Retrotransposon insertion polymorphisms in six rice genes and their evolutionary history

Retrotransposons are abundant in higher plant genomes. Although retrotransposons associated with plant genes have been identified, little is known about their evolutionary conservation at the level of species and subspecies. In the present study, we investigated the phylogenetic distribution of long terminal repeat (LTR) retrotransposon, long interspersed nuclear element (LINE) and short interspersed nuclear element (SINE) insertions in six genes in 95 cultivated and wild rice genotypes. These six genes are likely to be functional based on nonsynonymous (Ka) to synonymous (Ks) substitution ratios which were found to be significantly <1. Different conservation patterns of these retrotransposons in genes were observed in cultivated and wild rice species. Four out of seven retrotransposon insertions appear to predate the ancestral Oryza AA genome. Two of these insertions in genes 4 and 5 occurred early in the evolutionary history of Oryza. Two retrotransposon insertions in gene 1 arose after the divergence of Asian cultivated rice from its wild ancestor. Furthermore, the retrotransposon insertion in gene 3 appears to have occurred in the ancestral lineage leading to temperate japonicas. Conservation of retrotransposon insertions in genes in specific groups, species, and lineages might be related to their specific function.     

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.     

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.     

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.     

Analysis of genes associated with retrotransposons in the rice genome

Retrotransposons comprise a significant fraction of the rice genome. Despite their prevalence, the effects of retrotransposon insertions are not well understood, especially with regard to how they affect the expression of genes. In this study, we identified one-sixth of rice genes as being associated with retrotransposons, with insertions either in the gene itself or within its putative promoter region. Among genes with insertions in the promoter region, the likelihood of the gene being expressed was shown to be directly proportional to the distance of the retrotransposon from the translation start site. In addition, retrotransposon insertions in the transcribed region of the gene were found to be positively correlated with the presence of alternative splicing forms. Furthermore, preferential association of retrotransposon insertions with genes in several functional classes was identified. Some of the retrotransposons that are part of full-length cDNA (fl-cDNA) contribute splice sites and give rise to novel exons. Several interesting trends concerning the effects of retrotransposon insertions on gene expression were identified. Taken together, our data suggests that retrotransposon association with genes have a role in gene regulation. The data presented in this study provides a foundation for experimental studies to determine the role of retrotransposons in gene regulation.     

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.     

Chloroplast genome sequence confirms distinctness of Australian and Asian wild rice

Cultivated rice (Oryza sativa) is an AA genome Oryza species that was most likely domesticated from wild populations of O. rufipogon in Asia. O. rufipogon and O. meridionalis are the only AA genome species found within Australia and occur as widespread populations across northern Australia. The chloroplast genome sequence of O. rufipogon from Asia and Australia and O. meridionalis and O. australiensis (an Australian member of the genus very distant from O. sativa) was obtained by massively parallel sequencing and compared with the chloroplast genome sequence of domesticated O. sativa. Oryza australiensis differed in more than 850 sites single nucleotide polymorphism or indel from each of the other samples. The other wild rice species had only around 100 differences relative to cultivated rice. The chloroplast genomes of Australian O. rufipogon and O. meridionalis were closely related with only 32 differences. The Asian O. rufipogon chloroplast genome (with only 68 differences) was closer to O. sativa than the Australian taxa (both with more than 100 differences). The chloroplast sequences emphasize the genetic distinctness of the Australian populations and their potential as a source of novel rice germplasm. The Australian O. rufipogon may be a perennial form of O. meridionalis.     

