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.     

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.     

Fluorescence in situ hybridization of single copy transgenes in rice chromosomes

Summary Fluorescence in situ hybridization (FISH) is a powerful tool for visualizing the chromosomal location of targeted sequences and has been applied in many areas, including karyotyping, breeding and characterization of genes introduced into the plant genome. A simple, routine and sensitive FISH procedure was developed for localizing single copy genes in rice (Oryza sativa L.) metaphase chromosomes. We used digoxygenin-labeled endogenous or T-DNA sequences as small as 5.6 kb to probe corresponding endogenous sequences or the T-DNA insert in denatured rice metaphase chromosomes prepared from root meristem tissue. The hybridized probe sequence was labeled with cy3-conjugated anti-mouse IgG and visualized using fluorescence microscopy. Single copy and multiple copy introduced T-DNA sequences, as well as endogenous sequences, were localized on the chromosomes. The FISH protocol was effectively used to sereen the chromosomal location of introduced T-DNA and number of integration loci in rice.     

Variable copy number DNA sequences in rice

Summary We have cloned two types of variable copy number DNA sequences from the rice embryo genome. One of these sequences, which was cloned in pRB301, was amplified about 50-fold during callus formation and diminished in copy number to the embryonic level during regeneration. The other clone, named pRB401, showed the reciprocal pattern. The copy numbers of both sequences were changed even in the early developmental stage and eliminated from nuclear DNA along with growth of the plant. Sequencing analysis of the pRB301 insert revealed some open reading frames and direct repeat structures, but corresponding sequences were not identified in the EMBL and LASL DNA databases. Sequencing of the nuclear genomic fragment cloned in pRB401 revealed the presence of the 3rps12-rps7 region of rice chloroplast DNA. Our observations suggest that during callus formation (dedifferentiation), regeneration and the growth process the copy numbers of some DNA sequences are variable and that nuclear integrated chloroplast DNA acts as a variable copy number sequence in the rice genome. Based on data showing a common sequence in mitochondria and chloroplast DNA of maize (Stern and Lonsdale 1982) and that the rps12 gene of tobacco chloroplast DNA is a divided gene (Torazawa et al. 1986), it is suggested that the sequence on the inverted repeat structure of chloroplast DNA may have the character of a movable genetic element.     

Molecular and cytological characterization of repetitive DNA sequences in Brassica

Summary We isolated three different repetitive DNA sequences from B. campestris and determined their nucleotide sequences. In order to analyze organization of these repetitive sequences in Brassica, Southern blot hybridization and in situ hybridization with metaphase chromosomes were performed. The sequence cloned in the plasmid pCS1 represented a middle repetitive sequence present only in B. campestris and not detected in closely related B. Oleracea. This sequence was localized at centromeric regions of six specific chromosomes of B. campestris. The second plasmid, pBT4, contained a part of the 25S ribosomal RNA gene, and its copy number was estimated to be 1,590 and 1,300 per haploid genome for B. campestris and B. oleracea, respectively. In situ hybridization with this sequence showed a clear signal at the NOR region found in the second largest chromosome of B. Campestris. The third plasmid, pBT11, contained a 175-bp insert that belongs to a major family of tandem repeats found in all the Brassica species. This sequence was detected at centromeric regions of all the B. campestris chromosomes. Our study indicates that in situ hybridization with various types of repetitive sequences should give important information on the evolution of repetitive DNA in Brassica species.     

USE OF REPEATED DNA SEQUENCES AS CYTOLOGICAL MARKERS

The majority of DNA that is found in most of the flowering plants appears to be non-coding DNA. Much of this excess DNA consists of nucleotide sequences which exist as multiple copies throughout the genome and are designated as repetitive sequences. Those sequences which are found in moderately high to high numbers of copies are observed to be of the greatest value as cytological markers. Moderately high copies may exist as sequences which are dispersed throughout the chromosomes of some species and not dispersed in other more distantly related species. By taking advantage of this characteristic and the technique of in situ hybridization with biotinylated probes, breakpoints of chromosomal translocations may be observed between species such as wheat and rye. Many of the high copy number repetitive sequences are organized in a tandem fashion in specific loci in the chromosome. Chromosomal identification may be accomplished by using the in situ hybridization technique. Upon in situ hybridization with a repetitive sequence isolated from Aegilops squarrosa, the patterns of the sites of hybridization allowed the D-genome chromosomes to be identified. The sequence was also observed only on the D-genome chromosomes of several polyploid species indicating its usefulness as a genome specific marker. Using this genome specificity, assessment of the orientation of the D-genome chromosomal segments of hexaploid wheat carrying the sequence during interphase and prophase of mitotic root tip cells was possible. Repetitive DNA sequences, therefore, provide cytological markers necessary for studies of chromosomal identification, genome allocation, and genome orientation. The use of biotin-labeled DNA probes allows the technique of in situ hybridization to be performed much more rapidly and with a greater degree of safety and reliability.     

