The rice mitochondrial genomes and their variations

Based on highly redundant and high-quality sequences, we assembled rice (Oryza sativa) mitochondrial genomes for two cultivars, 93-11 (an indica variety) and PA64S (an indica-like variety with maternal origin of japonica), which are paternal and maternal strains of an elite superhybrid rice Liang-You-Pei-Jiu (LYP-9), respectively. Following up with a previous analysis on rice chloroplast genomes, we divided mitochondrial sequence variations into two basic categories, intravarietal and intersubspecific. Intravarietal polymorphisms are variations within mitochondrial genomes of an individual variety. Intersubspecific polymorphisms are variations between subspecies among their major genotypes. In this study, we identified 96 single nucleotide polymorphisms (SNPs), 25 indels, and three segmental sequence variations as intersubspecific polymorphisms. A signature sequence fragment unique to indica varieties was confirmed experimentally and found in two wild rice samples, but absent in japonica varieties. The intersubspecific polymorphism rate for mitochondrial genomes is 0.02% for SNPs and 0.006% for indels, nearly 2.5 and 3 times lower than that of their chloroplast counterparts and 21 and 38 times lower than corresponding rates of the rice nuclear genome, respectively. The intravarietal polymorphism rates among analyzed mitochondrial genomes, such as 93-11 and PA64S, are 1.26% and 1.38% for SNPs and 1.13% and 1.09% for indels, respectively. Based on the total number of SNPs between the two mitochondrial genomes, we estimate that the divergence of indica and japonica mitochondrial genomes occurred approximately 45,000 to 250,000 years ago.     

The Organelle Genomes of Hassawi Rice (Oryza sativa L.) and Its Hybrid in Saudi Arabia: Genome Variation, Rearrangement, and Origins

Hassawi rice (Oryza sativa L.) is a landrace adapted to the climate of Saudi Arabia, characterized by its strong resistance to soil salinity and drought. Using high quality sequencing reads extracted from raw data of a whole genome sequencing project, we assembled both chloroplast (cp) and mitochondrial (mt) genomes of the wild-type Hassawi rice (Hassawi-1) and its dwarf hybrid (Hassawi-2). We discovered 16 InDels (insertions and deletions) but no SNP (single nucleotide polymorphism) is present between the two Hassawi cp genomes. We identified 48 InDels and 26 SNPs in the two Hassawi mt genomes and a new type of sequence variation, termed reverse complementary variation (RCV) in the rice cp genomes. There are two and four RCVs identified in Hassawi-1 when compared to 93-11 (indica) and Nipponbare (japonica), respectively. Microsatellite sequence analysis showed there are more SSRs in the genic regions of both cp and mt genomes in the Hassawi rice than in the other rice varieties. There are also large repeats in the Hassawi mt genomes, with the longest length of 96,168 bp and 96,165 bp in Hassawi-1 and Hassawi-2, respectively. We believe that frequent DNA rearrangement in the Hassawi mt and cp genomes indicate ongoing dynamic processes to reach genetic stability under strong environmental pressures. Based on sequence variation analysis and the breeding history, we suggest that both Hassawi-1 and Hassawi-2 originated from the Indonesian variety Peta since genetic diversity between the two Hassawi cultivars is very low albeit an unknown historic origin of the wild-type Hassawi rice.     

Genome-wide intraspecific DNA-sequence variations in rice

Genome-wide comparative analysis of the DNA sequences of two major cultivated rice subspecies, Oryza sativa L. ssp indica and Oryza sativa L. ssp japonica, have revealed their extensive microcolinearity in gene order and content. However, deviations from colinearity are frequent owing to insertions or deletions. Intraspecific sequence polymorphisms commonly occur in both coding and non-coding regions. These variations often affect gene structures and may contribute to intraspecific phenotypic adaptations.     

