Complete chloroplast genome sequences of Hordeum vulgare , Sorghum bicolor and Agrostis stolonifera , and comparative analyses with other grass genomes

Comparisons of complete chloroplast genome sequences of Hordeum vulgare, Sorghum bicolor and Agrostis stolonifera to six published grass chloroplast genomes reveal that gene content and order are similar but two microstructural changes have occurred. First, the expansion of the IR at the SSC/IRa boundary that duplicates a portion of the 5′ end of ndhH is restricted to the three genera of the subfamily Pooideae (Agrostis, Hordeum and Triticum). Second, a 6 bp deletion in ndhK is shared by Agrostis, Hordeum, Oryza and Triticum, and this event supports the sister relationship between the subfamilies Erhartoideae and Pooideae. Repeat analysis identified 19-37 direct and inverted repeats 30 bp or longer with a sequence identity of at least 90%. Seventeen of the 26 shared repeats are found in all the grass chloroplast genomes examined and are located in the same genes or intergenic spacer (IGS) regions. Examination of simple sequence repeats (SSRs) identified 16–21 potential polymorphic SSRs. Five IGS regions have 100% sequence identity among Zea mays, Saccharum officinarum and Sorghum bicolor, whereas no spacer regions were identical among Oryza sativa, Triticum aestivum, H. vulgare and A. stolonifera despite their close phylogenetic relationship. Alignment of EST sequences and DNA coding sequences identified six C–U conversions in both Sorghum bicolor and H. vulgare but only one in A. stolonifera. Phylogenetic trees based on DNA sequences of 61 protein-coding genes of 38 taxa using both maximum parsimony and likelihood methods provide moderate support for a sister relationship between the subfamilies Erhartoideae and Pooideae. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Phylogenetic study of Oryzoideae species and related taxa of the Poaceae based on <Emphasis Type="Italic">atpB</Emphasis>-<Emphasis Type="Italic">rbcL</Emphasis> and <Emphasis Type="Italic">ndhF</Emphasis> DNA sequences

Oryzoideae (Poaceae) plants have economic and ecological value. However, the phylogenetic position of some plants is not clear, such as Hygroryza aristata (Retz.) Nees. and Porteresia coarctata (Roxb.) Tateoka (syn. Oryza coarctata). Comprehensive molecular phylogenetic studies have been carried out on many genera in the Poaceae. The different DNA sequences, including nuclear and chloroplast sequences, had been extensively employed to determine relationships at both higher and lower taxonomic levels in the Poaceae. Chloroplast DNA ndhF gene and atpB-rbcL spacer were used to construct phylogenetic trees and estimate the divergence time of Oryzoideae, Bambusoideae, Panicoideae, Pooideae and so on. Complete sequences of atpB-rbcL and ndhF were generated for 17 species representing six species of the Oryzoideae and related subfamilies. Nicotiana tabacum L. was the outgroup species. The two DNA datasets were analyzed, using Maximum Parsimony and Bayesian analysis methods. The molecular phylogeny revealed that H. aristata (Retz.) Nees was the sister to Chikusichloa aquatica Koidz. Moreover, P. coarctata (Roxb.) Tateoka was in the genus Oryza. Furthermore, the result of evolution analysis, which based on the ndhF marker, indicated that the time of origin of Oryzoideae might be 31 million years ago.     

Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

A software tool was developed for the identification of simple sequence repeats (SSRs) in a barley ( Hordeum vulgare L.) EST (expressed sequence tag) database comprising 24,595 sequences. In total, 1,856 SSR-containing sequences were identified. Trimeric SSR repeat motifs appeared to be the most abundant type. A subset of 311 primer pairs flanking SSR loci have been used for screening polymorphisms among six barley cultivars, being parents of three mapping populations. As a result, 76 EST-derived SSR-markers were integrated into a barley genetic consensus map. A correlation between polymorphism and the number of repeats was observed for SSRs built of dimeric up to tetrameric units. 3'-ESTs yielded a higher portion of polymorphic SSRs (64%) than 5'-ESTs did. The estimated PIC (polymorphic information content) value was 0.45 +/- 0.03. Approximately 80% of the SSR-markers amplified DNA fragments in Hordeum bulbosum, followed by rye, wheat (both about 60%) and rice (40%). A subset of 38 EST-derived SSR-markers comprising 114 alleles were used to investigate genetic diversity among 54 barley cultivars. In accordance with a previous, RFLP-based, study, spring and winter cultivars, as well as two- and six-rowed barleys, formed separate clades upon PCoA analysis. The results show that: (1) with the software tool developed, EST databases can be efficiently exploited for the development of cDNA-SSRs, (2) EST-derived SSRs are significantly less polymorphic than those derived from genomic regions, (3) a considerable portion of the developed SSRs can be transferred to related species, and (4) compared to RFLP-markers, cDNA-SSRs yield similar patterns of genetic diversity.     

Sequence subfamilies of satellite repeats related to rDNA intergenic spacer are differentially amplified on<Emphasis Type="Italic"> Vicia sativa</Emphasis> chromosomes

We cloned and sequenced the Vicia sativa 25S-18S rDNA intergenic spacer (IGS) and the satellite repeat S12, thought to be related to the spacer sequence. The spacer was shown to contain three types of subrepeats (A, B, and C) with monomers of 173 bp (A), 10 bp (B), and 66 bp (C), separated by unique or partially duplicated sequences. Two spacer variants were detected in V. sativa that differed in length (2990 and 3168 bp) owing to an extra copy of the subrepeat A. The A subrepeats were also shown to be highly homologous to the satellite repeat S12, which is located in large clusters on chromosomes 4, 5, and 6, and is not associated with the rDNA loci. Sequencing of additional S12 clones retrieved from a shotgun genomic library allowed definition of three subfamilies of this repeat based on minor differences in their nucleotide sequences. Two of these subfamilies could be discriminated from the rest of the S12 sequences as well as from the IGS A subrepeats using specific oligonucleotide primers that labeled only a subset of the S12 loci when used in the primed in situ DNA labeling (PRINS) reaction on mitotic chromosomes. These experiments showed that, in spite of the high overall similarity of the IGS A subrepeats and the S12 satellite repeats, there are S12 subfamilies that are divergent from the common consensus and are present at only some of the chromosomes containing the S12 loci. Thus, the subfamilies may have evolved at these loci following the spreading of the A subrepeats from the IGS to genomic regions outside the rDNA clusters.Electronic Supplementary Material Supplementary material is available in the online version of this article at Accession numbers: GenBank AY234364–AY234374. The monomer sequences and additional information about the family of IGS-like repeat S12 will also appear in the PlantSat database (Macas et al. 2002, ) under Accession name Vicia_sativa_IGS-like     

Development of ten microsatellite markers for <Emphasis Type="Italic">Quercus mongolica</Emphasis> var. <Emphasis Type="Italic">crispula</Emphasis> by database mining

Simple sequence repeats (SSRs) in the NCBI dbEST database were surveyed to identify potential SSR markers for Quercus mongolica. In total, 2,691 gene sequences, mainly from expressed sequence tags (ESTs) for Q. robur and Q. petraea had been registered. Twenty-two PCR primers were designed for SSRs in these sequences and screened for polymorphisms in 16 Q. mongolica trees. Ten loci were easily genotyped and showed polymorphism, with numbers of alleles and expected heterozygosity ranging from 3 to 15 and 0.28 to 0.94, respectively. These EST-SSR markers should be useful for studying the genetic diversity of Quercus species.     