A molecular and cytogenetic survey of major repeated DNA sequences in tomato (Lycopersicon esculentum)

Summary The major families of repeated DNA sequences in the genome of tomato (Lycopersicon esculentum) were isolated from a sheared DNA library. One thousand clones, representing one million base pairs, or 0.15% of the genome, were surveyed for repeated DNA sequences by hybridization to total nuclear DNA. Four major repeat classes were identified and characterized with respect to copy number, chromosomal localization by in situ hybridization, and evolution in the family Solanaceae. The most highly repeated sequence, with approximately 77000 copies, consists of a 162 bp tandemly repeated satellite DNA. This repeat is clustered at or near the telomeres of most chromosomes and also at the centromeres and interstitial sites of a few chromosomes. Another family of tandemly repeated sequences consists of the genes coding for the 45 S ribosomal RNA. The 9.1 kb repeating unit in L. esculentum was estimated to be present in approximately 2300 copies. The single locus, previously mapped using restriction fragment length polymorphisms, was shown by in situ hybridization as a very intense signal at the end of chromosome 2. The third family of repeated sequences was interspersed throughout nearly all chromosomes with an average of 133 kb between elements. The total copy number in the genome is approximately 4200. The fourth class consists of another interspersed repeat showing clustering at or near the centromeres in several chromosomes. This repeat had a copy number of approximately 2100. Sequences homologous to the 45 S ribosomal DNA showed cross-hybridization to DNA from all solanaceous species examined including potato, Datura, Petunia, tobacco and pepper. In contrast, with the exception of one class of interspersed repeats which is present in potato, all other repetitive sequences appear to be limited to the crossing-range of tomato. These results, along with those from a companion paper (Zamir and Tanksley 1988), indicate that tomato possesses few highly repetitive DNA sequences and those that do exist are evolving at a rate higher than most other genomic sequences.     

Isolation of a new retrotransposon-like DNA sequence and its use in analysis of diversity within the Oryza officinalis

To better understand the genetic diversity of the wild relatives of rice (Oryza sativa L.) in the O. officinalis species complex repetitive DNA markers were obtained from the diploid species of this complex. One cloned sequence from O. eichingeri gave intense hybridization signals with all species of the O. officinalis complex. This 242 bp clone, named pOe.49, has a copy number from 0.9 to 4.0 × 10⁴ in diploid species of this complex. Analysis of the primary structure and database searches revealed homology of pOe.49 to a number of sequences representing part of the integrase coding domain of retroviruses and gypsy-like retrotransposons. Sequencing of specific PCR products confirmed that pOe.49 is part of a gypsy-like retrotransposon. RFLP analysis was used to study the genomic organisation of pOe.49 among 30 accessions of the O. officinalis complex using 10 restriction enzymes. Diversity analysis based on 120 polymorphic fragments obtained from the RFLP assay grouped the O. officinalis complex accessions by genome, species and eco-geographic groups. The results suggest that, with further characterization, this retrotransposon-like DNA sequence may be useful for phylogenetic analysis of species in the O. officinalis complex. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Microarray-based survey of repetitive genomic sequences in Vicia spp.

A modified DNA microarray-based technique was devised for preliminary screening of short fragment genomic DNA libraries from three Vicia species (V. melanops, V. narbonensis, and V. sativa) to isolate representative highly abundant DNA sequences that show different distribution patterns among related legume species. The microarrays were sequentially hybridized with labeled genomic DNAs of thirteen Vicia and seven other Fabaceae species and scored for hybridization signals of individual clones. The clones were then assigned to one of the following groups characterized by hybridization to: (1) all tested species, (2) most of the Vicia and Pisum species, (3) only a few Vicia species, and (4) preferentially a single Vicia species. Several clones from each group, 65 in total, were sequenced. All Group I clones were identified as rDNA genes or fragments of chloroplast genome, whereas the majority of Group II clones showed significant homologies to retroelement sequences. Clones in Groups III and IV contained novel dispersed repeats with copy numbers 10²–10⁶/1C and two genus-specific tandem repeats. One of these belongs to the VicTR-B repeat family, and the other clone (S12) contains an amplified portion of the rDNA intergenic spacer. In situ hybridization using V. sativa metaphase chromosomes revealed the presence of the S12 sequences not only within rDNA genes, but also at several additional loci. The newly identified repeats, as well as the retroelement-like sequences, were characterized with respect to their abundance within individual genomes. Correlations between the repeat distributions and the current taxonomic classification of these species are discussed.     