Characterization of chloroplast DNA microsatellites from Saccharum spp and related species

Microsatellites, or simple sequence repeats (SSRs), and their flanking regions in chloroplast genomes (plastomes) of some species of the family Poaceae were analyzed in silico to look for DNA sequence variations. Comparison of the complete chloroplast DNA sequences (cpDNAs) of sugarcane (Saccharum hybrid cv. SP-80-3280 and S. officinarum cv. NCo310) and related species, Agrostis stolonifera, Brachypodium distachyon, Hordeum vulgare subsp vulgare, Lolium perenne, Oryza nivara, O. sativa subsp indica, O. sativa subsp japonica, Sorghum bicolor, Triticum aestivum, Zea mays, and Z. mays cv. B73, allowed us to examine the organization of chloroplast SSRs (cpSSRs) in genic and intergenic regions. We identified 204 cpSSRs in the sugarcane cpDNA; 22.5% were in genic regions. The ndh, rps, trn, and rpl gene clusters of the chloroplasts had the most repeats. Mononucleotide repeats were the most abundant cpSSRs in these species; however, di-, tri-, tetra-, penta-, and hexanucleotide repeats were also identified. Many base substitutions and deletions/insertions were identified in the cpSSR loci and their flanking regions. Multiple alignments of all cpSSR sequences of Poaceae species made identification of nucleotide variability possible; repeat motifs are not uniformly distributed across the Poaceae plastomes, but are mostly confined to intergenic regions. Phylogeny was determined by maximum parsimony and neighbor-joining inference methods. The cpSSRs of these species were found to be polymorphic. It appears that individual cpSSRs in the Poaceae are stable, at least over short periods of evolutionary time. We conclude that the plastome database can be exploited for phylogenetic analysis and biotechnological development.     

Chloroplast genome variation in upland and lowland switchgrass

Switchgrass (Panicum virgatum L.) exists at multiple ploidies and two phenotypically distinct ecotypes. To facilitate interploidal comparisons and to understand the extent of sequence variation within existing breeding pools, two complete switchgrass chloroplast genomes were sequenced from individuals representative of the upland and lowland ecotypes. The results demonstrated a very high degree of conservation in gene content and order with other sequenced plastid genomes. The lowland ecotype reference sequence (Kanlow Lin1) was 139,677 base pairs while the upland sequence (Summer Lin2) was 139,619 base pairs. Alignments between the lowland reference sequence and short-read sequence data from existing sequence datasets identified as either upland or lowland confirmed known polymorphisms and indicated the presence of other differences. Insertions and deletions principally occurred near stretches of homopolymer simple sequence repeats in intergenic regions while most Single Nucleotide Polymorphisms (SNPs) occurred in intergenic regions and introns within the single copy portions of the genome. The polymorphism rate between upland and lowland switchgrass ecotypes was found to be similar to rates reported between chloroplast genomes of indica and japonica subspecies of rice which were believed to have diverged 0.2-0.4 million years ago.     

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.     

Development of genome-wide DNA polymorphism database for map-based cloning of rice genes

DNA polymorphism is the basis to develop molecular markers that are widely used in genetic mapping today. A genome-wide rice (Oryza sativa) DNA polymorphism database has been constructed in this work using the genomes of Nipponbare, a cultivar of japonica, and 93-11, a cultivar of indica. This database contains 1,703,176 single nucleotide polymorphisms (SNPs) and 479,406 Insertion/Deletions (InDels), approximately one SNP every 268 bp and one InDel every 953 bp in rice genome. Both SNPs and InDels in the database were experimentally validated. Of 109 randomly selected SNPs, 107 SNPs (98.2%) are accurate. PCR analysis indicated that 90% (97 of 108) of InDels in the database could be used as molecular markers, and 68% to 89% of the 97 InDel markers have polymorphisms between other indica cultivars (Guang-lu-ai 4 and Long-te-pu B) and japonica cultivars (Zhong-hua 11 and 9522). This suggests that this database can be used not only for Nipponbare and 93-11, but also for other japonica and indica cultivars. While validating InDel polymorphisms in the database, a set of InDel markers with each chromosome 3 to 5 marker was developed. These markers are inexpensive and easy to use, and can be used for any combination of japonica and indica cultivars used in this work. This rice DNA polymorphism database will be a valuable resource and important tool for map-based cloning of rice gene, as well as in other various research on rice (http://shenghuan.shnu.edu.cn/ricemarker).     

