ITS1 Sequences of Nuclear Ribosomal DNA in Wild Rices and Cultivated Rices of China and Their Phylogenetic Implications

he first internal transcribed spacer (ITS1) of nuclear ribosomal DNA of three wild rice species and two subspecies of cultivated rice, which are distributed in China, was amplified using PCR technique and sequenced with automated fluorescent sequencing. The sequences of ITS1 ranged from 193 bp to 218 bp in size and G/C content varied from 69.3%to 72.7%. In pairwise comparison among the five taxa, sequence site divergence ranged from 1.5 % to 10.6%. Phylogenetic analysis of ITS1 sequences using Wagner parsimony generated a single well-resolved tree, which revealed that Oryza rufipogon was much more closely related to cultivated rice species than to the other two wild species. Oryza granulata was less closely related to either cultivated rice species or the other two wild species, and might be a unique and isolated taxon in the genus Oryza. The phylogenetic relationships of the three wild rice species and two cultivated rice subspecies inferred from ITS1 sequences is highly concordant with those based on the molecular evidence from isozyme, chloroplast DNA (cpDNA), mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) of the genus Oryza.     

基于线粒体基因片段核苷酸多态性的亚洲栽培稻起源进化研究

亚洲栽培稻的祖先是普通野生稻,已成为世界公认的观点,然而亚洲栽培稻的2个亚种:粳稻和籼稻是一次起源还是二次起源仍存在很大争议,其起源地是国内还是国外依然是国际学者间争论的焦点。本文通过对184份亚洲栽培稻和203份普通野生稻3段基因序列cox3、cox1、orf 224和2段基因间序列ssv-39/178、rps2-trnfM的多样性研究,验证了以下观点:1)粳稻起源于中国,籼稻起源于中国和国外;2)亚洲栽培稻的起源为二次起源,即普通野生稻存在偏籼和偏粳2种类型,亚洲栽培稻的2个亚种籼稻和粳稻在进化过程中分别由偏籼型的普通野生稻和偏粳型的普通野生稻进化而来。     

Extraordinarily polymorphic ribosomal DNA in wild and cultivated rice.

A collection of 481 rice accessions was surveyed for ribosomal DNA (rDNA) intergenic spacer length polymorphism to assess the extent of genetic diversity in Chinese and Asian rice germplasm. The materials included 83 accessions of common wild rice, Oryza rufipogon, 75 of which were from China; 348 entries of cultivated rice (Oryza sativa), representing almost all the rice growing areas in China; and 50 cultivars from South and East Asia. A total of 42 spacer length variants (SLVs) were detected. The size differences between adjacent SLVs in the series were very heterogeneous, ranging from ca. 21 to 311 bp. The 42 SLVs formed 80 different rDNA phenotypic combinations. Wild rice displayed a much greater number of rDNA SLVs than cultivated rice, while cultivated rice showed a larger number of rDNA phenotypes. Indica and japonica groups of O. sativa contained about equal numbers of SLVs, but the SLV distribution was significantly differentiated: indica rice was preferentially associated with longer SLVs and japonica rice with shorter ones. The results may have significant implications regarding the origin and evolution of cultivated rice, as well as the inheritance and molecular evolution of rDNA intergenic spacers in rice. Key words : rDNA, Oryza rufipogon, Oryza sativa, germplasm diversity, evolution.     

中国野生稻及栽培稻核糖体DNA第一转录间隔区序列分析及其系统学意义

用 PCR技术从产于我国的 3种野生稻和亚洲栽培稻的 2个亚种中特异地扩增和测序了 r DNA的第一转录间隔区。普通野生稻 (Oryza rufipogon)、药用野生稻 (O.officinalis)、疣粒野生稻 (O.granu-lata)和栽培稻的两个亚种 (O.sativa ssp.indica,O.sativa ssp.japonica)的 ITS1序列为 1 93bp、1 94bp、2 1 8bp、1 94bp和 1 94bp,它们的 G/ C含量为 69.3%～ 72 .7% ,序列中位点趋异率为 1 .5%～ 1 0 .6%。序列的相似性比较和简约性分支分析的结果表明 ,普通野生稻与栽培稻的两个亚种之间的亲缘关系最为密切 ;药用野生稻与普通野生稻和与栽培稻的两个亚种的相似性都为 82 % ,说明它与 AA基因组有一定的亲缘关系 ;疣粒野生稻与普通野生稻、药用野生稻和栽培稻两个亚种的亲缘关系相对较远 ,它在稻属中可能是一个系统地位较独特的类群。以 ITS1序列构建的 3种野生稻和 2个栽培稻亚种的系统发育关系与前人用同工酶、叶绿体 DNA、线粒体 DNA和核 DNA资料重建的稻属的系统发育关系基本一致     

