Dual‐color oligo‐FISH can reveal chromosomal variations and evolution in Oryza species

Fluorescence in situ hybridization using probes based on oligonucleotides (oligo‐FISH) is a useful tool for chromosome identification and karyotype analysis. Here we developed two oligo‐FISH probes that allow the identification of each of the 12 pairs of chromosomes in rice (Oryza sativa). These two probes comprised 25 717 (green) and 25 215 (red) oligos (45 nucleotides), respectively, and generated 26 distinct FISH signals that can be used as a barcode to uniquely label each of the 12 pairs of rice chromosomes. Standard karyotypes of rice were established using this system on both mitotic and meiotic chromosomes. Moreover, dual‐color oligo‐FISH was used to characterize diverse chromosomal abnormalities. Oligo‐FISH analyses using these probes in various wild Oryza species revealed that chromosomes from the AA, BB or CC genomes generated specific and intense signals similar to those in rice, while chromosomes with the EE genome generated less specific signals and the FF genome gave no signal. Together, the oligo‐FISH probes we established will be a powerful tool for studying chromosome variations and evolution in the genus Oryza.     

Chromosomal polymorphism of ribosomal genes in the genus Oryza

The genes encoding for 18S–5.8S–28S ribosomal RNA (rDNA) are both conserved and diversified. We used rDNA as probe in the fluorescent in situ hybridization (rDNA-FISH) to localized rDNAs on chromosomes of 15 accessions representing ten Oryza species. These included cultivated and wild species of rice, and four of them are tetraploids. Our results reveal polymorphism in the number of rDNA loci, in the number of rDNA repeats, and in their chromosomal positions among Oryza species. The numbers of rDNA loci varies from one to eight among Oryza species. The rDNA locus located at the end of the short arm of chromosome 9 is conserved among the genus Oryza. The rDNA locus at the end of the short arm of chromosome 10 was lost in some of the accessions. In this study, we report two genome specific rDNA loci in the genus Oryza. One is specific to the BB genome, which was localized at the end of the short arm of chromosome 4. Another may be specific to the CC genome, which was localized in the proximal region of the short arm of chromosome 5. A particular rDNA locus was detected as stretched chromatin with bright signals at the proximal region of the short arm of chromosome 4 in O. grandiglumis by rDNA-FISH. We suggest that chromosomal inversion and the amplification and transposition of rDNA might occur during Oryza species evolution. The possible mechanisms of cyto-evolution in tetraploid Oryza species are discussed.     

A lineage‐specific centromere retrotransposon in Oryza brachyantha

Most eukaryotic centromeres contain large quantities of repetitive DNA, such as satellite repeats and retrotransposons. Unlike most transposons in plant genomes, the centromeric retrotransposon (CR) family is conserved over long evolutionary periods among a majority of the grass species. CR elements are highly concentrated in centromeres, and are likely to play a role in centromere function. In order to study centromere evolution in the Oryza (rice) genus, we sequenced the orthologous region to centromere 8 of Oryza sativa from a related species, Oryza brachyantha. We found that O. brachyantha does not have the canonical CRR (CR of rice) found in the centromeres of all other Oryza species. Instead, a new Ty3‐gypsy (Metaviridae) retroelement (FRetro3) was found to colonize the centromeres of this species. This retroelement is found in high copy numbers in the O. brachyantha genome, but not in other Oryza genomes, and based on the dating of long terminal repeats (LTRs) of FRetro3 it was amplified in the genome in the last few million years. Interestingly, there is a high level of removal of FRetro3 based on solo‐LTRs to full‐length elements, and this rapid turnover may have played a role in the replacement of the canonical CRR with the new element by active deletion. Comparison with previously described ChIP cloning data revealed that FRetro3 is found in CENH3‐associated chromatin sequences. Thus, within a single lineage of the Oryza genus, the canonical component of grass centromeres has been replaced with a new retrotransposon that has all the hallmarks of a centromeric retroelement.     