光敏核不育水稻农垦58S与其衍生不育系的叶绿体DNA的比较

扩增了光敏核不育系农垦58S及从它衍生出来的5个两用核不育系叶绿体DNA的ORF（open reading frame）100、ORF29-TrnC^GCA、rps16（ribosomal proteins16）基因内含子和TrnT^UGU-TrnL^UAA（tRNA^Thr（UGU）-tRNA^-Leu（UAA）等4个片段,并测定了其序列。研究结果表明,粳型光敏核不系农垦58S的叶绿体为粳型。农垦58S衍生的核不育系中,粳型核不育系7001S以及3个籼型核不育系1103S、培矮64S和广占63S的叶绿体DNA为粳型,与选育者提供的细胞质系谱一致。籼型核不育系W6154S的叶绿体DNA为籼型,与选育者提供的细胞质系谱不一致,推断选育者在选育过程中更换过细胞质（曾用不育系作过父本）。5个粳型叶绿体DNA的rps16基因内含子和TrnT^UGU-TrnL^UAA间区的序列相互之间有1-2个单核苷酸的变异。     

Pattern and rate of indel evolution inferred from whole chloroplast intergenic regions in sugarcane, maize and rice.

Microstructural changes such as insertions and deletions (=indels) are a major driving force in the evolution of non-coding DNA sequences. To better understand the mechanisms by which indel mutations arise, as well as the molecular evolution of non-coding regions, the number and pattern of indels and nucleotide substitutions were compared in the whole chloroplast genomes. Comparisons were made for a total of over 38 kb non-coding DNA sequences from 126 intergenic regions in two data sets representing species with different divergence times: sugarcane and maize and Oryza sativa var. indica and japonica. The main findings of this study are: (i) Approximately half of all indels are single nucleotide indels. This observation agrees with previous studies in various organisms. (ii) The distribution and number of indels was different between two data sets, and different patterns were observed for tandem repeat and non-repeat indels. (iii) Distribution pattern of tandem repeat indels showed statistically significant bias towards A/T-rich. (iv) The rate of indel mutation was estimated to be approximately 0.8 +/- 0.04 x 10(-9) per site per year, which was similar to previous estimates in other organisms. (v) The frequencies of nucleotide substitutions and indels were significantly lower in inverted repeat (IR).     

Polymorphisms and evolutionary history of retrotransposon insertions in rice promoters

Retrotransposons are ubiquitous in higher plant genomes. The presence or absence of retrotransposons in whole genome and high throughput genomic sequence (HTGS) from cultivated and wild rice was investigated to understand the organization and evolution of retrotransposon insertions in promoter regions. Approximately half of the Oryza sativa subsp. japonica 'Nipponbare' promoters with retrotransposons conserved in Oryza sativa subsp. indica '93-11' and four wild rice species showed higher sequence conservation in retrotransposon than nonretrotransposon regions. We further investigated, in detail, the evolutionary dynamics of five retrotransposons in the promoter regions of 95 rice genotypes. Our data suggest that four of five insertions (Rp2-Rp5) occurred in the ancestor of AA genome, while the other insertion (Rp1) predates the ancestral divergence of Oryza officinalis (CC genome). Four retrotransposons (Rp2-Rp5) were present in 52% (Rp2), 29% (Rp3), 53% (Rp4), and 43% (Rp5) of the rice genotypes with AA genome type, and the fifth retrotransposon (Rp1) was present in 95% of the rice genotypes with AA, BBCC, or CC genome types. Furthermore, most of these retrotransposons were found to evolve slower than flanking promoter regions, suggesting a role in promoter function for regulating downstream genes.     

