Amplification of sequence tagged sites in five avian species using heterologous oligonucleotides

Short of a complete genomic DNA sequence, sequence tagged sites (STSs) have emerged as major genomic reagents for the genetic analysis of little-studied ecologically and agriculturally important organisms. Here, we report STS developed for the turkey (Meleagris gallopavo), guinea fowl (Numidea meleagris), Japanese quail (Coturnix coturnix) and pigeon using primers specific for reference DNA sequences of two chicken (Gallus gallus) genes, aggrecan (agc1) and type X collagen (col10). Additional STSs were also developed for turkey, quail and chicken using primers specific for the human apobec-1 gene. The total length of the STSs developed was 5990, 2522, 4127, 1539 and 6600 bp for the turkey, guinea fowl, Japanese quail, pigeon and chicken, respectively. Based on splice site consensus GT and AG sequences, four of the seven agc1-based chicken STS appear to contain introns. The human gene-based STSs showed no significant sequence identity with the reference GenBank sequences. Maximum likelihood, maximum parsimony and neighbour-joining analysis of an agc1-based STS that was common to all five species showed phylogenetic relationships consistent with those previously defined using mitochondria DNA sequences and nuclear gene restriction maps. Additionally, several putative single nucleotide polymorphisms (SNPs) were detected within the STSs, including eight in the turkey, two in the quail, and two in the chicken when multiple sequences were evaluated from each species. This report describes new STSs that are resources for genetic and physical mapping and genome analysis within and among avian species. These resources should further aid in our understanding of the biology of agriculturally important but little-studied guinea fowl and turkey. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transfer of sequence tagged site PCR markers between wheat and barley.

Transfer of mapping information between related species has facilitated the development of restriction fragment length polymorphism (RFLP) maps in the cereals. Sequence tagged site (STS) primer sets for use in the polymerase chain reaction may be developed from mapped RFLP clones. For this study, we mapped 97 STS primer sets to chromosomes in wheat and barley to determine the potential transferability of the primer sets and the degree of correspondence between RFLP and STS locations. STS products mapped to the same chromosome group in wheat and barley 75% of the time. RFLP location predicted STS location 69% of the time in wheat and 56% of the time in barley. Southern hybridizations showed that most primer sets amplified sequences homologous to the RFLP clone, although additional sequences were often amplified that did not hybridize to the RFLP clone. Nontarget sequences were often amplified when primer sets were transferred across species. In general, results suggest a good probability of success in transferring STSs between wheat and barley, and that RFLP location can be used to predict STS location. However, transferability of STSs cannot be assumed, suggesting a need for recombinational mapping of STS markers in each species as new primer sets are developed. Key words : sequence tagged sites, PCR, wheat, barley.     

Sequence specificity of tRNA-modifying enzymes: An analysis of 258 tRNA sequences

The specificity and recognition of tRNA-modifying enzymes may be accounted for in part by nucleotide sequences which are localized next to the modifiable nucleoside. In order to determine the sequence specificity of tRNA-modifying enzymes, we have surveyed 55 published tRNA sequences from Escherichia coli, Salmonella typhimurium and T4 phage. For each modified nucleoside, the nucleotide sequence surrounding the modification site was determined for all tRNAs known to contain the modified nucleoside. Subsequently all tRNAs not containing the modified nucleoside were examined for the absence of the putative recognition site. We present the detailed analysis of 12 modified nucleosides for which we found a strong correlation between the modified nucleoside and the local nucleotide sequence. This suggests that these sequences may be recognition sites for tRNA-modifying enzymes. For each of the 12 modified nucleosides we have indentified a recognition sequence present in the tRNA set containing the modification and not in the set without it. All 203 other published tRNA sequences were then examined to see if the sequence specificity rules apply to other organisms, including both prokaryotes and eukaryotes. In several cases a good adherence was found, indicating conservation of the putative recognition sequences.     