重复DNA顺序pRRD9在稻属中的拷贝数测定

本文应用狭缝印渍杂交方法,把水稻基因组总DNA和含水稻中度重复顺序片段的质粒(pRRD9)DNA分别转移到尼龙膜上形成狭缝印渍、然后用³²P标记的 pRRD9插入片段进行杂交、根据各狭缝印渍的放射性强度,测定水稻(Oryza)一些栽培种和野生种基因组中重复DNA顺序的拷贝数,并就拷贝数与水稻进化关系及基因组型的联系进行讨论.     

重复DNA顺序pRRD9在稻属中的拷贝数测定

本文应用狭缝印渍杂交方法,把水稻基因组总DNA和含水稻中度重复顺序片段的质粒(pRRD9)DNA分别转移到尼龙膜上形成狭缝印渍、然后用~(32)P标记的 pRRD9插入片段进行杂交、根据各狭缝印渍的放射性强度,测定水稻(Oryza)一些栽培种和野生种基因组中重复DNA顺序的拷贝数,并就拷贝数与水稻进化关系及基因组型的联系进行讨论.     

Evolutionary Genomics of Weedy Rice in the USA

Red rice is an interfertiie, weedy form of cultivated rice (Oryza sativa L.) that competes aggressively with the cropin the southern US, reducing yields and contaminating harvests. No wild Oryza species occur In North America andthe weed has been proposed to have evolved through multiple mechanisms, including "de-domestication" of UScrop cultivars, accidental introduction of Asian weeds, and hybridization between US crops and Asian wild/weedyOryza strains. The phenotype of US red rice ranges from "crop mimics", which share some domestication traitswith the crop, to strains closely resembling Asian wild Oryza species. Assessments of genetic diversity haveindicated that many weed strains are closely related to Asian taxa (including indica and aus rice varieties, whichhave never been cultivated in the US, and the Asian crop progenitor O. rufipogon), whereas others show geneticsimilarity to the tropical japonica varieties cultivated in the southern US. Herein, we review what is known aboutthe evolutionary origins and genetic diversity of US red rice and describe an ongoing research project to furthercharacterize the evolutionary genomics of this aggressive weed.     

野生稻和栽培稻的随机多态DNA(RAPD)分析

应用 RAPD方法对药用野生稻、普通野生稻、粳稻和籼稻进行基因组多态性分析。 1 2个随机引物共扩增出 1 3 2条 RAPD带 ,片段大小在 3 0 0～ 3 5 0 0 bp之间 ,其中有 1 0 6条表现出多态性 ,占总扩增片段的86.4%。根据遗传距离分析 ,用 UPGMA法构建了聚类树状图 ,结果表明 ,普通野生稻的遗传特性比药用野生稻更接近于栽培稻。     

Genetic control of a transition from black to straw-white seed hull in rice domestication

The genetic mechanism involved in a transition from the black-colored seed hull of the ancestral wild rice (Oryza rufipogon and Oryza nivara) to the straw-white seed hull of cultivated rice (Oryza sativa) during grain ripening remains unknown. We report that the black hull of O. rufipogon was controlled by the Black hull4 (Bh4) gene, which was fine-mapped to an 8.8-kb region on rice chromosome 4 using a cross between O. rufipogon W1943 (black hull) and O. sativa indica cv Guangluai 4 (straw-white hull). Bh4 encodes an amino acid transporter. A 22-bp deletion within exon 3 of the bh4 variant disrupted the Bh4 function, leading to the straw-white hull in cultivated rice. Transgenic study indicated that Bh4 could restore the black pigment on hulls in cv Guangluai 4 and Kasalath. Bh4 sequence alignment of all taxa with the outgroup Oryza barthii showed that the wild rice maintained comparable levels of nucleotide diversity that were about 70 times higher than those in the cultivated rice. The results from the maximum likelihood Hudson-Kreitman-Aguade test suggested that the significant reduction in nucleotide diversity in rice cultivars could be caused by artificial selection. We propose that the straw-white hull was selected as an important visual phenotype of nonshattered grains during rice domestication.     