New families of site-specific repetitive DNA sequences that comprise constitutive heterochromatin of the Syrian hamster (Mesocricetus auratus, Cricetinae, Rodentia)

We molecularly cloned new families of site-specific repetitive DNA sequences from BglII- and EcoRI-digested genomic DNA of the Syrian hamster (Mesocricetus auratus, Cricetrinae, Rodentia) and characterized them by chromosome in situ hybridization and filter hybridization. They were classified into six different types of repetitive DNA sequence families according to chromosomal distribution and genome organization. The hybridization patterns of the sequences were consistent with the distribution of C-positive bands and/or Hoechst-stained heterochromatin. The centromeric major satellite DNA and sex chromosome-specific and telomeric region-specific repetitive sequences were conserved in the same genus (Mesocricetus) but divergent in different genera. The chromosome-2-specific sequence was conserved in two genera, Mesocricetus and Cricetulus, and a low copy number of repetitive sequences on the heterochromatic chromosome arms were conserved in the subfamily Cricetinae but not in the subfamily Calomyscinae. By contrast, the other type of repetitive sequences on the heterochromatic chromosome arms, which had sequence similarities to a LINE sequence of rodents, was conserved through the three subfamilies, Cricetinae, Calomyscinae and Murinae. The nucleotide divergence of the repetitive sequences of heterochromatin was well correlated with the phylogenetic relationships of the Cricetinae species, and each sequence has been independently amplified and diverged in the same genome.     

Genetic mapping with dispersed repeated sequences in the rice blast fungus: mapping the SMO locus

Summary The SMO genetic locus in strains of the fungus Magnaporthe grisea that infect weeping lovegrass, directs the formation of correct cell shapes in asexual spores, infection structures, and asci. We have identified and characterized a Smo^– strain of M. grisea that infects rice. The smo mutation in this strain segregates as a single gene mutation and is allelic to previously identified smo alleles. A marked reduction in pathogenicity co-segregates with the Smo^– phenotype, suggesting that the SMO locus plays a role in rice pathogenicity. A family of dispersed repeated DNA sequences, called MGR, have been discovered in the nuclear DNA of M. grisea rice pathogens. Genetic crosses between Smo^– rice pathogens and Smo⁺ non-rice pathogens were used to follow the segregation of the SMO locus and individual MGR sequences. Using DNA blot analysis with cloned MGR hybridization probes, we mapped the SMO locus to a chromosomal region flanked by two closely linked MGR sequences. We demonstrated that the copy number of MGR sequences could be reduced in subsequent crosses to non-rice pathogens of M. grisea, and that new MGR sequences did not occur following meiosis indicating that these sequences are stable in the genome. We conclude that restriction fragment polymorphism mapping with cloned MGR sequences as hybridization probes is an effective way to map genes in the rice blast fungus.     

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.     

Centromeric repetitive DNA sequences in the genus Brassica

Representatives of two major repetitive DNA sequence families from the diploid Brassica species B. campestris and B. oleracea were isolated, sequenced and localized to chromosomes by in situ hybridization. Both sequences were located near the centromeres of many chromosome pairs in both diploid species, but major sites of the two probes were all on different chromosome pairs. Such chromosome specificity is unusual for plant paracentromeric repetitive DNA. Reduction of stringency of hybridization gave centromeric hybridization sites on more chromosomes, indicating that there are divergent sequences present on other chromosomes. In tetraploid species derived from the diploids, the number of hybridization sites was different from the sum of the diploid ancestors, and some chromosomes had both sequences, indicating relatively rapid homogenization and copy number evolution since the origin of the tetraploid species.     