Evolutionary trajectory of phytoalexin biosynthetic gene clusters in rice

Plants frequently possess operon‐like gene clusters for specialized metabolism. Cultivated rice, Oryza sativa, produces antimicrobial diterpene phytoalexins represented by phytocassanes and momilactones, and the majority of their biosynthetic genes are clustered on chromosomes 2 and 4, respectively. These labdane‐related diterpene phytoalexins are biosynthesized from geranylgeranyl diphosphate via ent‐copalyl diphosphate or syn‐copalyl diphosphate. The two gene clusters consist of genes encoding diterpene synthases and chemical‐modification enzymes including P450s. In contrast, genes for the biosynthesis of gibberellins, which are labdane‐related phytohormones, are scattered throughout the rice genome similar to other plant genomes. The mechanism of operon‐like gene cluster formation remains undefined despite previous studies in other plant species. Here we show an evolutionary insight into the rice gene clusters by a comparison with wild Oryza species. Comparative genomics and biochemical studies using wild rice species from the AA genome lineage, including Oryza barthii, Oryza glumaepatula, Oryza meridionalis and the progenitor of Asian cultivated rice Oryza rufipogon indicate that gene clustering for biosynthesis of momilactones and phytocassanes had already been accomplished before the domestication of rice. Similar studies using the species Oryza punctata from the BB genome lineage, the distant FF genome lineage species Oryza brachyantha and an outgroup species Leersia perrieri suggest that the phytocassane biosynthetic gene cluster was present in the common ancestor of the Oryza species despite the different locations, directions and numbers of their member genes. However, the momilactone biosynthetic gene cluster evolved within Oryza before the divergence of the BB genome via assembly of ancestral genes.     

Homologous recombination mediated by two palindromic repeated sequences in the mitochondrial genome of Oryza

Palindromic repeated sequences (PRSs) are distributed in at least ten regions of the mitochondrial (mt) genome of rice and are, apparently, mobile. In the present study, we examined the possibility of homologous recombination via some PRSs during the course of evolution of Oryza. We first performed Southern hybridization of the DNA from 11 species (18 strains) of Oryza in order to identify the distribution of PRSs in the mitochondrial genome of Oryza. The hybridization patterns revealed genome type-specific and/or species-specific variations. We speculated that homologous recombination via some PRSs might have made a contribution to such variations. After subsequent polymerase chain reaction, Southern hybridization and sequencing, we concluded that homologous recombination mediated by two PRSs occurred in the mtDNA of Oryza after divergence of the BB genome type and the other genome types of Oryza. Evidence was obtained that some PRSs were involved in both insertion and recombination events during the evolution of Oryza. Our results indicate, therefore, that PRSs have contributed considerably to the polymorphism of Oryza mtDNAs.     

Comparative molecular cytogenetics of major repetitive sequence families of three Dendrobium species (Orchidaceae) from Bangladesh

Background and Aims

Dendrobium species show tremendous morphological diversity and have broad geographical distribution. As repetitive sequence analysis is a useful tool to investigate the evolution of chromosomes and genomes, the aim of the present study was the characterization of repetitive sequences from Dendrobium moschatum for comparative molecular and cytogenetic studies in the related species Dendrobium aphyllum, Dendrobium aggregatum and representatives from other orchid genera.

Methods

In order to isolate highly repetitive sequences, a c₀t-1 DNA plasmid library was established. Repeats were sequenced and used as probes for Southern hybridization. Sequence divergence was analysed using bioinformatic tools. Repetitive sequences were localized along orchid chromosomes by fluorescence in situ hybridization (FISH).

Key Results

Characterization of the c₀t-1 library resulted in the detection of repetitive sequences including the (GA)_n dinucleotide DmoO11, numerous Arabidopsis-like telomeric repeats and the highly amplified dispersed repeat DmoF14. The DmoF14 repeat is conserved in six Dendrobium species but diversified in representative species of three other orchid genera. FISH analyses showed the genome-wide distribution of DmoF14 in D. moschatum, D. aphyllum and D. aggregatum. Hybridization with the telomeric repeats demonstrated Arabidopsis-like telomeres at the chromosome ends of Dendrobium species. However, FISH using the telomeric probe revealed two pairs of chromosomes with strong intercalary signals in D. aphyllum. FISH showed the terminal position of 5S and 18S–5·8S–25S rRNA genes and a characteristic number of rDNA sites in the three Dendrobium species.