New SINE families from rice, OsSN, with poly(A) at the 3' ends

A database search of the sequences flanking a member of rice retrotransposon RIRE7 revealed that a 298-bp sequence in the region downstream of the member is a repetitive sequence interspersed in the genome of Oryza sativa cv. Nipponbare. Most of the repetitive sequences were flanked by a direct repeat of a target-site sequence, about 14 bp in length. The consensus sequence, 293 bp in length, had no regions encoding any proteins but had sequence motifs of an internal promoter of RNA polymerase III. These indicate that the sequence is a retroposon SINE, designated OsSN1 (Oryza sativa SINE1). OsSN1 is a new rice SINE, because it has no homology with any of the three p-SINE families previously identified from rice, and because it has a stretch of A at the 3' end, unlike p-SINE and any other Gramineae SINEs which have a stretch of T at the 3' end. The Nipponbare genome was found to have many members related to OsSN1, forming two additional new SINE families (designated OsSN2 and OsSN3). OsSN2 and OsSN3 are highly homologous to the 3' and 5' regions of OsSN1, respectively. This suggests that OsSN1 has a mosaic structure, which is generated by sequence exchange (or shuffling) between ancestral OsSN2 and OsSN3. Despite the absence of homology in the 3' regions between OsSN1 (or OsSN2) and OsSN3, a sequence, 5'-TTCTC-3', is commonly present in the region preceding the A stretch at the 3' end. This sequence together with the A stretch and a stem-loop structure found in the region near the A stretch are assumed to be important for retroposition. OsSN members were present in strains of Oryza species, as were p-SINE members. Some of the members showed insertion polymorphism at the respective loci among the rice strains. p-SINE had such polymorphic members, which are useful for classification and phylogenetic analysis of various strains of Oryza species. The polymorphic members of OsSN were more frequently found than those of p-SINE, and therefore, such members are likely to be useful for extensive taxonomic and phylogenetic studies on various rice strains.     

Simple sequence repeat analyses of interspecific hybrids and MAALs of <Emphasis Type="Italic">Oryza </Emphasis><Emphasis Type="Italic">officinalis</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

Wild rice is a valuable resource for the genetic improvement of cultivated rice (Oryza sativa L., AA genome). Molecular markers are important tools for monitoring gene introgression from wild rice into cultivated rice. In this study, Simple sequence repeat (SSR) markers were used to analyze interspecific hybrids of O. sativa-O. officinalis (CC genome), the backcrossing progenies and the parent plants. Results showed that most of the SSR primers (335 out of 396, 84.6%) developed in cultivated rice successfully amplified products from DNA samples of wild rice O. officinalis. The polymorphism ratio of SSR bands between O. sativa and O. officinalis was as high as 93.9%, indicating differences between the two species with respect to SSRs. When the SSR markers were applied in the interspecific hybrids, only a portion of SSR primers amplified O. officinalis-specific bands in the F(1) hybrid (52.5%), BC(1) (52.5%), and MAALs (37.0%); a number of the bands disappeared. Of the 124 SSR loci that detected officinalis-specific bands in MAAL plants, 96 (77.4%) showed synteny between the A and C-genomes, and 20 (16.1%) showed duplication in the C-genome. Sequencing analysis revealed that indels, substitution and duplication contribute to the diversity of SSR loci between the genomes of O. sativa and O. officinalis.     

Two new SINE elements, p-SINE2 and p-SINE3, from rice

p-SINE1 was the first plant SINE element identified in the Waxy gene in Oryza sativa, and since then a large number of p-SINE1-family members have been identified from rice species with the AA or non-AA genome. In this paper, we report two new rice SINE elements, designated p-SINE2 and p-SINE3, which form distinct families from that of p-SINE1. Each of the two new elements is significantly homologous to p-SINE1 in their 5'-end regions with that of the polymerase III promoter (A box and B box), but not significantly homologous in the 3'-end regions, although they all have a T-rich tail at the 3' terminus. Despite the three elements sharing minimal homology in their 3'-end regions, the deduced RNA secondary structures of p-SINE1, p-SINE2 and p-SINE3 were found to be similar to one another, such that a stem-loop structure seen in the 3'-end region of each element is well conserved, suggesting that the structure has an important role on the p-SINE retroposition. These findings suggest that the three p-SINE elements originated from a common ancestor. Similar to members of the p-SINE1 family, the members of p-SINE2 or p-SINE3 are almost randomly dispersed in each of the 12 rice chromosomes, but appear to be preferentially inserted into gene-rich regions. The p-SINE2 members were present at respective loci not only in the strains of the species with the AA genome in the O. sativa complex, but also in those of other species with the BB, CC, DD, or EE genome in the O. officinalis complex. The p-SINE3 members were, however, only present in strains of species in the O. sativa complex. These findings suggest that p-SINE2 originated in an ancestral species with the AA, BB, CC, DD and EE genomes, like p-SINE1, whereas p-SINE3 originated in an ancestral strain of the species with the AA genome. The nucleotide sequences of p-SINE1 members are more divergent than those of p-SINE2 or p-SINE3, indicating that p-SINE1 is likely to be older than p-SINE2 and p-SINE3. This suggests that p-SINE2 and p-SINE3 have been derived from p-SINE1.     