Efficiency of microsatellite enrichment in<Emphasis Type="Italic">Prosopis chilensis</Emphasis> using magnetic capture

Microsatellites (i.e., simple sequence repeats [SSRs]) are highly variable genetic markers that are widely used at an intraspecific level in population genetic studies. Here we employed an enrichment strategy for microsatellite isolation by using microsatellite oligoprobes and magnetic capture of the fragments (Fischer and Bachmann, 1998) inProsopis chilensis (Mol.) Stuntz (Fabaceae). We analyzed the obtained level of enrichment by sequencing 120 enriched genomic fragments. A total of 521 SSR motives were detected. According to specific search criteria (SSR motifs ≥3 repeat units and ≥6 bp length), 95.8% of the clones contained SSR motifs. Of these, 7.8% showed homology to chloroplast sequences and 92.2% to nuclear sequences. When regarding only nuclear SSRs with 5 or more repeat units and a minimum length of 10 bp, the level of enrichment was 30.8%. A FASTA search against the European Molecular Biology Laboratory (EMBL) database univocally revealed 4 clones in transcribed regions, 102 clones in genomic regions with unknown function, and 9 clones in chloroplast regions. Among the loci with longer repeat units (≥10 bp, ≥5 repeat units), 3 were in transcribed regions and 65 were in other genomic regions. We discuss the applicability of these markers for population genetic studies.     

Structure and organization of the rDNA intergenic spacer region in <Emphasis Type="Italic">Pythium ultimum</Emphasis>

Nucleotide sequences of the rDNA intergenic spacer (IGS) region in Pythium ultimum were determined in 16 clones obtained from three isolates differing in production of sexual organs. Several sequences with different lengths were detected in each isolate, showing heterogeneity in the IGS region. In addition, several tandem repeat regions were detected in all the clones. The sequences, length, and number of each copy largely varied among repeat regions. Length heterogeneity arose from the complex combination of the number of copy within the repeat regions. Furthermore, the nucleotide sequence of each copy and the number of repetition varied not only between isolates but also between clones from an isolate. Based on the sequence similarity and the number of copies in repeat regions, specific patterns different between homothallic P. ultimum and the Pythium group HS (hyphal swellings) were recognized in a few regions. These results suggest that these two groups have slight genetic differences in the IGS region, although the differences in most of the repeat regions were not enough to identify each group.     

The chloroplast genome sequence from <Emphasis Type="Italic">Eugenia uniflora</Emphasis>, a Myrtaceae from Neotropics

Eugenia uniflora is a plant native to tropical America that holds great ecological and economic importance. The complete chloroplast (cp) genome sequence of Eugenia uniflora, a member of the Neotropical Myrtaceae family, is reported here. The genome is 158,445 bp in length and exhibits a typical quadripartite structure of the large (LSC, 87,459 bp) and small (SSC, 18,318 bp) single-copy regions, separated by a pair of inverted repeats (IRs, 26,334 bp). It contains 111 unique genes, including 77 protein-coding genes, 30 tRNAs and 4 rRNAs. The genome structure, gene order, GC content and codon usage are similar to the typical angiosperm cp genomes. Comparison of the entire cp genomes of E. uniflora L. and three other Myrtaceae revealed an expansion of 43 bp in the intergenic spacer located between the IRA/large single-copy (LSC) border and the first gene of LSC region. Simple sequence repeat (SSR) analysis revealed that most SSRs are AT rich, which contribute to the overall AT richness of the cp genome. Additionally, fewer SSRs are distributed in the protein-coding sequences compared to the noncoding regions. Phylogenetic analysis among 58 species based on 57 cp genes demonstrated a closer relationship between E. uniflora L. and Syzygium cumini (L). Skeels compared to the Eucalyptus clade in the Myrtaceae family. The complete cp genome sequence of E. uniflora reported here has importance for population genetics, as well as phylogenetic and evolutionary studies in this species and other Myrtaceae species from Neotropical regions.     

