic structure and intraspecific differentiation of O. sativa, and the directional evolution of SSR. The genetic structure was investigated by model-based structure analysis and construction of neighbor-joining phylogenetic tree. Comparison between genetic structure and predefined populations according to Ting’s taxonomic system revealed a hierarchical genetic structure: two distinct subspecies, each with three ecotypes and different numbers of geo-ecogroups within each ecotype. Two subspecies evidently resulted from adaptation to different environments. The different cropping systems imposed on the subspecies led to further differentiation, but the variation within each subspecies resulted from different causes. Indica, under tropical-like or lowland-like environments, exhibited clear differentiation among seasonal ecotypes, but not among soil-watery ecotypes; and japonica showed clear differences between soil water regime ecotypes, but not among seasonal ecotypes. Chinese cultivated rice took on evident directional evolution in microsatellite allele size at several aspects, such as subspecies and geographical populations. Japonica has smaller allele sizes than indica, and this may partly be the result of their different domestication times. Allele size was also negatively correlated with latitude and altitude, and this may be interpreted by different mutation rates, selection pressures, and population size effects under different environments and cropping systems.     

Multiple introgression events surrounding the Hd1 flowering-time gene in cultivated rice, Oryza sativa L.

Flowering time is a major determinant for the local adaptation of crops. Hd1 is a key flowering-time gene in rice and is orthologous to the Arabidopsis CONSTANS gene. To elucidate the role of Hd1 in selection, we examined the Hd1 alleles of 60 landraces of Asian cultivated rice (Oryza sativa L.) originating from all regions of Asia, which comprised three cultivar groups, indica, japonica, and aus. The identified alleles were classified into four allele groups. The functional Hd1 alleles in allele groups I and II corresponded to indica and japonica, respectively. Non-functional alleles in these groups were not clearly associated with cultivar groups or locations. Allele groups III and IV corresponded to the aus cultivar group. The ancestry of each cultivar group was identified by the coalescent approach for Hd1 molecular evolution using the haplotype patterns of 14 regions over the 1.1 Mb chromosomal region surrounding Hd1 and the pSINE patterns of two loci, 1.4 and 4.4 Mb apart from Hd1. The haplotype patterns clearly revealed that Hd1 allele migration was caused by multiple and complex introgression events between cultivar groups. The Hd1 haplotypes among dozens of accessions of the wild species O. rufipogon were strongly divergent and only two of the haplotype clusters in O. rufipogon were closely related to those in cultivated rice. This strongly suggested that multiple introgression events have played an important role in the shaping and diversification of adaptation in addition to primary selection steps at the beginning of domestication.     

Comparative analysis of microsatellite DNA polymorphism in landraces and cultivars of rice

Genetic polymorphisms of ten microsatellite DNA loci were examined among 238 accessions of landraces and cultivars that represent a significant portion of the distribution range for both indica and japonica groups of cultivated rice. In all, 93 alleles were identified with these ten markers. The number of alleles varied from a low of 3 or 4 at each of four loci, to an intermediate value of 9–14 at five loci, and to an extra-ordinarily high 25 at one locus. The numbers of alleles per locus are much larger than those detected using other types of markers. The number of alleles detected at a locus is significantly correlated with the number of simple sequence repeats in the targeted microsatellite DNA. Indica rice has about 14% more alleles than japonica rice, and such allele number differences are more pronounced in landraces than in cultivars. The indica-japonica differentiation component accounted for about 10% of the diversity in the total sample, and twice as much differentiation was detected in cultivars as in landraces. About two-thirds as many alleles were observed in cultivars as in landraces; another two-thirds of the alleles in the cultivar group were found in modern elite cultivars or parents of hybrid rice. The majority of the simple sequence repeat (SSR) alleles that were present in high or intermediate frequencies in landraces ultimately survived into modern elite cultivars and hybrids. The greater resolving power and the efficient production of massive amounts of SSR data may be particularly useful for germplasm assessment and evolutionary studies of crop plants.