Molecular Evolution of the TAC1 Gene from Rice （Oryza sativa L.）

Tiller angle is a key feature of the architecture of cultivated rice(Oryza sativa),since it determines planting density and influences rice yield.Our previous work identified Tiller Angle Control 1(TACl) as a major quantitative trait locus that controls rice tiller angle.To further clarify the evolutionary characterization of the TACl gene,we compared a TACl-containing 3164-bp genomic region among 113 cultivated varieties and 48 accessions of wild rice,including 43 accessions of O.rufipogon and five accessions of O.nivara.Only one single nucleotide polymorphism(SNP),a synonymous substitution,was detected in TACl coding regions of the cultivated rice varieties, whereas one synonymous and one nonsynonymous SNP were detected among the TACl coding regions of wild rice accessions.These data indicate that little natural mutation and modification in the TACl coding region occurred within the cultivated rice and its progenitor during evolution.Nucleotide diversities in the TACl gene regions of O.sativa and O.rufipogon of 0.00116 and 0.00112,respectively, further indicate that TACl has been highly conserved during the course of rice domestication.A functional nucleotide polymorphism (FNP) of TACl was only found in the japonica rice group.A neutrality test revealed strong selection,especially in the 3’-flanking region of the TACl coding region containing the FNP in the japonica rice group.However,no selection occurred in the indica and wild-rice groups.A phylogenetic tree derived from TACl sequence analysis suggests that the indica and japonica subspecies arose independently during the domestication of wild rice.     

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.     

Comparative proteomic analysis of indica and japonica rice varieties

Indica and japonica are two main subspecies of Asian cultivated rice (Oryza sativa L.) that differ clearly in morphological and agronomic traits, in physiological and biochemical characteristics and in their genomic structure. However, the proteins and genes responsible for these differences remain poorly characterized. In this study, proteomic tools, including two-dimensional electrophoresis and mass spectrometry, were used to globally identify proteins that differed between two sequenced rice varieties (93–11 and Nipponbare). In all, 47 proteins that differed significantly between 93–11 and Nipponbare were identified using mass spectrometry and database searches. Interestingly, seven proteins were expressed only in Nipponbare and one protein was expressed specifically in 93–11; these differences were confirmed by quantitative real-time PCR and proteomic analysis of other indica and japonica rice varieties. This is the first report to successfully demonstrate differences in the protein composition of indica and japonica rice varieties and to identify candidate proteins and genes for future investigation of their roles in the differentiation of indica and japonica rice.     

The Birth of a Black Rice Gene and Its Local Spread by Introgression

The origin and spread of novel agronomic traits during crop domestication are complex events in plant evolution. Wild rice (Oryza rufipogon) has red grains due to the accumulation of proanthocyanidins, whereas most cultivated rice (Oryza sativa) varieties have white grains induced by a defective allele in the Rc basic helix-loop-helix (bHLH) gene. Although the events surrounding the origin and spread of black rice traits remain unknown, varieties with black grains due to anthocyanin accumulation are distributed in various locations throughout Asia. Here, we show that the black grain trait originated from ectopic expression of the Kala4 bHLH gene due to rearrangement in the promoter region. Both the Rc and Kala4 genes activate upstream flavonol biosynthesis genes, such as chalcone synthase and dihydroflavonol-4-reductase, and downstream genes, such as leucoanthocyanidin reductase and leucoanthocyanidin dioxygenase, to produce the respective specific pigments. Genome analysis of 21 black rice varieties as well as red- and white-grained landraces demonstrated that black rice arose in tropical japonica and its subsequent spread to the indica subspecies can be attributed to the causal alleles of Kala4. The relatively small size of genomic fragments of tropical japonica origin in some indica varieties indicates that refined introgression must have occurred by natural crossbreeding in the course of evolution of the black trait in rice.     

Independent Losses of Function in a Polyphenol Oxidase in Rice: Differentiation in Grain Discoloration between Subspecies and the Role of Positive Selection under Domestication

Asian rice (Oryza sativa) cultivars originated from wild rice and can be divided into two subspecies by several criteria, one of which is the phenol reaction (PHR) phenotype. Grains of indica cultivars turn brown in a phenol solution that accelerates a similar process that occurs during prolonged storage. By contrast, the grains of japonica do not discolor. This distinction may reflect the divergent domestication of these two subspecies. The PHR is controlled by a single gene, Phr1; here, we report the cloning of Phr1, which encodes a polyphenol oxidase. The Phr1 gene is indeed responsible for the PHR phenotype, as transformation with a functional Phr1 can complement a PHR negative cultivar. Phr1 is defective in all japonica lines but functional in nearly all indica and wild strains. Phylogenetic analysis showed that the defects in Phr1 arose independently three times. The multiple recent origins and rapid spread of phr1 in japonica suggest the action of positive selection, which is further supported by several population genetic tests. This case may hence represent an example of artificial selection driving the differentiation among domesticated varieties.     