Complete chloroplast genome sequence of <Emphasis Type="Italic">Capsicum</Emphasis><Emphasis Type="Italic">baccatum</Emphasis> var. <Emphasis Type="Italic">baccatum</Emphasis>

Molecular markers derived from the complete chloroplast genome can provide effective tools for species identification and phylogenetic resolution. Complete chloroplast (cp) genome sequences of Capsicum species have been reported. We herein report the complete chloroplast genome sequence of Capsicum baccatum var. baccatum, a wild Capsicum species. The total length of the chloroplast genome is 157,145 bp with 37.7 % overall GC content. One pair of inverted repeats, 25,910 bp in length, was separated by a small single-copy region (17,974 bp) and large single-copy region (87,351 bp). This region contains 86 protein-coding genes, 30 tRNA genes, 4 rRNA genes, and 11 genes contain one or two introns. Pair-wise alignments of chloroplast genome were performed for genome-wide comparison. Analysis revealed a total of 134 simple sequence repeat (SSR) motifs and 282 insertions or deletions variants in the C. baccatum var. baccatum cp genome. The types and abundances of repeat units in Capsicum species were relatively conserved, and these loci could be used in future studies to investigate and conserve the genetic diversity of the Capsicum species.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Sequence analysis of rDNA intergenic spacer (IGS) of <Emphasis Type="Italic">Porphyra haitanensis</Emphasis>

The intergenic spacer region (IGS) has been used for the first time to analyze the genetic variability of Porphyra haitanensis from different areas. In order to determine that whether the IGS sequences could be used for classification and identification in intraspecies of Porphyra, the partial IGS sequences of cultivated strains of P. haitanensis (isolated from Putian-Fujian Province, Shantou-Guangdong Province and Ningbo-Zhejiang Province), were amplified, sequenced and analyzed. The sequence analysis indicated that the partial IGS sequences from the three stains were the external transcribed spacers (ETS) of 3′ end of the IGS gene. In the three stains, the length of IGS sequences ranged from 1,085 to 1,100 bp and the G + C content varied from 50.88% to 51.27%. There were 55 variable sites which occupied approximately 5% of the ETS sequences. Similarity analysis and multisequencing alignment of sequences indicated that the partial IGS sequences of the three stains of P. haitanensis had notable variabilities. Therefore, the IGS sequence could be used as the critical genetic marker in intraspecies of P. haitanensis. Furthermore, IGS sequence analysis will be a powerful tool for genetic diversity and classification in intraspecies of other Porphyra species.     

Map and analysis of microsatellites in the genome of <Emphasis Type="Italic">Populus</Emphasis>: The first sequenced perennial plant

We mapped and analyzed the microsatellites throughout 284295605 base pairs of the unambiguously assembled sequence scaffolds along 19 chromosomes of the haploid poplar genome. Totally, we found 150985 SSRs with repeat unit lengths between 2 and 5 bp. The established microsatellite physical map demonstrated that SSRs were distributed relatively evenly across the genome of Populus. On average, These SSRs occurred every 1883 bp within the poplar genome and the SSR densities in intergenic regions, introns, exons and UTRs were 85.4%, 10.7%, 2.7% and 1.2%, respectively. We took di-, tri-, tetra-and pentamers as the four classes of repeat units and found that the density of each class of SSRs decreased with the repeat unit lengths except for the tetranucleotide repeats. It was noteworthy that the length diversification of microsatellite sequences was negatively correlated with their repeat unit length and the SSRs with shorter repeat units gained repeats faster than the SSRs with longer repeat units. We also found that the GC content of poplar sequence significantly correlated with densities of SSRs with uneven repeat unit lengths (tri-and penta-), but had no significant correlation with densities of SSRs with even repeat unit lengths (di-and tetra-). In poplar genome, there were evidences that the occurrence of different microsatellites was under selection and the GC content in SSR sequences was found to significantly relate to the functional importance of microsatellites.     

