Variation in the nucleotide sequence of a prolamin gene family in wild rice

Variation in the DNA sequence of the 10 kDa prolamin gene family within the wild rice species Oryza rufipogon was probed using the direct sequencing of PCR-amplified genes. A comparison of the nucleotide and deduced amino-acid sequences of eight Asian strains of O. rufipogon and one strain of the related African species O. longistaminata is presented.     

A tourist element in the 5'-flanking region of the catalase gene CatA reveals evolutionary relationships among Oryza species with various genome types

Tourist-OsaCatA, a transposable element, was found in the 5′-flanking region of the rice gene CatA. The characteristics of this element are similar to those of the other Tourist elements so far found in Oryza sativa. PCR and sequence analyses of 37 accessions of 18 species revealed that all the Oryza species examined, except for one accession, have either a full-length or a partial Tourist element at this locus. Unlike the Tourist elements previously reported, this Tourist element is found in all four Oryza species complexes in the Oryzeae tribe. All AA genome Oryza species, except O. longistaminata, contain the full-length Tourist element. O. longistaminata and the species of the O. officinalis, O. meyeriana and O. ridleyi complexes contain the partial element. A phylogenetic tree of Oryza species based on the nucleotide sequences of these Tourist elements was constructed. The O. longistaminata accessions were placed near the neighboring cluster of the officinalis complex. We propose that the ancestor of O. longistaminata and that of other species with the AA genome diverged, and the ancestor(s) of the O. officinalis, O. ridleyi and O. meyeriana complexes then diverged from the ancestor of O. longistaminata in the course of the evolution of the Oryza species. The Tourist elements associated with CatA and its orthologs thus provide useful tools for examining evolutionary relationships among Oryza species. Received: 12 March 1999 / Accepted: 7 July 1999     

Distinct evolutionary patterns of Oryza glaberrima deciphered by genome sequencing and comparative analysis

Here we present the genomic sequence of the African cultivated rice, Oryza glaberrima, and compare these data with the genome sequence of Asian cultivated rice, Oryza sativa. We obtained gene‐enriched sequences of O. glaberrima that correspond to about 25% of the gene regions of the O. sativa (japonica) genome by methylation filtration and subtractive hybridization of repetitive sequences. While patterns of amino acid changes did not differ between the two species in terms of the biochemical properties, genes of O. glaberrima generally showed a larger synonymous–nonsynonymous substitution ratio, suggesting that O. glaberrima has undergone a genome‐wide relaxation of purifying selection. We further investigated nucleotide substitutions around splice sites and found that eight genes of O. sativa experienced changes at splice sites after the divergence from O. glaberrima. These changes produced novel introns that partially truncated functional domains, suggesting that these newly emerged introns affect gene function. We also identified 2451 simple sequence repeats (SSRs) from the genomes of O. glaberrima and O. sativa. Although tri‐nucleotide repeats were most common among the SSRs and were overrepresented in the protein‐coding sequences, we found that selection against indels of tri‐nucleotide repeats was relatively weak in both African and Asian rice. Our genome‐wide sequencing of O. glaberrima and in‐depth analyses provide rice researchers not only with useful genomic resources for future breeding but also with new insights into the genomic evolution of the African and Asian rice species.     

Phylogenetic relationships of annual and perennial wild rice: probing by direct DNA sequencing

Summary The phylogenetic relationships between Asian wild rice strains were analyzed by direct sequencing of PCR-amplified DNA fragments. The sequence of three introns located in the phytochrome gene was determined for eight strains of the Asian wild rice, Oryza rufipogon, and one strain of the related African species, Oryza longistaminata. The number of nucleotide substitutions per site between various strains within a single species, O. rufipogon, ranged between 0.0017 and 0.0050, while those between two related species, O. rufipogon and O. longistaminate, were 0.043–0.049 (23–26 within 532 bp). Taken together with the sequence differences of the 10-kDa prolamin gene, a model is proposed for the phylogenetic relationships and evolutionary history of annuals and perennials within O. rufipogon.     