Single-strand conformation polymorphism of sequence-tagged site markers based on partial sequences of cDNA clones in Cryptomeria japonica

Sixty-seven sequence-tagged site (STS) markers were identified from partial sequences of cDNA clones obtained from the inner bark of Cryptomeria japonica. Polymorphisms of the STSs were investigated for the parental clones of a mapping population, Haara and Kumotooshi, using both single-strand conformation polymorphism (SSCP) and sequencing analysis. Twenty-two STSs showed nucleotide differences between Haara and Kumotooshi, of which 19 STS differences were detectable under the electrophoresis conditions we used here. We also analyzed SSC-polymorphism in 10 additional clones derived from various Japanese regions to evaluate the usefulness of the STSs developed here among other populations of C. japonica. Twenty-five, about 40%, of the STSs showed polymorphism under selected electrophoresis conditions. The genotype segregation for 19 STSs was investigated among the Haara x Kumotooshi F(1) population, and these STS markers were mapped on a linkage map. SSCP analysis of STSs was efficient in terms of cost and time, and it allows detection of a sufficiently high proportion of polymorphisms to provide a convenient means for mapping of expressed sequences on a linkage map and for studying various aspects of population genetics.     

Evaluation of cleaved amplified polymorphic sequence markers for <Emphasis Type="Italic">Chamaecyparis obtusa</Emphasis> based on expressed sequence tag information from <Emphasis Type="Italic">Cryptomeria japonica</Emphasis>

We have developed and evaluated sequence-tagged site (STS) primers based on expressed sequence-tag information derived from sugi (Cryptomeria japonica) for use in hinoki (Chamaecyparis obtusa), a species that belongs to a different family (although it appears to be fairly closely related to sugi). Of the 417 C. japonica STS primer pairs we screened, 120 (~30%) were transferable and provided specific PCR amplification products from 16 C. obtusa plus trees. We used haploid megagametophytes to investigate the homology of 80 STS fragments between C. obtusa and C. japonica and to identify orthologous loci. Nearly 90% of the fragments showed high (>70%) degrees of similarity between the species, and 35 STSs indicated homology to entries with the same putative function in a public DNA database. Of the 120 STS fragments amplified, 72 showed restriction fragment length polymorphisms; in addition, the CC2430 primers detected amplicon length polymorphism. We assessed the inheritance pattern of 27 cleaved amplified polymorphic sequence markers, using 20 individuals from the segregation population. All the markers analyzed were consistent with the marker inheritance patterns obtained from the screening panel, and no markers (except CC2716) showed significant (P<0.01) deviation from the expected segregation ratio. In total, 136 polymorphic markers were developed using C. japonica-based STS primers without any sequence modification. In addition, the applicability of STS-based markers developed in one species to other species was found to closely reflect the evolutionary distance between the species, which is roughly concordant with the difference between their rbcL sequences. We plan to use these markers for genetic studies in C. obtusa. Most of the markers should also provide reliable anchor loci for comparative mapping studies of the C. obtusa and C. japonica genomes.     

Identification and characterization of an acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) gene from the microalga O. tauri

In order to identify novel genes encoding enzymes involved in the terminal step of triacylglycerol (TAG) formation, a database search was carried out in the genome of the unicellular photoautotrophic green alga Ostreococcus tauri. The search led to the identification of three putative type 2 acyl-CoA:diacylglycerol acyltransferase-like sequences (DGAT; EC 2.3.1.20), and revealed the absence of any homolog to type 1 or type 3 DGAT sequence in the genome of O. tauri. For two of the cDNA sequences (OtDGAT2A and B) enzyme activity was detected by heterologous expression in Saccharomyces cerevisiae mutant strains with impaired TAG metabolism. However, activity of OtDGAT2A was too low for further analysis. Analysis of their amino acid sequences showed that they share limited identity with other DGAT2 from different plant species, such as Ricinus communis and Vernicia fordii with ～25 to 30% identity. Lipid analysis of the mutant yeast cells revealed that OtDGAT2B showed broad substrate specificity accepting saturated as well as mono- and poly-unsaturated acyl-CoAs as substrates.     

