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。     

A chromosome-level genome assembly of the wild rice Oryza rufipogon facilitates tracing the origins of Asian cultivated rice

Oryza rufipogon Griff. is a wild progenitor of the Asian cultivated rice Oryza sativa. To better understand the genomic diversity of the wild rice, high-quality reference genomes of O. rufipogon populations are needed, which also facilitate utilization of the wild genetic resources in rice breeding. In this study, we generated a chromosome-level genome assembly of O. rufipogon using a combination of short-read sequencing, single-molecule sequencing, BioNano and Hi-C platforms. The genome sequence(399.8 Mb) was assembled into 46 scaffolds on the 12 chromosomes, with contig N50 and scaffold N50 of 13.2 Mb and 20.3 Mb,respectively. The genome contains 36,520 protein-coding genes, and 49.37% of the genome consists of repetitive elements. The genome has strong synteny with those of the O. sativa subspecies indica and japonica, but containing some large structural variations. Evolutionary analysis unveiled the polyphyletic origins of O. sativa, in which the japonica and indica genome formations involved different divergent O. rufipogon(including O. nivara) lineages, accompanied by introgression of genomic regions between japonica and indica. This high-quality reference genome provides insight on the genome evolution of the wild rice and the origins of the O. sativa subspecies, and valuable information for basic research and rice breeding.     

RAPD Determinations on the Genetic Differentiation of Chinese Common Wild Rice

Ninety accessions which included Chinese common wild rice (Oryza rufipogon) from 8 provinces and traditional cultivars from lower and middle basins of Yangtze River, southeast of China and Yunnan Province as well as some commercial varieties were analyzed by RAPD with 24 primers. A scattered figure suggesting the indica-japonica and wild-domestication differentiations among 90 rice accessions was generated based on RAPD data. The results indicated that Chinese common wild rice, indica and japonica accessions were divided into 3 groups respectively. Chinese common wild rice were somewhat closer to the japonica type than the indica type.     

Cloning and characterization of repetitive DNA sequences from genomes of Oryza minuta and Oryza australiensis

The value of genome-specific repetitive DNA sequences for use as molecular markers in studying genome differentiation was investigated. Five repetitive DNA sequences from wild species of rice were cloned. Four of the clones, pOm1, pOm4, pOmA536, and pOmPB10, were isolated from Oryza minuta accession 101141 (BBCC genomes), and one clone, pOa237, was isolated from Oryza australiensis accession 100882 (EE genome). Southern blot hybridization to different rice genomes showed strong hybridization of all five clones to O. minuta genomic DNA and no cross hybridization to genomic DNA from Oryza sativa (AA genome). The pOm1 and pOmA536 sequences showed cross hybridization only to all of the wild rice species containing the C genome. However, the pOm4, pOmPB10, and pOa237 sequences showed cross hybridization to O. australiensis genomic DNA in addition to showing hybridization to the O. minuta genomic DNA.     

中国普通野生稻遗传分化的RAPD研究

多数学者已认定亚洲栽培稻（ＯｒｙｚａｓａｔｉｖａＬ．）的祖先是普通野生稻（Ｏ．ｒｕｆｉｐｏｇｏｎ）。然而栽培稻的籼、粳分化是发生在驯化之前还是在驯化之后,也即普通野生稻是否存在籼、粳分化的问题,是十几年来稻作起源研究中争论的热点之一。Ｓｅｃｏｎｄ［１］用多个同工酶位点的分析结果得出结论,普通野生稻在驯化为栽培稻之前就已经发生了籼、粳分化,即有籼型普通野生稻和粳型普通野生稻之分。Ｍｏｒｉｓｈｉｍａ和Ｇａｄｒｉｎａｂ［２］用２４个形态和生理性状及１２个同工酶位点和杂交亲合力等方法证明普通野生稻没有发…     