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     

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     

Analysis of T-DNA-<Emphasis Type="Italic">Xa21</Emphasis> loci and bacterial blight resistance effects of the transgene <Emphasis Type="Italic">Xa21</Emphasis> in transgenic rice

The genetic loci and phenotypic effects of the transgene Xa21, a bacterial blight (BB) resistance gene cloned from rice, were investigated in transgenic rice produced through an Agrobacterium-mediated transformation system. The flanking sequences of integrated T-DNAs were isolated from Xa21 transgenic rice lines using thermal asymmetric interlaced PCR. Based on the analysis of 24 T-DNA- Xa21 flanking sequences, T-DNA loci in rice could be classified into three types: the typical T-DNA integration with the definite left and right borders, the T-DNA integration linked with the adjacent vector backbone sequences and the T-DNA integration involved in a complicated recombination in the flanking sequences. The T-DNA integration in rice was similar to that in dicotyledonous genomes but was significantly different from the integration produced through direct DNA transformation approaches. All three types of integrated transgene Xa21 could be stably inherited and expressed the BB resistance through derived generations in their respective transgenic lines. The flanking sequences of the typical T-DNA integration consisted of actual rice genomic DNA and could be used as probes to locate the transgene on the rice genetic map. A total of 15 different rice T-DNA flanking sequences were identified. They displayed restriction fragment length polymorphisms (RFLPs) between two rice varieties, ZYQ8 and JX17, and were mapped on rice chromosomes 1, 3, 4, 5, 7, 9, 10, 11 and 12, respectively, by using a double haploid population derived from a cross between ZYQ8 and JX17. The blast search and homology comparison of the rice T-DNA flanking sequences with the rice chromosome-anchored sequence database confirmed the RFLP mapping results. On the basis of genetic mapping of the T-DNA- Xa21 loci, the BB resistance effects of the transgene Xa21 at different chromosome locations were investigated using homozygous transgenic lines with only one copy of the transgene. Among the transgenic lines, no obvious position effects of the transgene Xa21 were observed. In addition, the BB resistance levels of the Xa21 transgenic plants with different transgene copy numbers and on different genetic backgrounds were also investigated. It was observed that genetic background (or genome) effects were more obvious than dosage effects and position effects on the BB resistance level of the transgenic plants.     

Arrangement and size distribution of repeat and single copy DNA sequences in four species ofCucurbitaceae

DNA sequence organization patterns have been studied in fourCucurbitaceae plant species, namely,Luffa cylindrica (sponge gourd),L. acutangula (ridge gourd),Benincasa hispida (ash gourd) andCoccinia indica (ivy gourd). Extensive interspersion of repeat and single copy sequences has been observed in sponge gourd and ridge gourd. In ash gourd and ivy gourd, however, there is a limited interspersion of these sequences and a large portion of the single copy DNA remains uninterspersed. The interspersed repetitive sequences are composed of a major class (75–80%) of short repeats (300 base pairs long) and a minor class (15–20%) of long repeats (2 000–4 000 base pairs) in all the four species. The average length of single copy sequences dispersed among repeats is 1 800–2 900 base pairs. In spite of these gross similarities in the genome organization in the four species, the fraction of repeats and single copy sequences involved in short and long period interspersion patterns, and fraction of single copy sequences remaining uninterrupted by repeats are vastly different. The probable implications of these differences with respect to speciation events and rates of genome evolution are discussed.Molecular Analysis ofCucurbitaceae Genomes, III. — NCL Communication No.: 3595.     

Involvement of transposition in dispersion of tandem repeat sequences (TrsA) in rice genomes

We describe a method to identify and characterize DNA fragments containing the junction of AA genome-specific tandem repeat sequences (here called TrsA) with adjacent chromosomal sequences of rice by the polymerase chain reaction (PCR) using a pair of primers that hybridize with TrsAs and a flanking non-TrsA sequence. With this method, we obtained results suggesting that TrsA sequences present at two loci (here called trsA1 and trsA2) are flanked by direct repeats of chromosomal sequences of 172 by and about 440 by in length, respectively. These results support the idea that the TrsA sequences have been inserted into each locus by transposition, resulting in duplication of the chromosomal sequence used as target. We also describe a method to identify and characterize TrsA sequences repeated in only a few copies in the rice genome by PCR, using a pair of primers that hybridize with two different portions in the TrsA sequence, and demonstrate that TrsA sequences are present not only in rice strains with the AA genome, but also in those with non-AA genomes. The TrsA sequences were present at the trsA1 locus in all the rice strains examined, indicating that TrsA was inserted and amplified at the locus before the divergence of the various species of rice in the Oryza genus. TrsA sequences were present at the trsA2 locus, however, only in an O. sativa IR36 strain, indicating that TrsA was inserted and amplified at this locus during divergence of rice strains with the AA genome.