Genome-specific repetitive sequences in the genus Oryza

Summary Repetitive DNA sequences are useful molecular markers for studying plant genome evolution and species divergence. In this paper, we report the isolation and characterization of four genome-type specific repetitive DNA sequences in the genus Oryza. Sequences specific to the AA, CC, EE or FF genome types are described. These genome-type specific repetitive sequences will be useful in classifying unknown species of wild or domestic rice, and in studying genome evolution at the molecular level. Using an AA genome-specific repetitive DNA sequence (pOs48) as a hybridization probe, considerable differences in its copy number were found among different varieties of Asian-cultivated rice (O. sativa) and other related species within the AA genome type. Thus, the relationship among some of the members of AA genome type can be deduced based on the degree of DNA sequence similarity of this repetitive sequence.     

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.     

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.     

Isolation and identification of a non-specific tandemly repeated DNA sequence in Oryza species

A tandemly repeated DNA sequence (RRS7) was isolated from Oryza alta (CCDD). RRS7-related sequences were also found tandemly arrayed in genomes AA, BB, BBCC, CC, and EE, and a small amount of RRS7-related sequences were detected in genome FF and the Oryza species with unknown genomes. DNA sequence analysis of the 1844-bp insert of RRS7 revealed that it contained six tandemly repeated units, of which five were 155 bp in length and one was 194 bp in length and contained an imperfect internal 39-bp duplication. Southern blot analysis showed that the boundary sequence contained in RRS7 is a single-copy sequence. A 155-bp consensus sequence derived from the six monomeric repeats contained no internal repeat and showed no significant homology to other currently known sequences. The results of Southern blot and sequence analysis revealed that there are at least two subfamilies present in the RRS7 family; these are represented by the DraI site and the MspI site, respectively. Restriction digestion with two pairs of isoschizomers MboI/Sau3A and MspI/HpaII demonstrated that most of the C residues in the GATC sites and the internal C in the CCGG sites of the RRS7 family in O. Alta were methylated. The usefulness of the RRS7 family in determining the evolutionary relationship of the genome DD and other Oryza genomes is discussed.     

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.     

Distribution and Organization of AA Genome- specific and Tandemly Repetitive Sequences in Chinese Common Wild Rice and Cultivated Rice

Repetitive DNA sequences are useful molecular markers for studying plant genome evolution and species diversity. The authors report the isolation and characterization of repetitive DNA sequences (pOs139) from Oryza sativa cuhivars "Zhaiyeqing". By Southern blot analysis, the authors discovered that pOs139 sequences were organized not only tandemly, but also highly specifc for the AA genome of Oryza genus. Sequence analysis revealed that the clone pOs139 contains a 355 bp repetitive unit. The genomic DNA of 29 Chinese common wild accessions, and 43 cultivated rice accessions, were analyzed by Southern blot with pOs139 as a probe. The results illustrated that there was significant difference in hybridization patterns between japonica and indica subspecies. Hybridization bands of indica subspecies were much more than those of japonica, and the Chinese common wild rice was similar to indica in hybridization patterns. The copy number estimated by dot blot hybridization analysis indicated that a considerable degree of variation existed among different accessions of O. sativa and the Chinese common wild rice. It is interesting to note that japonica subspecies contains relatively low copy numbers of pOs139-related repetitive DNA sequences, while the indica and Chinese common wild rice contain relatively high copy numbers.     

稻属不同染色体组着丝粒BAC克隆的分离和鉴定

着丝粒在真核生物有丝分裂和减数分裂染色体正常的分离和传递中起着重要的作用。通过构建5个稻属二倍体野生种的基因组BAC文库, 采用菌落杂交和FISH技术, 筛选和鉴定了各染色体组着丝粒克隆, 并且分析了这些克隆在不同基因组间的共杂交情况, 结果表明: (1) C染色体组的野生种O. officinalis 和F染色体组的野生种O. brachyantha具有各自着丝粒特异的卫星DNA序列, 并且O. brachyantha着丝粒还具有特异的逆转座子序列; (2) A、B和E染色体组的野生稻O. glaberrima、O. punctata和O. australiensis着丝粒区域都含有与栽培稻着丝粒重复序列CentO和CRR同源的序列; (3) C染色体组野生稻O. officinalis的2条体细胞染色体着丝粒具有CentO的同源序列, 同时也发现其所有着丝粒区域都包含栽培稻CRR的同源序列。这些结果对克隆稻属不同染色体组的着丝粒序列、研究不同染色体组间着丝粒的进化关系和稻属不同着丝粒DNA序列与功能之间的关系均具有重要意义。     