普通野生稻和亚洲栽培稻线粒体DNA的RFLP分析

通过７个探针、１７种内切酶探针组合对１１８份普通野生稻和７６份亚洲栽培稻的线粒体ＤＮＡ（ｍｔＤＮＡ）ＲＦＬＰ分析表明,籼粳分化是亚洲栽培稻线粒体基因组分化的主流,７６个栽培稻中,３６个品种ｍｔＤＮＡ为籼型,４０个品种ｍｔＤＮＡ为粳型。普通野生稻ｍｔＤＮＡ以籼型为主（８６份）,粳型较少（７份）,１份类型难以确定,还有２４份没有籼粳分化。     

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。     

An RFLP species-specific DNA sequence for the A genome of rice.

A DNA sequence, pOs6.20, was cloned from the nuclear genome of cultivated rice (Oryza sativa L.) by hybridization with a human minisatellite sequence. At high hybridization stringency, a subfragment of the rice sequence, pOs6.20.3, detected low-copy restriction fragment length polymorphisms (RFLPs), which behaved as Mendelian genetic markers. This subfragment detected multicopy RFLPs between an indica and a javanica cultivar at medium hybridization stringency. The sequences detected by pOs6.20.3 at high hybridization stringency did not occur in rice species possessing the B, C, D, E, and F genomes. These results suggest that the RFLPs detected by this sequence can be placed on a genetic map of rice and that this sequence can be used as a species-specific probe for the A genome of rice.     

Genome sequencing of a 239-kb region of rice chromosome 10L reveals a high frequency of gene duplication and a large chloroplast DNA insertion

In this study we describe a 239-kb region on the long arm of rice chromosome 10 that contains a high density (71%) of locally duplicated genes, including 24 copies of a glutathione S-transferase gene. Intriguingly, embedded within this cluster is a large insertion (approximately 33 kb) of rice (Oryza sativa) chloroplast DNA that is derived from two separate regions of the chloroplast genome. We used DNA fiber-based fluorescence in situ hybridization (fiber-FISH) analyses of O. sativa spp. japonica nuclei to confirm that the insertion of organellar DNA was not a cloning artifact. The sequence of the chloroplast insertion is nearly identical (99.7% identity) to the corresponding regions in the published rice chloroplast genome sequence, suggesting that the transfer event occurred recently. PCR amplification and sequence analysis in two subspecies of rice, O. sativa spp. japonica and spp. indica, indicates that the transfer event predated the divergence of these two subspecies. The chloroplast insertion is flanked by a 2.1-kb perfect direct repeat that is unique to this location in the rice genome.     

Genetic Variation and Indica Japonica Differentiation in Yunnan Indigenous Rice

Yunnan, in Southwest China, has long been recognized as part of a center of diversity for Asia cultivated rice (Oryza sativa L. ). The authors surveyed DNA restriction fragment length polymorphisms (RFLPs) in a sample of 87 accessions from Yunnan indigenous rice with 7 single copy probes. The results ihdicated that both indica and japonica rice are genetically highly diverse. The number of alleles and the level of genic diversity in japonica rice were higher than those in indica rice. The results also showed that indica and japonica rices were clearly differentiated and the extent of differentiation varies with chromosomal regions as represented by the RFLP markers. Authors' results appeared to be in favor of the diphyletic hypothesis concerning the origin of cultivated rice.     

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.     