Robustness and Strategies of Adaptation among Farmer Varieties of African Rice (Oryza glaberrima) and Asian Rice (Oryza sativa) across West Africa

This study offers evidence of the robustness of farmer rice varieties (Oryza glaberrima and O. sativa) in West Africa. Our experiments in five West African countries showed that farmer varieties were tolerant of sub-optimal conditions, but employed a range of strategies to cope with stress. Varieties belonging to the species Oryza glaberrima – solely the product of farmer agency – were the most successful in adapting to a range of adverse conditions. Some of the farmer selections from within the indica and japonica subspecies of O. sativa also performed well in a range of conditions, but other farmer selections from within these two subspecies were mainly limited to more specific niches. The results contradict the rather common belief that farmer varieties are only of local value. Farmer varieties should be considered by breeding programmes and used (alongside improved varieties) in dissemination projects for rural food security.     

Molecular Population Genetics of Rice Domestication

Domestication is a selection process that genetically modifies species to meet human needs. A most intriguing feature of domestication is the extreme phenotypic diversification among breeds. What could be the ultimate source of such genetic variations? Another notable outcome of artificial selection is the reduction in the fitness of domesticated species when they live in the wild without human assistance. The complete sequences of the two subspecies of rice cultivars provide an opportunity to address these questions. Between the two subspecies, we found much higher rates of non‐synonymous (N) than synonymous (S) substitutions and the N/S ratios are higher between cultivars than between wild species. Most interestingly, substitutions of highly dissimilar amino acids that are deleterious and uncommon between natural species are disproportionately common between the two subspecies of rice. We suggest strong selection in the absence of effective recombination may be the driving force, which we called the domestication‐associated Hill‐Robertson effect. These hitchhiking mutations may contribute to some fitness reduction in cultivars. Comparisons of the two genomes also reveal the existence of highly divergent regions in the genomes. Haplotypes in these regions often form highly polymorphic linkage blocks that are much older than speciation between wild species. Genes from such regions could contribute to the differences between indica and japonica and are likely to be involved in the diversifying selection under domestication. Their existence suggests that the amount of genetic variation within the single progenitor species Oryza rufipogon may be insufficient to account for the variation among rice cultivars, which may come from a more inclusive gene pool comprising most of the A‐genome wild species. Genes from the highly polymorphic regions also provide strong support for the independent domestication of the two subspecies. The genomic variation in rice has revealing implications for studying the genetic basis of indica‐japonica differentiation under rice domestication and subsequent improvement.     

A causal C–A mutation in the second exon of GS3 highly associated with rice grain length and validated as a functional marker

Comparative sequencing of GS3, the most important grain length (GL) QTL, has shown that differentiation of rice GL might be principally due to a single nucleotide polymorphism (SNP) between C and A in the second exon. A total of 180 varieties representing a wide range of rice germplasm were used for association analysis between C–A mutation and GL in order to confirm the potential causal mutation. A cleaved amplified polymorphic sequence (CAPS) marker, SF28, was developed based on the C–A polymorphism in the GS3 gene. A total of 142 varieties carried allele C with GL from 6.4 to 8.8 mm, while the remaining 38 varieties carried allele A with GL from 8.8 to 10.7 mm. Twenty-four unlinked SSR markers were selected to genotype 180 varieties for population structure analysis. Population structure was observed when the population was classified to three subpopulations. Average GL of either genotype A or genotype C within japonica among the three subpopulations had no significant difference from that in indica, respectively, although indica rice had longer grains on average than japonica in the 180 varieties. However, genotype C always had longer grain length on average than genotype A among three subpopulations. The mutation could explain 79.1, 66.4 and 34.7% of GL variation in the three subpopulations, respectively. These results clearly confirmed the mutation between C and A was highly associated with GL. The SF28 could be a functional marker for improvement of rice grain length.     