The Complete Chloroplast Genome of <Emphasis Type="Italic">Coix lacryma-jobi</Emphasis> and a Comparative Molecular Evolutionary Analysis of Plastomes in Cereals

Graminoid molecular evolution was investigated by chloroplast genome (plastome) scale analyses. A complete plastome from Coix lacryma-jobi (Poaceae) and a draft plastome from Joinvillea plicata (Joinvilleaceae) were sequenced and analyzed. The draft plastome included conserved protein-coding loci routinely analyzed in previous studies plus one additional locus of demonstrated phylogenetic utility. The methodological approach was to directly sequence overlapping amplicons from known plastome regions. Over 100 pairs of amplification and sequencing primers were designed and positioned to flank overlapping 1,200-base pair fragments around the entire plastome. Newly determined sequences were analyzed with published plastomes from representatives of Panicoideae, Ehrhartoideae, and Pooideae. Considerable variation was found for studies within the family and even within Andropogoneae. Readily interpreted mutation patterns were observed, such as small inversions in hairpin-loop regions and indels, which were common in intergenic spacers. Maximum or near-maximum bootstrap support was observed in all analyses resolving relationships between subfamilies. However, the addition of characters from noncoding regions increased the number of parsimony-informative characters and lengthened short internal branches (Andropogoneae), better defining intergeneric relationships. Thus, characters in complete plastomes can be used over a wide scope of phylogenetic studies.     

<Emphasis Type="Italic">Sr33</Emphasis> and <Emphasis Type="Italic">Sr35</Emphasis> gene homolog identification in genomes of cereals related to <Emphasis Type="Italic">Aegilops tauschii</Emphasis> and <Emphasis Type="Italic">Triticum monococcum</Emphasis>

Using bioinformatics analysis, the homologs of genes Sr33 and Sr35 were identified in the genomes of Triticum aestivum, Hordeum vulgare, and Triticum urartu. It is known that these genes confer resistance to highly virulent wheat stem rust races (Ug99). To identify amino acid sites important for this resistance, the found homologs were compared with the Sr33 and Sr35 protein sequences. It was found that sequences S5DMA6 and E9P785 are the closest homologs of protein RGAle, a Sr33 gene product, and sequences M7YFA9 (CNL-C) and F2E9R2 are homologs of protein CNL9, a Sr35 gene product. It is assumed that the homologs of genes Sr33 and Sr35, which were obtained from the wild relatives of wheat and barley, can confer resistance to various forms of stem rust and can be used in the future breeding programs aimed at improvement of national wheat varieties.     

Complete Chloroplast Genome Sequence of <Emphasis Type="Italic">Glycine max</Emphasis> and Comparative Analyses with other Legume Genomes

Lack of complete chloroplast genome sequences is still one of the major limitations to extending chloroplast genetic engineering technology to useful crops. Therefore, we sequenced the soybean chloroplast genome and compared it to the other completely sequenced legumes, Lotus and Medicago. The chloroplast genome of Glycine is 152,218 basepairs (bp) in length, including a pair of inverted repeats of 25,574 bp of identical sequence separated by a small single copy region of 17,895 bp and a large single copy region of 83,175 bp. The genome contains 111 unique genes, and 19 of these are duplicated in the inverted repeat (IR). Comparisons of Glycine, Lotus and Medicago confirm the organization of legume chloroplast genomes based on previous studies. Gene content of the three legumes is nearly identical. The rpl22 gene is missing from all three legumes, and Medicago is missing rps16 and one copy of the IR. Gene order in Glycine, Lotus, and Medicago differs from the usual gene order for angiosperm chloroplast genomes by the presence of a single, large inversion of 51 kilobases (kb). Detailed analyses of repeated sequences indicate that many of the Glycine repeats that are located in the intergenic spacer regions and introns occur in the same location in the other legumes and in Arabidopsis, suggesting that they may play some functional role. The presence of small repeats of psbA and rbcL in legumes that have lost one copy of the IR indicate that this loss has only occurred once during the evolutionary history of legumes.