SUB1A‐dependent and ‐independent mechanisms are involved in the flooding tolerance of wild rice species

Crop tolerance to flooding is an important agronomic trait. Although rice (Oryza sativa) is considered a flood‐tolerant crop, only limited cultivars display tolerance to prolonged submergence, which is largely attributed to the presence of the SUB1A gene. Wild Oryza species have the potential to unveil adaptive mechanisms and shed light on the basis of submergence tolerance traits. In this study, we screened 109 Oryza genotypes belonging to different rice genome groups for flooding tolerance. Oryza nivara and Oryza rufipogon accessions, belonging to the A‐genome group, together with Oryza sativa, showed a wide range of submergence responses, and the tolerance‐related SUB1A‐1 and the intolerance‐related SUB1A‐2 alleles were found in tolerant and sensitive accessions, respectively. Flooding‐tolerant accessions of Oryza rhizomatis and Oryza eichingeri, belonging to the C‐genome group, were also identified. Interestingly, SUB1A was absent in these species, which possess a SUB1 orthologue with high similarity to O. sativa SUB1C. The expression patterns of submergence‐induced genes in these rice genotypes indicated limited induction of anaerobic genes, with classical anaerobic proteins poorly induced in O. rhizomatis under submergence. The results indicated that SUB1A‐1 is not essential to confer submergence tolerance in the wild rice genotypes belonging to the C‐genome group, which show instead a SUB1A‐independent response to submergence.     

Sequence diversity of a domesticated transposase gene, <Emphasis Type="Italic">MUG1</Emphasis>, in <Emphasis Type="Italic">Oryza</Emphasis> species

MUG1 is a MULE transposon-related domesticated gene in plants. We assessed the sequence diversity, neutrality, expression, and phylogenetics of the MUG1 gene among Oryza ssp. We found MUG1 expression in all tissues analyzed, with different levels in O. sativa. There were 408 variation sites in the 3886 bp of MUG1 locus. The nucleotide diversity of the MUG1 was higher than functionally known genes in rice. The nucleotide diversity (π) in the domains was lower than the average nucleotide diversity in whole coding region. The π values in nonsynonymous sites were lower than those of synonymous sites. Tajima D and Fu and Li D* values were mostly negative values, suggesting purifying selection in MUG1 sequences of Oryza ssp. Genome-specific variation and phylogenetic analyses show a general grouping of MUG1 sequences congruent with Oryza ssp. biogeography; however, our MUG1 phylogenetic results, in combination with separate B and D genome studies, might suggest an early divergence of the Oryza ssp. by continental drift of Gondwanaland. O. longistaminata MUG1 divergence from other AA diploids suggests that it might not be a direct ancestor of the African rice species. These authors contributed equally to this work.     

A Tourist element in the 5′-flanking region of the catalase gene CatA reveals evolutionary relationships among Oryza species with various genome types

Tourist-OsaCatA, a transposable element, was found in the 5′-flanking region of the rice gene CatA. The characteristics of this element are similar to those of the other Tourist elements so far found in Oryza sativa. PCR and sequence analyses of 37 accessions of 18 species revealed that all the Oryza species examined, except for one accession, have either a full-length or a partial Tourist element at this locus. Unlike the Tourist elements previously reported, this Tourist element is found in all four Oryza species complexes in the Oryzeae tribe. All AA genome Oryza species, except O.?longistaminata, contain the full-length Tourist element. O. longistaminata and the species of the O. officinalis, O. meyeriana and O.?ridleyi complexes contain the partial element. A phylogenetic tree of Oryza species based on the nucleotide sequences of these Tourist elements was constructed. The O.?longistaminata accessions were placed near the neighboring cluster of the officinalis complex. We propose that the ancestor of O.?longistaminata and that of other species with the AA genome diverged, and the ancestor(s) of the O.?officinalis, O.?ridleyi and O.?meyeriana complexes then diverged from the ancestor of O.?longistaminata in the course of the evolution of the Oryza species. The Tourist elements associated with CatA and its orthologs thus provide useful tools for examining evolutionary relationships among Oryza species.     