A chromosome 5-specific repetitive DNA sequence in rice (Oryza sativa L)

Repetitive DNA sequences in the rice genome comprise more than half of the nuclear DNA. The isolation and characterization of these repetitive DNA sequences should lead to a better understanding of rice chromosome structure and genome organization. We report here the characterization and chromosome localization of a chromosome 5-specific repetitive DNA sequence. This repetitive DNA sequence was estimated to have at least 900 copies. DNA sequence analysis of three genomic clones which contain the repeat unit indicated that the DNA sequences have two sub-repeat units of 37 bp and 19 bp, connected by 30-to 90-bp short sequences with high similarity. RFLP mapping and physical mapping by fluorescence in situ hybridization (FISH) indicated that almost all copies of the repetitive DNA sequence are located in the centromeric heterochromatic region of the long arm of chromosome 5. The strategy for cloning such repetitive DNA sequences and their uses in rice genome research are discussed.     

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.     

Localization of specific repetitive DNA sequences in individual rice chromosomes

This paper describes the characterization and chromosomal distribution of three different rice (Oryza sativa) repetitive DNA sequences. The three sequences were characterized by sequence analysis, which gave 355, 498 and 756 bp for the length of the repeat unit in Os48, OsG3-498 and OsG5-756, respectively. Copy number determination by quantitative DNA slot-blot hybridization analysis showed 4000, 1080 and 920 copies, respectively, per haploid rice genome for the three sequences. In situ DNA hybridization analysis revealed that 95% of the silver grains detected with the Os48 probe were localized to euchromatic ends of seven long arms and one short arm out of the 12 rice chromosomes. For the OsG3-498 repetitive sequence, the majority of silver grains (58%) were also clustered at the same chromosomal ends as that of Os48. The minority (28%) of silver grains were located at heterochromatic short arms and centromeric regions. For the OsG5-756 repetitive sequence, 81% of the silver grains labeled the heterochromatic short arms and regions flanking all of the 12 centromeres. Thus, each of these three repetitive sequences was distributed at specific defined chromosomal locations rather than randomly at many chromosomal locations. The approximate copy number of a given repetitive DNA sequence at any specific chromosomal location was calculated by combining the information from in situ DNA hybridization analysis and the total copy number as determined by DNA slot-blot hybridization.by J. Huberman     

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.     

Repetitive Sequences in Plant Nuclear DNA:Types,Distribution, Evolution and Function

Repetitive DNA sequences are a major component of eukaryotic genomes and may account for up to 90% of the genome size. They can be divided into minisatellite, microsatellite and satellite sequences. Satellite DNA sequences are considered to be a fast-evolving component of eukaryotic genomes, comprising tandemly-arrayed, highly-repetitive and highly-conserved monomer sequences. The monomer unit of satellite DNA is 150–400 base pairs(bp) in length.Repetitive sequences may be species- or genus-specific, and may be centromeric or subtelomeric in nature. They exhibit cohesive and concerted evolution caused by molecular drive, leading to high sequence homogeneity. Repetitive sequences accumulate variations in sequence and copy number during evolution, hence they are important tools for taxonomic and phylogenetic studies, and are known as ‘‘tuning knobs' ' in the evolution. Therefore, knowledge of repetitive sequences assists our understanding of the organization, evolution and behavior of eukaryotic genomes. Repetitive sequences have cytoplasmic, cellular and developmental effects and play a role in chromosomal recombination. In the post-genomics era, with the introduction of next-generation sequencing technology, it is possible to evaluate complex genomes for analyzing repetitive sequences and deciphering the yet unknown functional potential of repetitive sequences.     

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.     

CXCR2基因多态性与奶牛乳房炎和乳品质的关联

采用PCR-SSCP技术研究了荷斯坦牛、西门塔尔牛和通江黄牛3个品种共160头个体CXCR2基因多态性与乳房炎抗性性状和对牛奶品质性状的遗传效应。结果表明: CXCR2基因有3个SNP多态位点, 分别位于序列的第685 bp、777 bp和861 bp位点, 确定了5个等位基因A、B、C、E和F。685 bp位点表现为BC和CC基因型, 777 bp位点表现为AA和AB基因型, 861 bp位点为CC和BC基因型。与奶牛乳房炎敏感性有关的基因型主要是BC、CC和FF, 可能对乳房炎有抗性的是AA、AB和EE基因型。据不同基因型对乳品质的遗传效应分析来看, AA、AB和EE基因型的乳品质性状极显著或显著优于其他基因型。     