A novel type of sequence variation: multiple-nucleotide length polymorphisms discovered in the bovine genome

Three types of sequence variations--single-nucleotide polymorphisms (SNPs), insertions and deletions (indels), and short tandem repeats (STRs)--have been extensively reported in mammalian genomes. In this study, we discovered a novel type of sequence variation, i.e., multiple-nucleotide length polymorphisms (MNLPs) in bovine UCN3 (Urocortin 3) and its receptor CRHR2 (corticotropin-releasing hormone receptor 2) genes. Both MNLPs featured involvement of multiple-nucleotide length polymorphisms (5-18 bases), low sequence identity, and 1.7- to 11-fold changes in promoter activity between two alleles. Therefore, this novel genetic complexity would contribute significantly to the evolutionary, functional, and phenotypic complexity of genomes within or among species.     

Primary structure of the crossover region in the genome of two intertype poliovirus recombinant

The nucleotide sequence of the crossover region on genomes of two intertypic (type 3/type 1) poliovirus recombinant, has been determined by the primer extension method. No deletions, insertions or rearrangements have been observed. Identical contiguous sequences, 7 or 11 nucleotides in length, respectively, have been found in two regions of the parental genomes, involved in the recombination.     

Characterization of the flanking regions of <Emphasis Type="Italic">Zea mays</Emphasis> microsatellites reveals a large number of useful sequence polymorphisms

Sequence characterization of the flanking regions of 52 sequence-tagged microsatellite loci and two gene fragments from 11 Zea mays inbred lines identified a total of 324 sequence polymorphisms. The sequence polymorphisms consisted of both single-nucleotide polymorphisms and insertions/deletions in a ratio of approximately two to one. The level of sequence variation within the flanking regions of microsatellites linked to expressed sequence tags was lower than microsatellites that were unlinked to expressed sequence tags. However, both types of microsatellites generated a similar number of sequence-based alleles across the 11 genotypes surveyed. In two out of 20 microsatellites examined in detail, evidence was found for size-based allele homoplasy. Conversion of the observed sequence polymorphisms into allele-specific oligonucleotides followed by covalent binding to glass slides allowed the sequence polymorphisms to be used in a simple hybridization-based genotyping procedure. This procedure enabled us to discriminate between different inbred lines and allowed variations within a single inbred to be identified. The sequence information presented in this report could be used as a starting point for other programmes in the further development of a non-gel based, multi-locus, multi-allele screen for large-scale maize genotyping.     

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。     

Nucleotide sequence of a Euglena gracilis chloroplast gene coding for the 16S rRNA: homologies to E. coli and Zea mays chloroplast 16S rRNA

The nucleotide sequence of 16S rDNA from Euglena gracilis chloroplasts has been determined representing the first complete sequence of an algal chloroplast rRNA gene. The structural part of the 16S rRNA gene has 1491 nucleotides according to a comparative analysis of our sequencing results with the published 5'- and 3'-terminal "T1-oligonucleotides" from 16S rRNA from E. gracilis. Alignment with 16S rDNA from Zea mays chloroplasts and E. coli reveals 80 to 72% sequence homology, respectively. Two deletions of 9 and 23 nucleotides are found which are identical in size and position with deletions observed in 16S rDNA of maize and tobacco chloroplasts and which seem to be characteristic for all chloroplast rRNA species. We also find insertions and deletions in E. gracilis not seen in 16S rDNA of higher plant chloroplasts. The 16S rRNA sequence of E. gracilis chloroplasts can be folded by base pairing according to the general 16S rRNA secondary structure model.     