Development and validation of a PCR-based functional marker system for the major wide-compatible gene locus S5 in rice

The major wide-compatibility gene locus S5 in rice (Oryza sativa L.) located on chromosome 6 has been recently cloned and a 136-bp deletion in the candidate gene encoding aspartyl protease has been characterized to be specific for wide-compatible varieties, while many single nucleotide polymorphisms have been identified at S5 between indica and japonica rice types. In the present study, we designed a PCR-based multiplex functional marker system targeting the deletion and the SNPs for precise determination of the allelic status at S5. By deploying the marker system, the allelic status at the S5 locus in 584 rice genotypes has been assayed. A total of 116 genotypes, including 11 cultivars, two known wide-compatible varieties, 48 IRRI germplasm lines, 12 Indian aromatic rice genotypes, 37 restorer lines and six breeding lines, have been identified to possess the 136-bp deletion specific for the neutral allele at S5. The marker system was able to clearly distinguish indica and japonica alleles from the neutral allele and has been validated in a mapping population derived from the three-way cross IR36/Dular//Akihikari, which segregated for spikelet sterility/fertility. The functional marker system targeting S5 developed in the present study will be very useful in rapid identification of wide-compatible genotypes, in predicting the success of inter-subspecific crosses and in targeted introgression of the wide-compatible allele of S5 into elite indica and japonica rice varieties.     

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.     

Genome-wide selection and introgression of Chinese rice varieties during breeding

China is the largest rice-producing country, but the genomic landscape of rice diversity has not yet been clarified. In this study, we re-sequence 1070 rice varieties collected from China (400) and other regions in Asia (670). Among the six major rice groups (aus, indica-I, indica-II, aromatic, temperate japonica, and tropical japonica), almost all Chinese varieties belong to the indica-II or temperate japonica group. Most Chinese indica varieties belong to indica-II, which consists of two subgroups developed during different phases of rice breeding. The genomic segments underlying the differences between these subgroups span 36.32 Mb. The Chinese japonica rice varieties fall into the temperate japonica group, consisting of two subgroups based on their geographical distribution. The genomic segments underlying the differences between these subgroups span 27.69 Mb. These differentiated segments in the Chinese indica varieties span 45 genes with nonsynonymous mutations that are closely related to variations in plant height and grain width. Fifty-four genes with nonsynonymous mutations are associated with the differences in heading date between the two Chinese japonica subgroups. These findings provide new insights into rice diversity in China that will facilitate the molecular breeding.     

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.     

Subspecies — Specific Microsatellite Markers for Rice (Oryza sativa L)

Subspecific classification of Asian rice (Oryza sativa L) into indica and japonica has always been a subject of interest althrough for rice breeders and geneticists. The present study aims at identifying subspecies specific microsatellite markers in six genotypes, each of indica and japonica using 372 microsatellite primers covering the entire genome. Only 36 primers gave clear polymorphism on 3% agarose gel and these can be used as diagnostic markers for routine and easy identification of the subspecies.     

Genetic Analysis of Cold Tolerance at the Germination and Booting Stages in Rice by Association Mapping

Low temperature affects the rice plants at all stages of growth. It can cause severe seedling injury and male sterility resulting in severe yield losses. Using a mini core collection of 174 Chinese rice accessions and 273 SSR markers we investigated cold tolerance at the germination and booting stages, as well as the underlying genetic bases, by association mapping. Two distinct populations, corresponding to subspecies indica and japonica showed evident differences in cold tolerance and its genetic basis. Both subspecies were sensitive to cold stress at both growth stages. However, japonica was more tolerant than indica at all stages as measured by seedling survival and seed setting. There was a low correlation in cold tolerance between the germination and booting stages. Fifty one quantitative trait loci (QTLs) for cold tolerance were dispersed across all 12 chromosomes; 22 detected at the germination stage and 33 at the booting stage. Eight QTLs were identified by at least two of four measures. About 46% of the QTLs represented new loci. The only QTL shared between indica and japonica for the same measure was qLTSSvR6-2 for SSvR. This implied a complicated mechanism of old tolerance between the two subspecies. According to the relative genotypic effect (RGE) of each genotype for each QTL, we detected 18 positive genotypes and 21 negative genotypes in indica, and 19 positive genotypes and 24 negative genotypes in japonica. In general, the negative effects were much stronger than the positive effects in both subspecies. Markers for QTL with positive effects in one subspecies were shown to be effective for selection of cold tolerance in that subspecies, but not in the other subspecies. QTL with strong negative effects on cold tolerance should be avoided during MAS breeding so as to not cancel the effect of favorable QTL at other loci.     

Different non-host resistance responses of two rice subspecies,japonica and indica,to Puccinia striiformis f. sp. tritici

Key message

Japonica and indica have different non-host resistance (NHR) abilities to Puccinia striiformis f. sp. tritici ( Pst ), and hydrogen peroxide (H ₂ O ₂ ) has a positive function in NHR to japonica against Pst.