插入玉米Ds转座因子的水稻转化群体及其分子分析

转座子标签法是一种利用转座因子插入高等植物基因组中造成基因突变,然后通过分离转座因子插入的旁邻顺序,进而克隆出突变基因的策略。这种策略在高等植物功能基因组学的研究中是十分有用的。为此目的,将玉米的Ds因子及bar基因连接至载体pCAMBIA1300的T-DNA区域中,构建成重组Ti质粒pDsBar1300。pDsBar1300中T-DNA区域中的潮霉素抗性基因可在转化过程中用作水稻转化植株的选择标记。插入在Ds因子中的bar基因可追踪转化后代的Ds因子。pDsBar1300通过根瘤农杆茵介导引入水稻品种中花11号的幼胚组织。从各转化愈伤组织中获得了1400株独立的Ds水稻转化植株。通过PPT抗性检测和PCR分析证明了水稻转化植株中Ds因子的整合。Southernblot分析了转化植株基因组中Ds因子的插入拷贝数,其中单拷贝插入比率约占70％。这些插有Ds因子的水稻转化植株,当引入自主型的Ac因子反式活化Ds因子后,可使Ds因子跳跃到不同位点上,就可得到更多的突变植株。     

Subspecies-specific intron length polymorphism markers reveal clear genetic differentiation in common wild rice （Oryza rufipogon L.） in relation to the domestication of cultivated rice （O. sativa L.）

It is generally accepted that Oryza rufipogon is the progenitor of Asian cultivated rice （O. sativa）. However, how the two subspecies of O. sativa （indica and japonica） were domesticated has long been debated. To investigate the genetic differentiation in O. rufipogon in relation to the domestication of O. sativa, we developed 57 subspecies-specific intron length polymorphism （SSILP） markers by comparison between 10 indica cultivars and 10 japonica cultivars and defined a standard indica rice and a standard japonica rice based on these SSILP markers. Using these SSILP markers to genotype 73 O. rufipogon accessions, we found that the indica alleles and japonica alleles of the SSILP markers were predominant in the O. rufipogon accessions, suggesting that SSILPs were highly conserved during the evolution of O. sativa. Cluster analysis based on these markers yielded a dendrogram consisting of two distinct groups： one group （Group I） comprises all the O. rufipogon accesions from tropical （South and Southeast） Asia as well as the standard indica rice; the other group （Group II） comprises all the O. rufipogon accessions from Southern China as well as the standard japonica rice. Further analysis showed that the two groups have significantly higher frequencies of indica alleles and japonica alleles, respectively. These results support the hypothesis that indica rice and japonica rice were domesticated from the O. rufipogon of tropical Asia and from that of Southern China, respectively, and suggest that the indica-japonica differentiation should have formed in O. rufipogon long before the beginning of domestication. Furthermore, with an O. glaberrima accession as an outgroup, it is suggested that the indica-japonica differentiation in O. ruffpogon might occur after its speciation from other AA-genome species.     

MITE类转座子mPing在水稻不同亚种间的差异分析

mPing是水稻中第一个被鉴定出的有活性的MITE类转座子,为了探索mPing在水稻粳稻品种日本晴和籼稻品种93-11基因组中的分布差异,本研究首先运用Southern杂交的方法初步检测m Ping在两个亚种中拷贝数的差异,然后通过同源性探寻方法发现,m Ping在水稻亚种日本晴和93-11基因组中拷贝数分别为52和14,并且日本晴基因组中的m Ping均为m Ping-1,93-11中m Ping-1的拷贝数为3,m Ping-2的拷贝数为11。通过分析m Ping上下游5 kb侧翼序列发现m Ping在日本晴和93-11中分别与23和3个已知基因相关联。本研究为阐明以m Ping的分布多样性为主要原因的粳稻和籼稻之间的遗传差异提供初步理论基础。     

Characterization of Interspecific Hybrids Between Oryza sativa L. and Three Wild Rice Species of China by Genomic In Situ Hybridization