Molecular Cloning,Expression and Characterization of <Emphasis Type="Italic">Oenococcus oeni</Emphasis> Priming Glycosyltransferases

Oenococcus oeni is the main bacterial species that drives malolactic fermentation in wine. Most O. oeni strains produce capsular exopolysaccharides (EPS) that may contribute to protect them in the wine hostile environment. In O. oeni genome sequences, several genes are predicted to encode priming glycosyltransferases (pGTs). These enzymes are essential for EPS formation as they catalyze the first biosynthetic step through the formation of a phosphoanhydride bond between a hexose-1-phosphate and a lipid carrier undecaprenyl phosphate. In many microorganisms, mutations abolishing the pGT activity also abolish the EPS formation. We first made an in silico analysis of all the genes encoding putative pGT over 50 distinct O. oeni genome sequences. Two polyisoprenyl-phosphate-hexose-1-phosphate transferases, WoaA and WobA, and a glycosyltransferase (It3) were particularly examined for their topology and amino acid sequence. Several isoforms of these enzymes were then expressed in E. coli, and their substrate specificity was examined in vitro. The substrate specificity varied depending on the protein isoform examined, and several mutations were shown to abolish WobA activity but not EPS synthesis. Further analysis of woaA and wobA gene expression levels suggests that WoaA could replace the deficient WobA and maintain EPS formation.     

Microsatellite repeats in grapevine reveal DNA polymorphisms when analysed as sequence-tagged sites (STSs)

Microsatellite repeat sequences were investigated as sequenced-tagged site (STS) DNA markers to determine the potential for genetic analysis of the grapevine genome. The PCR-generated markers detect codominant alleles at a single locus or site in the genome. The marker type is very informative detecting high heterozygosity (69%–88%) within individual grapevine cultivars and high genetic variation between cultivars, making it a useful marker type for plant genome mapping and genome typing. For five loci a screening of 26 V. vinifera cultivars found 13, 12, 8, 5, and 4 different length alleles respectively with some alleles more common than others. The genomic DNA sequences surrounding microsatellite sequences were conserved within the genus permitting STS primers to amplify STSs from other Vitis species. These Vitis species were found to have some unique alleles not present in V. vinifera.     

A Sequence-Tagged Site Map of Human Chromosome 11

We report the construction of 370 sequence-tagged sites (STSs) that are detectable by PCR amplification under sets of standardized conditions and that have been regionally mapped to human chromosome 11. DNA sequences were determined by sequencing directly from cosmid templates using primers complementary to T3 and T7 promoters present in the cloning vector. Oligonucleotide PCR primers were predicted by computer and tested using a battery of genomic DNAs. Cosmids were regionally localized on chromosome 11 by using fluorescence in situ hybridization or by analyzing a somatic cell hybrid panel. Additional STSs corresponding to known genes and markers on chromosome 11 were also produced under the same series of standardized conditions. The resulting STSs provide uniform coverage of chromosome 11 with an average spacing of 340 kb. The DNA sequence determined for use in STS production corresponds to about 0.1% (116 kb) of chromosome 11 and has been analyzed for the presence of repetitive sequences, similarities to known genes and motifs, and possible exons. Computer analysis of this sequence has identified and therefore mapped at least eight new genes on chromosome 11.     

Genetic variation of petaloid male-sterile cytoplasm of carrots revealed by sequence-tagged sites (STSs)

The mitochondrial DNA of various carrot lines was characterized by random amplified polymorphic DNA (RAPD) analysis, and six sequence-tagged sites (STSs) led to identification of the petaloid type of cytoplasmic male sterility (CMS). Using six STS primer combinations, we were able to classify five CMS lines into two groups and eight fertile carrots into six groups. Both the STS1 and the STS4 primer combinations differentiated CMS cytoplasms from the fertile cytoplasms, and the STS2 primer combination revealed two different types of CMS cytoplasms – of Wisconsin Wild and Cornell origins. Cybrid carrot lines with petaloid flowers which had been obtained by asymmetric cell fusion could also be separated from fertile cybrids by the STS1 primer combination. The STS1 fragment contained a homologous sequence with the orfB gene. DNA gel blot analysis indicated that homologous regions to the STS1 fragment existed in fertile types as well as the CMS types, although the restriction fragment size patterns differed. These observations demonstrate that rearrangements involving this region occurred in the mitochondrial genome. The STS4 fragment had a more complicated gene structure, including retrotransposon-like sequences and small segments of chloroplast genome. Received: 10 September 1998 / Accepted: 24 February 1999     