Abstract

Non-host interactions between Puccinia striiformis f. sp. tritici (Pst) and two rice subspecies were characterized using 23 rice varieties, including 11 japonica and 12 indica. Results showed that the infected fungal structures were easily produced in the leaves of indica, whereas only several substomatal vesicles and primary infection hyphae were observed in the leaves of japonica. This result indicated that indica is less resistant or more susceptible to Pst than japonica. Hydrogen peroxide accumulated in the initial phase of japonica–Pst interaction but not in indica–Pst interaction. A set of reactive oxygen species (ROS)-related genes was also induced in response to Pst infection, suggesting that ROS activation is one of the major mechanisms of non-host resistance of rice to Pst.     

A natural mutation in rc reverts white-rice-pericarp to red and results in a new, dominant, wild-type allele: Rc-g

The Rc locus regulates pigmentation of the rice bran layer, and selection for the rc allele (white pericarp) occurred during domestication of the crop. White bran is now ubiquitous among cultivated varieties throughout rice growing regions of the world. We identified a new allele that arose by natural mutation within the rc pseudogene of the cultivar 'Wells'. The mutation restored the reading frame of the gene, and reverted the bran layer pigmentation to red (wild-type). By sequencing the Rc locus in plants derived from red seeds, and linkage analysis in a segregating population, we were able to demonstrate that mutation within rc resulted in the new, dominant, wild-type allele Rc-g.     

Geographic distribution and multilocus organization of isozyme variation of rice (Oryza sativa L.)

Genetic organization of isozyme variation in rice (Oryza sativa L.) was investigated based on 17 polymorphic isozyme loci using a sample of 511 accessions of worldwide origin. The genetic diversity within the species was very high (H=0.36 with 4.82 alleles per locus), as compared with most selfing plant species. Three diversity centers were detected for isozyme variation including South Asia, China and Southeast Asia. The accessions were classified into three well-differentiated cultivar groups corresponding to the indica and japonica subspecies, and a new unnamed group. Variation within the cultivar groups accounted for 80% of the total isozyme variation. Within-country variation accounted for 58% of the total variation while among-region and among-country variation within the cultivar groups accounted for only 14% and 8% of the total variation. Analyses using log-linear models revealed that pronounced non-random associations between and among alleles at many unlinked isozyme loci were organized in a non-hierarchical pattern, and subspecific and macro-geographic differentiation was much more pronounced in multilocus phenotype frequencies than in allelic frequencies at individual loci. These results suggest that selection on multilocus gene complexes was largely responsible for the maintenance of the extensive isozyme variation within the species and the indica-japonica differentiation. Our results further suggest the independent domestication of indica and japonica, the dual origins of the indica rice from China and South Asia (India), and the differentiation of the ecotypes ’javanica’ and the ’temperate japonica’ within the japonica subspecies. Received: 5 August 1999 / Accepted: 13 December 1999     

Recombinational and mutational analyses of subspecific differentiation in Oryza sativa L.

Recombinational and mutational analyses were performed in an attempt to elucidate the nature of the genic differences and the ancestral relationship between the indica and japonica subspecies of Oryza sativa L. by taking representative varieties from each group. The absence of cytological aberrations at meiosis in indica×japonica hybrids suggest that genetic differences are predominantly genic rather than chromosomal. The pattern of variation induced in the key characters, and the frequency and spectra of mutations obtained in each group, failed to support the hypothesis that indica has given rise to japonica, and suggested that the two subspecies have originated from a common ancestor.     

Genome re-sequencing suggested a weedy rice origin from domesticated indica-japonica hybridization: a case study from southern China

Main conclusion

Whole-genome re-sequencing of weedy rice from southern China reveals that weedy rice can originate from hybridization of domesticated indica and japonica rice.

Abstract

Weedy rice (Oryza sativa f. spontanea Rosh.), which harbors phenotypes of both wild and domesticated rice, has become one of the most notorious weeds in rice fields worldwide. While its formation is poorly understood, massive amounts of rice genomic data may provide new insights into this issue. In this study, we determined genomes of three weedy rice samples from the lower Yangtze region, China, and investigated their phylogenetics, population structure and chromosomal admixture patterns. The phylogenetic tree and principle component analysis based on 46,005 SNPs with 126 other Oryza accessions suggested that the three weedy rice accessions were intermediate between japonica and indica rice. An ancestry inference study further demonstrated that weedy rice had two dominant genomic components (temperate japonica and indica). This strongly suggests that weedy rice originated from indica-japonica hybridization. Furthermore, 22,443 novel fixed single nucleotide polymorphisms were detected in the weedy genomes and could have been generated after indica-japonica hybridization for environmental adaptation.