Tissue-Specific Transcriptomic Profiling of Sorghum propinquum using a Rice Genome Array

Sorghum (Sorghum bicolor) is one of the world''s most important cereal crops. S. propinquum is a perennial wild relative of S. bicolor with well-developed rhizomes. Functional genomics analysis of S. propinquum, especially with respect to molecular mechanisms related to rhizome growth and development, can contribute to the development of more sustainable grain, forage, and bioenergy cropping systems. In this study, we used a whole rice genome oligonucleotide microarray to obtain tissue-specific gene expression profiles of S. propinquum with special emphasis on rhizome development. A total of 548 tissue-enriched genes were detected, including 31 and 114 unique genes that were expressed predominantly in the rhizome tips (RT) and internodes (RI), respectively. Further GO analysis indicated that the functions of these tissue-enriched genes corresponded to their characteristic biological processes. A few distinct cis-elements, including ABA-responsive RY repeat CATGCA, sugar-repressive TTATCC, and GA-responsive TAACAA, were found to be prevalent in RT-enriched genes, implying an important role in rhizome growth and development. Comprehensive comparative analysis of these rhizome-enriched genes and rhizome-specific genes previously identified in Oryza longistaminata and S. propinquum indicated that phytohormones, including ABA, GA, and SA, are key regulators of gene expression during rhizome development. Co-localization of rhizome-enriched genes with rhizome-related QTLs in rice and sorghum generated functional candidates for future cloning of genes associated with rhizome growth and development.     

Distribution and organization of a tandemly repeated 352-bp sequence in the oryzae family

Summary A 352-bp EcoRI fragment from rice DNA was cloned and shown to be a member of a tandem repeat. Sequence determination revealed homologies with human alpha satellite DNA and maize knob heterochromatin specific repeat. This 352-bp sequence is highly specific for the AA genome of rice. However, copy number and sequence organization are variable, depending on the accession analyzed. Several examples of amplification were observed in O. rufipogon and O. longistaminata. Use of resolutive polyacrylamide gel electrophoresis and 4-bp cutter enzymes allowed one to distinguish between the Indica and Japonica subtypes of O. sativa. The same method also discriminates between two groups of O. rufipogon, the presumed ancestor of O. sativa, suggesting that the present day Indica and Japonica subtypes originated independently from two O. rufipogon distinct populations.     

A comparative study of genetic relationships among the AA-genome<Emphasis Type="Italic"> Oryza</Emphasis> species using RAPD and SSR markers

In order to estimate genetic relationships of the AA-genome Oryza species, RAPD and SSR analyses were performed with 45 accessions, including 13 cultivated varieties (eight Oryza sativa and five Oryza glaberrima) and 32 wild accessions (nine Oryza rufipogon, seven Oryza nivara, three Oryza glumaepatula, four Oryza longistaminata, six Oryza barthii, and three Oryza meridionalis). A total of 181 clear and repeatable bands were amplified from 27 selected RAPD primers, and 101 alleles were detected from 29 SSR primer pairs. The dendrogram constructed using UPGMA from a genetic-similarity matrix based on the RAPD data supported the clustering of distinct five groups with a few exceptions: O. rufipogon/O. nivara/O. meridionalis, O. barthii/O. glaberrima, O. glumaepatula, O. sativa and O. longistaminata. The dendrogram based on the SSR analysis showed a more-complicated genetic variation pattern, but the O. longistaminata and O. barthii/O. glaberrima accessions were consistently separated from all other accessions, indicating significant differentiation of the African AA-genome Oryza species. For accessions in the O. rufipogon/O. nivara/O. sativa complex, it is apparent that geographical isolation has played an important role in differentiation of the Asian AA-genome Oryza taxa. It is also demonstrated from this study that both RAPD and SSR analyses are powerful methods for detecting polymorphisms among the different AA-genome Oryza accessions. However, the RAPD analysis provides a more-informative result in terms of the overall genetic relationships at the species level compared to the SSR analysis. The SSR analysis effectively reveals diminutive variation among accessions or individuals within the same species, given approximately the same number of primers or primer-pairs used in the studies.Communicated by Q. Zhang     

Evolutionary relationship of plant catalase genes inferred from exon-intron structures: isozyme divergence after the separation of monocots and dicots