Replication of nonautonomous retroelements in soybean appears to be both recent and common

Retrotransposons and their remnants often constitute more than 50% of higher plant genomes. Although extensively studied in monocot crops such as maize (Zea mays) and rice (Oryza sativa), the impact of retrotransposons on dicot crop genomes is not well documented. Here, we present an analysis of retrotransposons in soybean (Glycine max). Analysis of approximately 3.7 megabases (Mb) of genomic sequence, including 0.87 Mb of pericentromeric sequence, uncovered 45 intact long terminal repeat (LTR)-retrotransposons. The ratio of intact elements to solo LTRs was 8:1, one of the highest reported to date in plants, suggesting that removal of retrotransposons by homologous recombination between LTRs is occurring more slowly in soybean than in previously characterized plant species. Analysis of paired LTR sequences uncovered a low frequency of deletions relative to base substitutions, indicating that removal of retrotransposon sequences by illegitimate recombination is also operating more slowly. Significantly, we identified three subfamilies of nonautonomous elements that have replicated in the recent past, suggesting that retrotransposition can be catalyzed in trans by autonomous elements elsewhere in the genome. Analysis of 1.6 Mb of sequence from Glycine tomentella, a wild perennial relative of soybean, uncovered 23 intact retroelements, two of which had accumulated no mutations in their LTRs, indicating very recent insertion. A similar pattern was found in 0.94 Mb of sequence from Phaseolus vulgaris (common bean). Thus, autonomous and nonautonomous retrotransposons appear to be both abundant and active in Glycine and Phaseolus. The impact of nonautonomous retrotransposon replication on genome size appears to be much greater than previously appreciated.     

TEnest: automated chronological annotation and visualization of nested plant transposable elements

Organisms with a high density of transposable elements (TEs) exhibit nesting, with subsequent repeats found inside previously inserted elements. Nesting splits the sequence structure of TEs and makes annotation of repetitive areas challenging. We present TEnest, a repeat identification and display tool made specifically for highly repetitive genomes. TEnest identifies repetitive sequences and reconstructs separated sections to provide full-length repeats and, for long-terminal repeat (LTR) retrotransposons, calculates age since insertion based on LTR divergence. TEnest provides a chronological insertion display to give an accurate visual representation of TE integration history showing timeline, location, and families of each TE identified, thus creating a framework from which evolutionary comparisons can be made among various regions of the genome. A database of repeats has been developed for maize (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum), and barley (Hordeum vulgare) to illustrate the potential of TEnest software. All currently finished maize bacterial artificial chromosomes totaling 29.3 Mb were analyzed with TEnest to provide a characterization of the repeat insertions. Sixty-seven percent of the maize genome was found to be made up of TEs; of these, 95% are LTR retrotransposons. The rate of solo LTR formation is shown to be dissimilar across retrotransposon families. Phylogenetic analysis of TE families reveals specific events of extreme TE proliferation, which may explain the high quantities of certain TE families found throughout the maize genome. The TEnest software package is available for use on PlantGDB under the tools section (http://www.plantgdb.org/prj/TE_nest/TE_nest.html); the source code is available from (http://wiselab.org).     

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.     