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     

Molecular characterisation and chromosomal localisation of a telomere-like repetitive DNA sequence highly enriched in the C genome of Brassica

The aim of this work was to find C genome specific repetitive DNA sequences able to differentiate the homeologous A (B. rapa) and C (B. oleracea) genomes of Brassica, in order to assist in the physical identification of B. napus chromosomes. A repetitive sequence (pBo1.6) highly enriched in the C genome of Brassica was cloned from B. oleracea and its chromosomal organisation was investigated through fluorescent in situ hybridisation (FISH) in B. oleracea (2n = 18, CC), B. rapa (2n = 20, AA) and B. napus (2n = 38, AACC) genomes. The sequence was 203 bp long with a GC content of 48.3%. It showed up to 89% sequence identity with telomere-like DNA from many plant species. This repeat was clearly underrepresented in the A genome and the in situ hybridisation showed its B. oleracea specificity at the chromosomal level. Sequence pBo1.6 was localised at interstitial and/or telomeric/subtelomeric regions of all chromosomes from B. oleracea, whereas in B. rapa no signal was detected in most of the cells. In B. napus 18 to 24 chromosomes hybridised with pBo1.6. The discovery of a sequence highly enriched in the C genome of Brassica opens the opportunity for detailed studies regarding the subsequent evolution of DNA sequences in polyploid genomes. Moreover, pBo1.6 may be useful for the determination of the chromosomal location of transgenic DNA in genetically modified oilseed rape.     

Characterization of a novel satellite DNA sequence from Flying Dragon (Poncirus trifoliata)

Repetitive sequences constitute a significant component of most eukaryotic genomes, and the isolation and characterization of repetitive DNA sequences provide an insight into the organization of the genome of interest. Here, we report the isolation and the molecular analysis and methylation status of a novel tandemly organized repetitive DNA sequence from the genome of Poncirus trifoliata. Digestion of P. trifoliata DNA with Afa I produced a prominent fragment of approximately 400 bp. Southern blotting analysis of genomic DNA digested with the same enzyme revealed a ladder composed of DNA fragments that are multimers of the 400-bp Afa I band, indicating that the repetitive DNA is arrayed in tandem. This suggests that Afa I isolated a novel satellite that we have called Poncirus trifoliata satellite DNA 400 (PN400). This satellite composes 25% of the genome and it is also present in lemon, sour orange and kumquat. Analysis of the methylation status demonstrated that the cytosines in CCGG sequences in this satellite were methylated.     

Analysis of collinear regions of Oryza AA and CC genomes

Comparative analyses of genome structure and sequence of closely related species have yielded insights into the evolution and function of plant genomes. A total of 103,844 BAC end sequences delegated -73.8 Mb of O. officinalis that belongs to the CC genome type of the rice genus Oryza were obtained and compared with the genome sequences office cultivar, O. sativa ssp.japonica cv. Nipponbare. We found that more than 45% of O. officinalis genome consists of repeat sequences, which is higher than that of Nipponbare cultivar. To further investigate the evolutionary divergence of AA and CC genomes, two BAC-contigs of O. officinalis were compared with the collinear genomic regions of Nipponbare. Of 57 genes predicted in the AA genome orthologous regions, 39 had orthologs in the regions of the CC genome. Alignment of the orthologous regions indicated that the CC genome has undergone expansion in both genic and intergenic regions through primarily retroelement insertion. Particularly, the density of RNA transposable elements was 17.95% and 1.78% in O. officinalis and O. sativa, respectively. This explains why the orthologous region is about 100 kb longer in the CC genome in comparison to the AA genome.     

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     

Characterization and genetic mapping of a short, highly repeated, interspersed DNA sequence from rice (Oryza sativa L.).

Summary A short, highly repeated, interspersed DNA sequence from rice was characterized using a combination of techniques and genetically mapped to rice chromosomes by restriction fragment length polymorphism (RFLP) analysis. A consensus sequence (GGC)_n, where n varies from 13–16, for the repeated sequence family was deduced from sequence analysis. Southern blot analysis, restriction mapping of repeat element-containing genomic clones, and DNA sequence analysis indicated that the repeated sequence is interspersed in the rice genome, and is heterogeneous and divergent. About 200000 copies are present in the rice genome. Single copy sequences flanking the repeat element were used as RFLP markers to map individual repeat elements. Eleven such repeat elements were mapped to seven different chromosomes. The strategy for characterization of highly dispersed repeated DNA and its uses in genetic mapping, DNA fingerprinting, and evolutionary studies are discussed.