Conclusions

The repeated sequences isolated from D. moschatum c₀t-1 DNA constitute major DNA families of the D. moschatum, D. aphyllum and D. aggregatum genomes with DmoF14 representing an ancient component of orchid genomes. Large intercalary telomere-like arrays suggest chromosomal rearrangements in D. aphyllum while the number and localization of rRNA genes as well as the species-specific distribution pattern of an abundant microsatellite reflect the genomic diversity of the three Dendrobium species.     

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.     

A novel repetitive DNA sequence in the genus Oryza

Summary Repetitive DNA sequences in the genus Oryza (rice) represent a large fraction of the nuclear DNA. The isolation and characterization of major repetitive DNA sequences will lead to a better understanding of rice genome organization and evolution. Here we report the characterization of a novel repetitive sequence, CC-1, from the CC genome. This repetitive sequence is present as long tandem arrays with a repeat unit 194 bp in length in the CC-diploid genome but 172 bp in length in the BBCC and CCDD tetraploid genomes. This repetitive sequence is also present, though at lower copy numbers, in the AA and BB genomes, but is absent in the EE and FF genomes. Hybridization experiments revealed considerable differences both in copy numbers and in restriction fragment patterns of CC-1 both between and within rice species. The results support the hypothesis that the CC genome is more closely related to the AA genome than to the BB genome, and most distantly related to the EE and FF genomes.     

Genetic diversity and phylogenetic relationship as revealed by inter simple sequence repeat (ISSR) polymorphism in the genus Oryza

Inter simple sequence repeat (ISSR) polymorphism was used to determine genetic diversity and phylogenetic relationships in Oryza. Forty two genotypes including 17 wild species, representing AA,BB,CC,EE,FF,GG,BBCC,CCDD, and HHJJgenomes, two cultivated species, Oryza sativa (AA) and Oryza glaberrima (AA), and three related genera, Porteresia coarctata, Leersia and Rhynchoryza subulata, were used in ISSR analysis. A total of 30 ISSR primers were screened representing di-, tri-, tetra- and penta-nucleotide repeats, of which 11 polymorphic and informative patterns were selected to determine the genetic diversity. The consensus tree constructed using binary data from banding patterns generated by ISSR-PCR clustered 42 genotypes according to their respective genomes. ISSR analysis suggests that the genus Oryza may have evolved following a polyphyletic pathway; Oryza brachyantha (FF genome) is the most divergent species in Oryza and Oryza australiensis (EE genome) does not fall under the Officinalis complex. DNA profiles based on ISSR markers have revealed potential diagnostic fingerprints for various species and genomes, and also for individual accessions/cultivars. Additionally ISSR revealed 87 putative genome/species-specific molecular markers for eight of the nine genomes of Oryza. The ISSR markers are thus useful in the fingerprinting of cultivated and wild species germplasm, and in understanding the evolutionary relationships of Oryza. Received: 23 August 1999 / Accepted: 10 November 1999     

Genome-type-specific variation of the 19th intron sequence within the RNA polymerase I largest subunit gene in the genus Oryza

Rice PolA1 gene, encoding for the largest subunit of RNA polymerase I, spans ca. 15 kb containing 21 exons and presents as a single-copy-per-haploid genome. The genus Oryza comprises 22 wild species and 9 recognized genome types: AA, BB, CC, EE, FF, GG, BBCC, CCDD, and HHJJ. We analyzed sequences of the 19th intron (PI19) within PolA1 genes in 17 Oryza species. The AA species, containing two cultivated species, showed similar length of PI19 to that of CC species (287–296 bp). The longer PI19s were found in BB (502 bp) and FF (349 bp) species, although EE (217 bp) and GG (222 bp) species had shorter sequences. The size differences of the PI19s are particularly useful to discriminate between diploid (BB and CC) and allotetraploid (BBCC) species using simple PCR analysis. The evolutionary relationship among seven genomes was inferred based on the comparison of their PI19 sequences.     