Evolutionary Genomics of Weedy Rice in the USA

Red rice Is an Interfertlle, weedy form of cultivated rice （Oryza sativa L.） that competes aggressively with the crop In the southern US, reducing yields and contaminating harvests. No wild Oryza species occur In North America and the weed has been proposed to have evolved through multiple mechanisms, Including ＂de-domestication＂ of US crop cultlvars, accidental introduction of Asian weeds, and hybridization between US crops and Asian wild/weedy Oryza strains. The phenotype of US red rice ranges from ＂crop mimics＂, which share some domestication traits with the crop, to strains closely resembling Asian wild Oryza species. Assessments of genetic diversity have Indicated that many weed strains are closely related to Asian taxa （Including indica and aus rice varieties, which have never been cultivated In the US, and the Asian crop progenitor O. ruflpogon）, whereas others show genetic similarity to the tropical Japonica varieties cultivated In the southern US. Herein, we review what Is known about the evolutionary origins and genetic diversity of US red rice and describe an ongoing research project to further characterize the evolutionary genomlcs of this aggressive weed.     

稻属谷氨酰胺合成酶家族的系统分析

比较籼粳栽培稻和野生稻中谷氨酰胺合成酶(GS)基因和蛋白质的结果表明,水稻GS蛋白编码区序列高度保守,而非编码序列变异较大。GS2基因的进化比GS1基因保守。短药野生稻中GS基因进化主要是内含子的变异,但此种内含子的变异在籼粳栽培稻中幅度要小得多。     

Origins and population genetics of weedy red rice in the USA

Weedy red rice (Oryza sativa spontonea) is a persistent and problematic weed of rice culture worldwide. A major hypothesis for the mechanism of production of this weed in South and Southeast Asia is hybridization between cultivated rice (Oryza sativa) and wild rice (Oryza rufipogon). However, weedy red rice can often be found outside the range of O. rufipogon leaving questions on the origin and process behind weedy rice infestations. In the USA, weedy red rice was first documented as early as 1846 and has continued to affect rice production areas. In this study, we attempt to identify the origin and population structure of weedy red rice sampled from the USA using both DNA sequence data from a neutral nuclear locus as well as microsatellite genotype data. Results suggest that two major accessions of weedy rice exist, strawhull and blackhull, and these forms may both hybridize with the cultivated rice of the USA, O. sativa japonica. Using population assignment of multilocus genotype signatures with principal component analysis and structure, an Asian origin is supported for US weedy rice. Additionally, hybridization between strawhull and blackhull varieties was inferred and may present the opportunity for the production of new weedy forms in the future.     

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.     

Heading date 1 (Hd1), an ortholog of Arabidopsis CONSTANS, is a possible target of human selection during domestication to diversify flowering times of cultivated rice

During the domestication of rice (Oryza sativa L.), diversification of flowering time was important in expanding the areas of cultivation. Rice is a facultative short day (SD) plant and requires certain periods of dark to induce flowering. Heading date 1 (Hd1), a regulator of the florigen gene Hd3a, is one of the main factors used to generate diversity in flowering. Loss-of-function alleles of Hd1 are common in cultivated rice and cause the diversity of flowering time. However, it is unclear how these functional nucleotide polymorphisms of Hd1 accumulated in the course of evolution. Nucleotide polymorphisms within Hd1 and Hd3a were analyzed in 38 accessions of ancestral wild rice Oryza rufipogon and compared with those of cultivated rice. In contrast to cultivated rice, no nucleotide changes affecting Hd1 function were found in 38 accessions of wild rice ancestors. No functional changes were found in Hd3a in either cultivated or ancestral rice. A phylogenetic analysis indicated that evolution of the Hd1 alleles may have occurred independently in cultivars descended from various accessions of ancestral rice. The non-functional Hd1 alleles found in cultivated rice may be selected during domestication, because they were not found or very rare in wild ancestral rice. In contrast with Hd3a, which has been highly conserved, Hd1 may have undergone human selection to diversify the flowering times of rice during domestication or the early stage of the cultivation period.     