In order to understand the molecular evolution of catalase genes in higher plants, we compared the exon-intron structures of 12 genomic sequences from six plant species. It was assumed that the putative single primordial catalase gene had seven introns, because only those catalase genes having this structure are found in the monocotyledonae and dicotyledonae classes. After the evolutionary divergence of monocots from dicots, consecutive duplication of the primordial gene followed by the differential loss of introns occurred in each class to form three (or possibly four in dicots) diverse isozyme genes. In monocots, three ancestral isozyme genes were formed before the divergence of ancestral rice and maize. One of the rice genes, CatA, has an entirely new short intron which was not found in any other plant catalase gene examined. We have investigated the existence of the intron in the CatA homolog in other rice species by polymerase chain reaction (PCR) analysis. One major PCR product was found with the genomic DNAs from O. sativa (indica and japonica types), O. rufipogon and O. glaberrima. DNAs from several accessions of O. longistaminata showed variation in both the number and size of the DNA fragments amplified. PCR analyses and sequencing of the PCR products revealed that there are several CatA homologs having different sequences in some accessions of O. longistaminata. We have extended our study to other species in the Poaceae. The results suggest that the gain of the intron, most likely by insertion of a retroposon, took place in the ancestral genome of rice after its evolutionary divergence from other ancestral cereals such as barley, wheat and oat. Received: 20 November 1997 / Accepted: 5 January 1998     

A novel QTL <Emphasis Type="Italic">qSPP2.2</Emphasis> controlling spikelet per panicle identified from <Emphasis Type="Italic">Oryza longistaminata</Emphasis> (A. Chev. et Roehr.), mapped and transferred to <Emphasis Type="Italic">Oryza sativa</Emphasis> (L.)

Increasing the rice productivity from the current 10 to 12 tons/ha to meet the demand of estimated 8.8 billion people in 2035 is posing a major challenge. Wild relatives of rice contain some novel genes which can help in improving rice yield. Spikelet per panicle (SPP) is a valuable trait for determining yield potential in rice. In this study, a major QTL for increasing SPP has been identified, mapped, and transferred from African wild rice O. longistaminata to O. sativa (L.). The QTL was mapped on the long arm of chromosome 2 in a 167.1 kb region flanked by SSR markers RM13743 and RM13750, which are 1.0 cM apart, and is designated as qSPP2.2. The QTL explained up to 30% of phenotypic variance in different generations/seasons and showed positive additive effect of allele contributed by O. longistaminata. In addition, O. longistaminata allele in qSPP2.2 contributed to increase in grains per panicle, but decrease in the tillers per plant. The 167.1 kb region contains 23 predicted genes. Based on the functional annotation, three genes, LOC_Os02g44860, LOC_Os02g44990, and LOC_Os02g45010, were selected as putative candidates for characterization. Sequence analysis of the three genes revealed functional variations between the parental lines for LOC_Os02g44990 and a variation in 5′UTR for LOC_Os02g45010 which will help further to identify putative candidate gene(s). This is the first yield component QTL to be identified, mapped, and transferred from O. longistaminata.     

A genome-specific repetitive DNA sequence from Oryza eichingeri: characterization,localization, and introgression to O. sativa

In the course of transferring the brown planthopper resistance from a diploid, CC-genome wild rice species, Oryza eichingeri (IRGC acc. 105159 and 105163), to the cultivated rice variety 02428, we have isolated many alien addition and introgression lines. The O. eichingeri chromatin in some of these lines has previously been identified using genomic in situ hybridization and molecular-marker analysis. Here we cloned a tandemly repetitive DNA sequence from O. eichingeri IRGC acc105163, and detected it in 25 introgression lines. This repetitive DNA sequence showed high specificity to the rice CC genome, but was absent from all the four tetraploid species with BBCC or CCDD genomes. The monomer in this repetitive DNA sequence is 325–366-bp long, with a copy number of about 5,000 per 1 C of the O. eichingeri genome, showing 88% homology to a repetitive DNA sequence isolated from Oryza officinalis (2n=2x=24, CC). Fluorescent in situ hybridization revealed 11 signals distributed over eight O. eichingeri chromosomes, mostly in terminal or subterminal regions. Received: 28 November 2000 / Accepted: 3 April 2001     