Molecular diversity in the primary and secondary gene pools of genus Oryza

The objective of the present investigation was to assess the genetic relationships among the species of Oryza that belong to the primary gene pool (sativa complex) and the secondary gene pool (officinalis complex) using three marker systems such as RAPDs, ISSRs and SSRs. A total of 432 clear and reproducible bands were amplified from 18 RAPD primers; 113 bands were detected from 8 ISSR primers and 78 alleles were found to be amplified across the Oryza species from 13 SSR primer pairs. All the three dendrograms constructed, using UPGMA from the genetic similarity matrices based on the three marker data sets, were similar in their groupings. In all the three trees, two accessions of Oryza sativa formed an exclusive group indicating its genomic differentiation from its wild ancestors through the process of domestication. Distinctness between the wild species of the sativa and officinalis complexes was evident in all the trees derived from different markers. The groupings obtained among the species of the sativa complex were in perfect concordance with the species relationships established through classical crossability and cytogenetic analysis. This study has brought out some information on the species relationship between the diploid and tetraploid genomes of the officinalis complex possessing BB, CC and DD genomes. The higher level of similarity observed between the species possessing C and D genomes supports the view of many earlier authors that these two genomes might have originated from a single hybridization event. The results of this study also show that the diploid species possessing C genomes such as Oryza officinalis, Oryza rhizomatis and Oryza eichingeri are distinct from their allotetraploid counterparts possessing BBCC and CCDD genomes indicating a wider genomic differentiation in their evolutionary process.     

Genomic structure and evolution of the Pi2/9 locus in wild rice species

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is a devastating disease of rice worldwide. Among the 85 mapped resistance (R) genes against blast, 13 have been cloned and characterized. However, how these genes originated and how they evolved in the Oryza genus remains unclear. We previously cloned the rice blast R-genes Pi2, Pi9, and Piz-t, and analyzed their genomic structure and evolution in cultivated rice. In this study, we determined the genomic sequences of the Pi2/9 locus in four wild Oryza species representing three genomes (AA, BB and CC). The number of Pi2/9 family members in the four wild species ranges from two copies to 12 copies. Although these genes are conserved in structure and categorized into the same subfamily, sequence duplications and subsequent inversions or uneven crossing overs were observed, suggesting that the locus in different wild species has undergone dynamic changes. Positive selection was found in the leucine-rich repeat region of most members, especially in the largest clade where Pi9 is included. We also provide evidence that the Pi9 gene is more related to its homologues in the recurrent line and other rice cultivars than to those in its alleged donor species O. minuta, indicating a possible origin of the Pi9 gene from O. sativa. Comparative sequence analysis between the four wild Oryza species and the previously established reference sequences in cultivated rice species at the Pi2/9 locus has provided extensive and unique information on the genomic structure and evolution of a complex R-gene cluster in the Oryza genus.     

Genome-Wide Analysis in Wild and Cultivated <Emphasis Type="Italic">Oryza</Emphasis> Species Reveals Abundance of NBS Genes in Progenitors of Cultivated Rice

NBS-encoding genes play a critical role in the plant defense system. Wild relatives of crop plants are rich reservoirs of plant defense genes. Here, we performed a stringent genome-wide identification of NBS-encoding genes in three cultivated and eight wild Oryza species, representing three different genomes (AA, BB, and FF) from four continents. A total of 2688 NBS-encoding genes were identified from 11 Oryza genomes. All the three progenitor species of cultivated rice, namely O. barthii, O. rufipogon, and O. nivara, were the richest reservoir of NBS-encoding genes (214, 313, and 307 respectively). Interestingly, the two Asian cultivated species showed a contrasting pattern in the number of NBS-encoding genes. While indica subspecies maintained nearly equal number of NBS genes as its progenitor (309 and 313), the japonica subspecies had retained only two third in the course of evolution (213 and 307). Other major sources for NBS-encoding genes could be (i) O. longistaminata since it had the highest proportion of NBS-encoding genes and (ii) O. glumaepatula as it clustered distinctly away from the rest of the AA genome species. The present study thus revealed that NBS-encoding genes can be exploited from the primary gene pool for disease resistance breeding in rice.     