Life‐history traits and geographical divergence in wild rice (Oryza rufipogon) gene pool in Indochina Peninsula region

Indochina Peninsula is the primary centre of diversity of rice and lies partly in the centre of origin of cultivated rice (Oryza sativa) where the wild ancestor (Oryza rufipogon) is still abundant. The wild gene pool is potentially endangered by urbanisation and the expansion of agriculture, and by introgression hybridisation with locally cultivated rice varieties. To determine genetic diversity and structure of the wild rice of the region we genotyped nearly 1000 individuals using 20 microsatellite loci. We found ecological differentiation in 48 populations, distinguishable by their life‐history traits and the country of origin. Geographical divergence was suggested by isolation of the perennial Myanmar populations from those of Cambodia, Laos and Thailand. The annual types would be most likely to have lost genetic variation because of genetic drift and inbreeding. The growing of cultivated and wild rice together, however, gives ample opportunities for hybridisation, which already shows signs of genetic mixing, and will ultimately lead to replacement of the original wild rice gene pool. For conservation we suggest that wild rice should be conserved ex situ in order to prevent introgression from cultivated rice, along with in situ conservation in individual countries for the recurrent evolutionary process through local adaptation, but with sufficient isolation from cultivated rice fields to preserve genetic integrity of the wild populations.     

Genetic Variation and Evolution of the <Emphasis Type="Italic">Pi9</Emphasis> Blast Resistance Locus in the AA Genome <Emphasis Type="Italic">Oryza</Emphasis> Species

The rice nucleotide-binding site–leucine-rich repeat (NBS-LRR)-encoding resistance (R) gene Pi9 confers broad-spectrum resistance to the fungal pathogen Magnaporthe oryzae. The Pi9 locus comprises many NBS-LRR-like genes and is an ancient locus that is highly conserved in cultivated and wild rice species. To understand the genetic variation and molecular evolutionary mechanism of the Pi9 alleles in different rice species, we studied five AA genome Oryza species including two cultivated rice species (Oryza sativa and Oryza glaberrima) and three wild rice species (Oryza nivara, Oryza rufipogon, and Oryza barthii). A 2.9-kb fragment spanning the NBS-LRR core region of the Pi9 gene was amplified and sequenced from 40 accessions. Sequence comparison revealed that the Pi9 alleles had an intermediate-diversified nucleotide polymorphism among the AA genome Oryza species. Sequence variations were more abundant in the LRR region than in the NBS region, indicating that the LRR region has played a more important role for the evolution of the Pi9 alleles. Furthermore, positive selection was found to be the main force promoting the divergence of the Pi9 alleles, especially in the LRR region. Our results reveal the characteristics and evolutionary dynamics of the Pi9 alleles among the two cultivated and three wild rice species.     

Resequencing 50 accessions of cultivated and wild rice yields markers for identifying agronomically important genes

Rice is a staple crop that has undergone substantial phenotypic and physiological changes during domestication. Here we resequenced the genomes of 40 cultivated accessions selected from the major groups of rice and 10 accessions of their wild progenitors (Oryza rufipogon and Oryza nivara) to >15 × raw data coverage. We investigated genome-wide variation patterns in rice and obtained 6.5 million high-quality single nucleotide polymorphisms (SNPs) after excluding sites with missing data in any accession. Using these population SNP data, we identified thousands of genes with significantly lower diversity in cultivated but not wild rice, which represent candidate regions selected during domestication. Some of these variants are associated with important biological features, whereas others have yet to be functionally characterized. The molecular markers we have identified should be valuable for breeding and for identifying agronomically important genes in rice.     

云南元江普通野生稻中Pi-ta和Pib同源基因的克隆和分析

用高保真PCR技术从云南元江普通野生稻中克隆了抗稻瘟病Pi-ta同源基因的编码区及Pib基因的部分同源序列。Pi-ta同源基因的编码区序列与报道的栽培稻有99.7%的同源性。根据前人的结果,从元江普通野生稻的Pi-ta基因推导的氨基酸序列中918位点为丝氨酸,属于Pi-ta~-等位基因,不能对含有AVRPita基因的稻瘟病菌产生抗性。与Pi-ta基因相比,元江普通野生稻中的Pib同源基因第一外显子与栽培稻的相应序列间存在较大差异,其中有一段87 bp的DNA序列缺失,而且不能按正常的Pib基因序列的阅读框进行翻译。因此认为,元江普通野生稻不具有基于Pi-ta和Pib基因的抗稻瘟病遗传基础。     

野生稻与栽培稻及种间杂种F1叶表面亚显微结构的研究

用扫描电镜对原产中国的３种野生稻、２个栽培稻品种及栽培稻与野生稻间的杂种Ｆ１,就气孔频度、气孔器乳突、大瘤状乳突及本栓细胞乳突等叶片表面亚显微结构作了比较观察研究。结果显示这些性状具有种的特性,发现种间杂种的亚星微性状与两个亲本 特性及亲缘关系远近有关。