Genome-wide discovery of DNA polymorphism in Brassica rapa

Single nucleotide polymorphisms (SNPs) and/or insertion/deletions (InDels) are frequent sequence variations in the plant genome, which can be developed as molecular markers for genetic studies on crop improvement. The ongoing Brassica rapa genome sequencing project has generated vast amounts of sequence data useful in genetic research. Here, we report a genome-wide survey of DNA polymorphisms in the B. rapa genome based on the 557 bacterial artificial clone sequences of B. rapa ssp. pekinensis cv. Chiifu. We identified and characterized 21,311 SNPs and 6,753 InDels in the gene space of the B. rapa genome by re-sequencing 1,398 sequence-tagged sites (STSs) in eight genotypes. Comparison of our findings with a B. rapa genetic linkage map confirmed that STS loci were distributed randomly over the B. rapa whole genome. In the 1.4 Mb of aligned sequences, mean nucleotide polymorphism and diversity were θ = 0.00890 and π = 0.00917, respectively. Additionally, the nucleotide diversity in introns was almost three times greater than that in exons, and the frequency of observed InDel was almost 17 times higher in introns than in exons. Information regarding SNPs/InDels obtained here will provide an important resource for genetic studies and breeding programs of B. rapa.     

Proteomics and metabolomics analyses reveal the cucurbit sieve tube system as a complex metabolic space

The plant vascular system, and specifically the phloem, plays a pivotal role in allocation of fixed carbon to developing sink organs. Although the processes involved in loading and unloading of sugars and amino acids are well characterized, little information is available regarding the nature of other metabolites in the sieve tube system (STS) at specific sites along the pathway. Here, we elucidate spatial features of metabolite composition mapped with phloem enzymes along the cucurbit STS. Phloem sap (PS) was collected from the loading (source), unloading (apical sink region) and shoot–root junction regions of cucumber, watermelon and pumpkin. Our PS analyses revealed significant differences in the metabolic and proteomic profiles both along the source–sink pathway and between the STSs of these three cucurbits. In addition, metabolite profiles established for PS and vascular tissue indicated the presence of distinct compositions, consistent with the operation of the STS as a unique symplasmic domain. In this regard, at various locations along the STS we could map metabolites and their related enzymes to specific metabolic pathways. These findings are discussed with regard to the function of the STS as a unique and highly complex metabolic space within the plant vascular system.     

High-throughput genotyping in citrus accessions using an SNP genotyping array

We developed a 384 multiplexed SNP array, named CitSGA-1, for the genotyping of Citrus cultivars, and evaluated the performance and reliability of the genotyping. SNPs were surveyed by direct sequence comparison of the sequence tagged site (STS) fragment amplified from genomic DNA of cultivars representing the genetic diversity of citrus breeding in Japan. Among 1497 SNPs candidates, 384 SNPs for a high-throughput genotyping array were selected based on physical parameters of Illumina’s bead array criteria. The assay using CitSGA-1 was applied to a hybrid population of 88 progeny and 103 citrus accessions for breeding in Japan, which resulted in 73,726 SNP calls. A total of 351 SNPs (91 %) could call different genotypes among the DNA samples, resulting in a success rate for the assay comparable to previously reported rates for other plant species. To confirm the reliability of SNP genotype calls, parentage analysis was applied, and it indicated that the number of reliable SNPs and corresponding STSs were 276 and 213, respectively. The multiplexed SNP genotyping array reported here will be useful for the efficient construction of linkage map, for the detection of markers for marker-assisted breeding, and for the identification of cultivars.     

Sequence haplotypes revealed by sequence-tagged site fine mapping of the Ror1 gene in the centromeric region of barley chromosome 1H