Analysis of Starch Gene Diversity in the Wild Relatives of Oryza sativa

Genetic loci influencing traits important to humans have been selected during crop domestication. The starch properties of rice influence the ease of cooking and attractiveness of rice as a human food. Starch biosynthesis genes likely to influence starch properties in the grain were compared in wild and domesticated rice genotypes. Sequence variation was investigated in starch biosynthesis gene exons that have been reported to have a direct influence on rice amylose content, gelatinization temperature, and amylopectin chain length. Exons 6 and 10 of GBSSI, exon 8 of SSIIa and exons 11, 13, 14 and 16 of SBEIIb were amplified and sequenced from 13 wild Oryza species encompassing genome types AA to HHJJ. Thirty two single nucleotide polymorphisms (SNPs) were identified in the exons of GBSSI; 176 in exon 8 of SSIIa, and 43 in SBEIIb, giving a total of 251 SNPs among the species. Eighty six of these SNP caused changes in the encoded amino acid, of which 28 were missense mutations that affected highly conserved amino acids within the protein sequence of GBSSI, SSIIa or SBEIIb. Two indels were identified in Potamophila parviflora, a close relative of Zizania palustris, a North American native wild rice. Most of the nucleotide variations and non-conservative changes were observed in the genomes other than the AA genome species. This represents a genetic resource for use in rice starch manipulation. The impact of human selection at these loci can be deduced by comparison of modern cultivated genotypes with their wild progenitors.     

Phylogeny and Molecular Evolution of miR820 and miR396 microRNA Families in <Emphasis Type="Italic">Oryza</Emphasis> AA Genomes

The phylogeny and evolution of the microRNA families, miR820 and miR396, was analysed across the AA genomes of the Oryza species, the close relatives of domesticated rice. A highly dynamic evolution of the miR820 family was revealed. The number of copies of MIR820 genes, their chromosomal location and the mature microRNA sequence varied greatly with a total of 16 novel miR820 variants being identified. The phylogeny of pre-MIR820 sequences revealed that MIR820 genes of recently evolved Oryza AA genomes may have derived from sequence divergence of one or a few ancestral genes found in wild Australian perennial rice populations, Taxon B (jpn2)-MIR820 genes. Genomic scale duplication played an important role in the evolution of some of the miR396 family genes in AA genome Oryza species. miR396 family contained a MIR396 gene cluster (MIR396a and MIR396c) which was conserved across the cereal genomes. Nucleotide diversity analysis at these two MIR396 loci revealed that domesticated rice has retained less than 10% of the total diversity present in wild species. In contrast, the nucleotide sequence of four MIR396 loci remained almost conserved across domesticated and wild rices, indicating that they were under extreme functional constraint and may be involved in regulating some fundamental processes which are important both for wild and domesticated rices. Expression analysis demonstrated that miR820 variants were expressed in O. glaberrima O. barthi and O. longistaminata genome. These findings pose new challenges to explain the possible role of miR820 variants identified.     

The Oryza Map Alignment Project: The Golden Path to Unlocking the Genetic Potential of Wild Rice Species

The wild species of the genus Oryza offer enormous potential to make a significant impact on agricultural productivity of the cultivated rice species Oryza sativa and Oryza glaberrima. To unlock the genetic potential of wild rice we have initiated a project entitled the ‘Oryza Map Alignment Project’ (OMAP) with the ultimate goal of constructing and aligning BAC/STC based physical maps of 11 wild and one cultivated rice species to the International Rice Genome Sequencing Project’s finished reference genome – O. sativa ssp. japonica c. v. Nipponbare. The 11 wild rice species comprise nine different genome types and include six diploid genomes (AA, BB, CC, EE, FF and GG) and four tetrapliod genomes (BBCC, CCDD, HHKK and HHJJ) with broad geographical distribution and ecological adaptation. In this paper we describe our strategy to construct robust physical maps of all 12 rice species with an emphasis on the AA diploid O. nivara – thought to be the progenitor of modern cultivated rice.     