In the genus Oryza, interspecific hybrids are useful bridges for transferring the desired genes from wild species to cultivated rice （Oryza sativa L.）. In the present study, hybrids between O. sativa （AA genome） and three Chinese wild rices, namely O. rufipogon （AA genome）, O. officinalis （CC genome）, and O. meyeriana （GG genome）, were produced. Agricultural traits of the F1 hybrids surveyed were intermediate between their parents and appreciably resembled wild rice parents. Except for the O. sativa × O. rufipogon hybrid, the other F1 hybrids were completely sterile. Genomic in situ hybridization （GISH） was used for hybrid verification. Wild rice genomic DNAs were used as probes and cultivated rice DNA was used as a block. With the exception of O. rufipogon chromosomes, this method distinguished the other two wild rice and cultivated rice chromosomes at the stage of mitotic metaphase with different blocking ratios. The results suggest that a more distant phylogenetic relationship exists between O. meyeriana and O. sativa and that O. rufipogon and O. sativa share a high degree of sequence homology. The average mitotic chromosome length of O. officinalis and O. meyeriana was 1.25- and 1.51-fold that of O. sativa, respectively. 4＇,6＇-Diamidino- 2-phenylindole staining showed that the chromosomes of O. officinalis and O. meyeriana harbored more heterochromatin, suggesting that the C and G genomes were amplified with repetitive sequences compared with the A genome. Although chromocenters formed by chromatin compaction were detected with wild rice-specific signals corresponding to the C and G genomes in discrete domains of the F1 hybrid interphase nuclei, the size and number of O. meyeriana chromocenters were bigger and greater than those of O. officinalis. The present results provide an important understanding of the genomic relationships and a tool for the transfer of useful genes from three native wild rice species in China to cultivars.     

Independent domestication of Asian rice followed by gene flow from japonica to indica

Results from studies on the domestication process of Asian rice Oryza sativa have been controversial because of its complicated evolutionary history. Previous studies have yielded two alternative hypotheses about the origin(s) of the two major groups of O. sativa: japonica and indica. One study proposes a single common wild ancestor, whereas the other suggests that there were multiple domestication events of different types of wild rice. Here, we provide clear evidence of the independent domestication of japonica and indica obtained via high-throughput sequencing and a large-scale comparative analysis of two wild rice accessions (W1943 and W0106) and two cultivars (a japonica cultivar called "Nipponbare" and an indica cultivar called "Guangluai-4"). The different domestication processes of the two cultivar groups appear to have led to distinct patterns of molecular evolution in protein-coding regions. The intensity of purifying selection was relaxed only in the japonica group, possibly because of a bottleneck effect. Moreover, a genome-wide comparison between Nipponbare, Guangluai-4, and another indica cultivar (93-11) suggests multiple hybridization events between japonica and indica, both before and after the divergence of the indica cultivars. We found that a large amount of genomic DNA, including domestication-related genes, was transferred from japonica to indica, which might have been important in the development of modern rice. Our study provides an overview of the dynamic process of Asian rice domestication, including independent domestication events and subsequent gene flow.     

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.     

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.     

Genome-wide searching of single-nucleotide polymorphisms among eight distantly and closely related rice cultivars (Oryza sativa L.) and a wild accession (Oryza rufipogon Griff.).

We searched the genomes of eight rice cultivars (Oryza sativa L. ssp. japonica and ssp. indica) and a wild rice accession (Oryza rufipogon Griffith) for nucleotide polymorphisms, and identified 7805 polymorphic loci, including single-nucleotide polymorphisms (SNPs) and insertions/deletions (InDels), in predicted intergenic regions. Polymorphisms are useful as DNA markers for genetic analysis or positional cloning with segregating populations of crosses. Pairwise comparison between cultivars and a neighbor-joining tree calculated from SNPs agreed very well with relationships between rice strains predicted from pedigree data or calculated with other DNA markers such as p-SINE1 and simple sequence repeats (SSRs), suggesting that whole-genome SNP information can be used for analysis of evolutionary relationships. Using multiple SNPs to identify alleles, we drew a map to illustrate the alleles shared among the eight cultivars and the accession. The map revealed that most of the genome is mono- or di-allelic among japonica cultivars, whereas alleles well conserved among modern japonica paddy rice cultivars were often shared with indica cultivars or wild rice, suggesting that the genome structure of modern cultivars is composed of chromosomal segments from various genetic backgrounds. Use of allele-sharing analysis and association analysis were also tested and are discussed.