Genomic analysis of polycarpellary rice (Oryza sativa L.) through whole genome resequencing

There is a natural floral organ mutant of rice (var. Jugal) where the florets, popularly known as spikelet bear multiple carpels and produce multiple kernels in most of its grain. In our earlier work a detailed study has been done on its morpho-anatomical structure with allelic diversity and expression study of the major genetic loci associated with floral organ development. In present study high throughput whole genome sequencing was done which generated about of 3.7 million base pair genomic data for downstream analysis. The reads were about 101 bases long and mapped to the Oryza sativa var. Nipponbare as reference genome. Genome wide variant analysis detected 1,096,419 variants which included 943,033 SNPs and 153,386 InDels. A total of 24,920 non-synonymous SNPs were identified for 11,529 identified genes. Chromosome-wise distribution of uniquely mapped reads onto reference genome showed that maximum reads were mapped to 1st chromosome and least to 9th chromosome. 10th chromosome showed highest density of variations (about 325.6 per 100 kb genome sequence). Detailed sequence analysis of 23 floral organ developmental genes detected 419 potent variants where DL (Drooping Leaf) and OSH1 (Oryza sativa Homeobox1) genes showed highest number (32) of variants; whereas, MADS21 (Minichromosome Agamous Deficient Serum Factor 21) gene have lowest number (5) of variants. The information generated in this study will enrich the genomics of floral organ development in indica rice and cereal crops in general.

     

Non-LTR retrotransposons (LINEs) as ubiquitous components of plant genomes

During the course of work aimed at isolating a rice gene from Oryza australiensis by PCR, the oligonucleotide primers used were found to generate a fragment that showed sequence homology to the endonuclease (EN) region of the maize non-LTR retrotransposon (LINE) Cin4. We carried out further PCRs using oligonucleotide primers that hybridized to these sequences, and found that they amplified several fragments, each with homology to the EN regions, from Oryza sativa cv. Nipponbare as well as O. australiensis. We mapped the approximate locations of two rice LINE homologues by screening clones in a YAC library made from a rice (O. sativa) genome, and found that each homologue was present in a low copy number apparently at nonspecific regions on rice chromosomes. We then carried out PCR using degenerate oligonucleotide primers which hybridized to the rice LINE homologues and Cin4 to ascertain whether LINE homologues are present in a variety of members of the plant kingdom, including angiosperms, gymnosperms, bracken, horsetail and liverwort. Cloning and nucleotide sequencing revealed that 53 clones obtained from 27 out of 33 plant species contained LINE homologues. In addition to these homologues, we identified four homologues with EN regions in the Arabidopsis thaliana genome by a computer search of databases. The nucleotide sequences of almost all the LINE homologues were greatly diverged, but the derived amino acid sequences were well conserved, and all contained glutamic acid and tyrosine residues at almost the same relative positions as in the the active site regions of AP (apurinic/apyrimidinic)-endonucleases. The EN regions in the LINE homologues from closely related plant species show a closer phylogenetic relationship, indicating that sequence divergence during vertical transmission has been a major influence upon the evolution of plant LINEs. Received: 13 July 1998 / Accepted: 13 October 1998     

BAC library development for allotetraploid Leymus (Triticeae) wildryes enable comparative genetic analysis of lax-barrenstalk1 orthogene sequences and growth habit QTLs

Tall-caespitose basin wildrye (Leymus cinereus) and rhizomatous creeping wildrye (Leymus triticoides) are perennial Triticeae relatives of wheat and barley. Quantitative trait loci (QTLs) controlling rhizome proliferation were previously detected on homoeologous regions of LG3a and LG3b in two full-sib families derived from allotetraploid hybrids. Triticeae homoeologous group 3 aligns to rice chromosome 1, which contains the rice lax panicle and maize barrenstalk1 orthogene responsible for induction of axillary branch meristems, but this gene has not been mapped or sequenced in Triticeae. We developed bacterial artificial chromosome (BAC) libraries representing 6.1 haploid equivalents of the tetraploid Leymus genome (10.7 Mb). Overgo probes designed from the lax-barrenstalk1 orthogene hybridized to 12 Leymus BAC clones. Deduced amino-acid sequences from seven BAC clones were highly conserved with the rice, maize, and sorghum lax-barrenstalk1orthogenes. Gene specific primers designed from two of the most divergent BAC clones map to homoeologous regions of Leymus LG3a and LG3b and align with the lax-barrenstalk1 orthogene on rice 1L. Comparisons of genomic DNA sequences revealed two other conserved regions surrounding the Leymus LG3a, rice, and sorghum lax-barrenstalk1 ortholoci, and one of these regions was also present in maize and Leymus LG3b sequences. Comparisons of Leymus LG3a and LG3b lax-barrenstalk1 coding sequences and flanking genomic regions elucidate molecular differences between subgenomes.     