基因组原位杂交鉴定牛筋草AA基因组在穇子染色体上的分布及探针长度优化方法

采用基因组原位杂交(Genomic in situ hybridization,GISH)方法研究了牛筋草(Eleusine indica)AA基因组在穇子(E.coracana)染色体上的分布,并探讨了AA、BB基因组的同源关系。用超声波破碎法进行预剪切,以缺口平移法标记的牛筋草总DNA为探针,BB基因组的E.floccifolia(Forssk.)Spreng.总DNA为封阻,与AABB基因组穇子的中期染色体进行杂交。结果表明,牛筋草AA基因组分布在穇子的18条染色体上。不加封阻或加过量封阻均不能鉴别AA基因组,说明AA和BB基因组间的分化程度不大,双方共享的重复序列较多。牛筋草与E.floccifolia总DNA分别用超声波破碎2 min和3 min后,可得到峰值为300-750 bp的DNA片段,这说明不同物种的超声波破碎时间需要调整,以获得合适长度的探针。     

The contribution of short repeats of low sequence complexity to large conifer genomes

The abundance and genomic organization of six simple sequence repeats, consisting of di-, tri-, and tetranucleotide sequence motifs, and a minisatellite repeat have been analyzed in different gymnosperms by Southern hybridization. Within the gymnosperm genomes investigated, the abundance and genomic organization of micro- and minisatellite repeats largely follows taxonomic groupings. We found that only particular simple sequence repeat motifs are amplified in gymnosperm genomes, while others such as (CAC)₅ and (GACA)₄ are present in only low copy numbers. The variation in abundance of simple sequence motifs reflects a similar situation to that found in angiosperms. Species of the two- and three-needle pine section Pinus are relatively conserved and can be distinguished from Pinus strobus which belongs to the five-needle pine section Strobus. The hybridization pattern of Picea species, bald cypress and gingko were different from the patterns detected in the Pinus species. Furthermore, sequences with homology to the plant telomeric repeat (TTTAGGG)_n have been analyzed in the same set of gymnosperms. Telomere-like repeats are highly amplified within two- and three- needle pine genomes, such as slash pine (Pinus elliottii Engelm. var. elliottii), compared to P. strobus, Picea species, bald cypress and gingko. P. elliottii var. elliottii was used as a representative species to investigate the chromosomal organization of telomere-like sequences by fluorescence in situ hybridization (FISH). The telomere-like sequences are not restricted to the ends of chromosomes; they form large intercalary and pericentric blocks showing that they are a repeated component of the slash pine genome.Conifers have genomes larger than 20000 Mbp, and our results clearly demonstrate that repeats of low sequence complexity, such to (CA)₈, (GA)₈, (GGAT)₄ and (GATA)₄, and minisatellite- and telomere-like sequences represent a large fraction of the repetitive DNA of these species. The striking differences in abundance and genome organization of the various repeat motifs suggest that these repetitive sequences evolved differently in the gymnosperm genomes investigated. Received: 1 October 1999 / Accepted: 3 November 1999     

Genomic characterization of <Emphasis Type="Italic">Oryza</Emphasis> species-specific CACTA-like transposon element and its application for genomic fingerprinting of rice varieties

A repeated DNA fragment (pKRD) was isolated from the genomic library of weedy rice in Korea. The pKRD showed significant homology to Em/Spm CACTA-like transposon in whole genome sequences of rice released in the Blast rice sequence database of NCBI and was closely related to the TNP2 transposase group, including a TNP-like transposable element of rice. A Southern hybridization experiment demonstrated that the pKRD sequence is unique to the Oryza genome. The 126 sequences homologous to pKRD were evenly distributed in all 12 different chromosomes in rice genomes with multiple copy numbers. Different copy numbers ranging from 1,500 to 4,500 corresponding to rice species were detected by slot blot hybridization. In a DNA fingerprinting experiment, a pKRD probe was assessed to be the potential molecular marker for studying evolution and divergence, biodiversity and phylogenic analysis of rice species.