Sequence analysis of the complete mitochondrial DNA in 10 commonly used inbred rat strains

Rat remains a major biomedical model system for common, complex diseases. The rat continues to gain importance as a model system with the completion of its full genomic sequence. Although the genomic sequence has generated much interest, only three complete sequences of the rat mitochondria exist. Therefore, to increase the knowledge of the rat genome, the entire mitochondrial genomes (16,307–16,315 bp) from 10 inbred rat strains (that are standard laboratory models around the world) and 2 wild rat strains were sequenced. We observed a total of 195 polymorphisms, 32 of which created an amino acid change (nonsynonymous substitutions) in 12 of the 13 protein coding genes within the mitochondrial genome. There were 11 single nucleotide polymorphisms within the tRNA genes, six in the 12S rRNA, and 12 in the 16S rRNA including 3 insertions/deletions. We found 14 single nucleotide polymorphisms and 2 insertion/deletion polymorphisms in the D-loop. The inbred rat strains cluster phylogenetically into three distinct groups. The wild rat from Tokyo grouped closely with five inbred strains in the phylogeny, whereas the wild rat from Milwaukee was not closely related to any inbred strain. These data will enable investigators to rapidly assess the potential impact of the mitochondria in these rats on the physiology and the pathophysiology of phenotypes studied in these strains. Moreover, these data provide information that may be useful as new animal models, which result in novel combinations of nuclear and mitochondrial genomes, are developed. genome; mitochondria     

Patterns of Insertion and Deletion in Mammalian Genomes

Nucleotide insertions and deletions (indels) are responsible for gaps in the sequence alignments. Indel is one of the major sources of evolutionary change at the molecular level. We have examined the patterns of insertions and deletions in the 19 mammalian genomes, and found that deletion events are more common than insertions in the mammalian genomes. Both the number of insertions and deletions decrease rapidly when the gap length increases and single nucleotide indel is the most frequent in all indel events. The frequencies of both insertions and deletions can be described well by power law.Key Words: Insertion, deletion, gap, indel, mammalian genome.     

Analysis of complete nucleotide sequences of 12 gossypium chloroplast genomes: origin and evolution of allotetraploids

Background

Cotton (Gossypium spp.) is a model system for the analysis of polyploidization. Although ascertaining the donor species of allotetraploid cotton has been intensively studied, sequence comparison of Gossypium chloroplast genomes is still of interest to understand the mechanisms underlining the evolution of Gossypium allotetraploids, while it is generally accepted that the parents were A- and D-genome containing species. Here we performed a comparative analysis of 13 Gossypium chloroplast genomes, twelve of which are presented here for the first time.

Methodology/Principal Findings

The size of 12 chloroplast genomes under study varied from 159,959 bp to 160,433 bp. The chromosomes were highly similar having >98% sequence identity. They encoded the same set of 112 unique genes which occurred in a uniform order with only slightly different boundary junctions. Divergence due to indels as well as substitutions was examined separately for genome, coding and noncoding sequences. The genome divergence was estimated as 0.374% to 0.583% between allotetraploid species and A-genome, and 0.159% to 0.454% within allotetraploids. Forty protein-coding genes were completely identical at the protein level, and 20 intergenic sequences were completely conserved. The 9 allotetraploids shared 5 insertions and 9 deletions in whole genome, and 7-bp substitutions in protein-coding genes. The phylogenetic tree confirmed a close relationship between allotetraploids and the ancestor of A-genome, and the allotetraploids were divided into four separate groups. Progenitor allotetraploid cotton originated 0.43–0.68 million years ago (MYA).

Conclusion

Despite high degree of conservation between the Gossypium chloroplast genomes, sequence variations among species could still be detected. Gossypium chloroplast genomes preferred for 5-bp indels and 1–3-bp indels are mainly attributed to the SSR polymorphisms. This study supports that the common ancestor of diploid A-genome species in Gossypium is the maternal source of extant allotetraploid species and allotetraploids have a monophyletic origin. G. hirsutum AD1 lineages have experienced more sequence variations than other allotetraploids in intergenic regions. The available complete nucleotide sequences of 12 Gossypium chloroplast genomes should facilitate studies to uncover the molecular mechanisms of compartmental co-evolution and speciation of Gossypium allotetraploids.