Phylogenetic analysis of Oryza species, based on simple sequence repeats and their flanking nucleotide sequences from the mitochondrial and chloroplast genomes

Simple sequence repeats (SSR) and their flanking regions in the mitochondrial and chloroplast genomes were sequenced in order to reveal DNA sequence variation. This information was used to gain new insights into phylogenetic relationships among species in the genus Oryza. Seven mitochondrial and five chloroplast SSR loci equal to or longer than ten mononucleotide repeats were chosen from known rice mitochondrial and chloroplast genome sequences. A total of 50 accessions of Oryza that represented six different diploid genomes and three different allopolyploid genomes of Oryza species were analyzed. Many base substitutions and deletions/insertions were identified in the SSR loci as well as their flanking regions. Of mononucleotide SSR, G (or C) repeats were more variable than A (or T) repeats. Results obtained by chloroplast and mitochondrial SSR analyses showed similar phylogenetic relationships among species, although chloroplast SSR were more informative because of their higher sequence diversity. The CC genome is suggested to be the maternal parent for the two BBCC genome species (O. punctata and O. minuta) and the CCDD species O. latifolia, based on the high level of sequence conservation between the diploid CC genome species and these allotetraploid species. This is the first report of phylogenetic analysis among plant species, based on mitochondrial and chloroplast SSR and their flanking sequences.     

Construction of introgression lines carrying wild rice (Oryza rufipogon Griff.) segments in cultivated rice (Oryza sativa L.) background and characterization of introgressed segments associated with yield-related traits

Introgression lines (ILs) are useful tools for precise mapping of quantitative trait loci (QTLs) and the evaluation of gene action or interaction in theoretical studies. A set of 159 ILs carrying variant introgressed segments from Chinese common wild rice (Oryza rufipogon Griff.), collected from Dongxiang county, Jiangxi Province, in the background of Indica cultivar (Oryza sativa L.), Guichao 2, was developed using 126 polymorphic simple sequence repeats (SSR) loci. The 159 ILs represented 67.5% of the genome of O. rufipogon. All the ILs have the proportions of the recurrent parent ranging from 92.4 to 99.9%, with an average of 97.4%. The average proportion of the donor genome for the BC₄F₄ population was about 2.2%. The mean numbers of homozygous and heterozygous donor segments were 2 (ranging 0–8) and 1 (ranging 0–7), respectively, and the majority of these segments had sizes less than 10 cM. QTL analysis was conducted based on evaluation of yield-related traits of the 159 ILs at two sites, in Beijing and Hainan. For 6 out of 17 QTLs identified at two sites corresponding to three traits (panicles per plant, grains per panicle and filled grains per plant, respectively), the QTLs derived from O. rufipogon were usually associated with an improvement of the target trait, although the overall phenotypic characters of O. rufipogon were inferior to that of the recurrent parent. Of the 17 QTLs, 5 specific QTLs strongly associated with more than one trait were observed. Further analysis of the high-yielding and low-yielding ILs revealed that the high-yielding ILs contained relatively less introgressed segments than the low-yielding ILs, and that the yield increase or decrease was mainly due to the number of grain. On the other hand, low-yielding ILs contained more negative QTLs or disharmonious interactions between QTLs which masked trait-enchancing QTLs. These ILs will be useful in identifying the traits of yield, tolerance to low temperature and drought stress, and detecting favorable genes of common wild rice.