Genetic Mapping of Rice Using RFLP Markers and a Double Haploid Population of a Cross Between indica and japonica Varieties

A genetic linkage map of rice was constructed using a double haploid (DH) population from "Gui 630” (Oryza sativa subsp, indica)/"02428" (O. sativa subsp, japonica, wide compatibility variety) and RFLP markers. It consists of 233 loci and covers rice genomes about 2070 cM (centimorgan), and compares well with the other published rice maps. 25 RFLP markers, 2 telomeres and sh-2 (shattering ability) gene were first located on the molecular map of rice. RFLPs between "Gui 630' and "02428' mainly came from base substitution and a few DNA construction variance, not distributed evenly among chromosomes and on chromosome. This was probably resulted from the difference genetic stability among chromosomes and regions, in exchanging recombination ability in different segments of chromosome.     

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 new gypsy-type retrotransposon, RIRE7: preferential insertion into the tandem repeat sequence TrsD in pericentromeric heterochromatin regions of rice chromosomes

A portion of an insertion sequence present in a member of the RIRE3 family of retrotransposons in Oryza sativa L. cv. IR36 was found to have an LTR sequence followed by a PBS sequence complementary to the 3'-end region of tRNAMet, indicative of another rice retrotransposon (named RIRE7). Cloning and sequencing of PCR-amplified fragments that made up all parts of the RIRE7 sequence showed that RIRE7 is a gypsy-type retrotransposon with partial homology in the pol region to the rice gypsy-type retrotransposons RIRE2 and RIRE3 identified in rice previously. Interestingly, various portions of the RIRE7 sequence were homologous to several DNA segments present in the centromere regions of cereal chromosomes. Further cloning and nucleotide sequencing of fragments flanking RIRE7 copies showed that RIRE7 was inserted into a site within a tandem repeat sequence that has a unit length of 155 bp. The tandem repeat sequence, named TrsD, was homologous to tandem repeat sequences RCS2 and CentC, previously identified in the centromeric regions of rice and maize chromosomes. Fluorescence in situ hybridization (FISH) analysis of the metaphase chromosomes of O. sativa cv. Nipponbare showed that both RIRE7 and TrsD sequences were present in the centromere regions of the chromosomes. The presence of RIRE7 and the TrsD sequences in the centromere regions of several chromosomes was confirmed by the identification of several YAC clones whose chromosomal locations are known. Further FISH analysis of rice pachytene chromosomes showed that the TrsD sequences were located in a pericentromeric heterochromatin region. These findings strongly suggest that RIRE7 and TrsD are components of the pericentromeric heterochromatin of rice chromosomes.     

Evolutionary relationships among rice species with AA genome based on SINE insertion analysis

Previous studies based on morphological and molecular markers indicated that there are two cultivated and five wild rice species within the Oryza genus with the AA genome. In the cultivated rice species, Oryza sativa, a retroposon named p-SINE1 has been identified. Some of the p-SINE1 members characterized previously showed interspecific insertion polymorphisms in the species with the AA genome. In this study, we identified new p-SINE1 members showing interspecific insertion polymorphisms from representative strains of four wild rice species with the AA genome: O. barthii, O. glumaepatula, O. longistaminata, and O. meridionalis. Some of these members were present only in strains of one species, whereas the others were present in strains of two or more species. The p-SINE1 insertion patterns in the strains of the Asian and African cultivated rice species O. sativa and O. glaberrima were very similar to those of the Asian and African wild rice species O. rufipogon and O. barthii, respectively. This is consistent with the previous hypothesis that O. sativa and O. glaberrima are derived from specific wild rice species. Phylogenetic analysis based on the p-SINE1 insertion patterns showed that the strains of each of the five wild rice species formed a cluster. The strains of O. longistaminata appear to be distantly related to those of O. meridionalis. The strains of these two species appear to be distantly related to those of three other species, O. rufipogon, O. barthii and O. glumaepatula. The latter three species are closely related to one another with O. barthii and O. glumaepatula being most closely related. A phylogenetic tree including a hypothetical ancestor with all loci empty for p-SINE1 insertion showed that the strains of O. longistaminata are related most closely to the hypothetical ancestor. This indicates that O. longistaminata and O. meridionalis diverged early on, whereas the other species diverged relatively recently, and suggests that the Oryza genus with AA genome might have originated in Africa, rather than in Asia.     

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.