We describe the development of polymerase chain reaction-based, sequence-tagged site (STS) markers for fine mapping of the barley (Hordeum vulgare) Ror1 gene required for broad-spectrum resistance to powdery mildew (Blumeria graminis f. sp. hordei). After locating Ror1 to the centromeric region of barley chromosome 1H using a combined amplified fragment length polymorphism/restriction fragment-length polymorphism (RFLP) approach, sequences of RFLP probes from this chromosome region of barley and corresponding genome regions from the related grass species oat (Avena spp.), wheat, and Triticum monococcum were used to develop STS markers. Primers based on the RFLP probe sequences were used to polymerase chain reaction-amplify and directly sequence homologous DNA stretches from each of four parents that were used for mapping. Over 28,000 bp from 22 markers were compared. In addition to one insertion/deletion of at least 2.0 kb, 79 small unique sequence polymorphisms were observed, including 65 single nucleotide substitutions, two dinucleotide substitutions, 11 insertion/deletions, and one 5-bp/10-bp exchange. The frequency of polymorphism between any two barley lines ranged from 0.9 to 3.0 kb, and was greatest for comparisons involving an Ethiopian landrace. Haplotype structure was observed in the marker sequences over distances of several hundred basepairs. Polymorphisms in 16 STSs were used to generate genetic markers, scored by restriction enzyme digestion or by direct sequencing. Over 2,300 segregants from three populations were used in Ror1 linkage analysis, mapping Ror1 to a 0.2- to 0.5-cM marker interval. We discuss the implications of sequence haplotypes and STS markers for the generation of high-density maps in cereals.     

Distribution and evolution of circular miniproteins in flowering plants

Cyclotides are disulfide-rich miniproteins with the unique structural features of a circular backbone and knotted arrangement of three conserved disulfide bonds. Cyclotides have been found only in two plant families: in every analyzed species of the violet family (Violaceae) and in few species of the coffee family (Rubiaceae). In this study, we analyzed >200 Rubiaceae species and confirmed the presence of cyclotides in 22 species. Additionally, we analyzed >140 species in related plant families to Rubiaceae and Violaceae and report the occurrence of cyclotides in the Apocynaceae. We further report new cyclotide sequences that provide insights into the mechanistic basis of cyclotide evolution. On the basis of the phylogeny of cyclotide-bearing plants and the analysis of cyclotide precursor gene sequences, we hypothesize that cyclotide evolution occurred independently in various plant families after the divergence of Asterids and Rosids ( approximately 125 million years ago). This is strongly supported by recent findings on the in planta biosynthesis of cyclotides, which involves the serendipitous recruitment of ubiquitous proteolytic enzymes for cyclization. We further predict that the number of cyclotides within the Rubiaceae may exceed tens of thousands, potentially making cyclotides one of the largest protein families in the plant kingdom.     

Phylogenetic analysis of the UDP-glycosyltransferase multigene family of Arabidopsis thaliana

A class of UDP-glycosyltransferases (UGTs) defined by the presence of a C-terminal consensus sequence is found throughout the plant and animal kingdoms. Whereas mammalian enzymes use UDP-glucuronic acid, the plant enzymes typically use UDP-glucose in the transfer reactions. A diverse array of aglycones can be glucosylated by these UGTs. In plants, the aglycones include plant hormones, secondary metabolites involved in stress and defense responses, and xenobiotics such as herbicides. Glycosylation is known to regulate many properties of the aglycones such as their bioactivity, their solubility, and their transport properties within the cell and throughout the plant. As a means of providing a framework to start to understand the substrate specificities and structure-function relationships of plant UGTs, we have now applied a molecular phylogenetic analysis to the multigene family of 99 UGT sequences in Arabidopsis. We have determined the overall organization and evolutionary relationships among individual members with a surprisingly high degree of confidence. Through constructing a composite phylogenetic tree that also includes all of the additional plant UGTs with known catalytic activities, we can start to predict both the evolutionary history and substrate specificities of new sequences as they are identified. The tree already suggests that while the activities of some subgroups of the UGT family are highly conserved among different plant species, others subgroups shift substrate specificity with relative ease.     

Grass consensus STS markers: an efficient approach for detecting polymorphism in Lolium

For ryegrass and many forage crops, characterization of varieties is often difficult for two reasons: few of discriminant morphological traits and a great within-varieties variation. Futhermore, few molecular markers are publicly accessible. In this paper we describe two approaches for the development of 42 sequence-tagged-site (STS) markers. Firstly, 14 STS markers were developed from Lolium sequences found in data bases. Secondly, 28 STS markers were developed from sequences found in related species of Gramineae. Out of 42 STS markers developed, 85.8% yielded successfull amplification and 62% revealed a high level of polymorphism with an average of five alleles per locus. The analysis of amplicons reveals a high STS marker specificity, a high conservation in gene structure and a strong intron sequence homology between allelic forms. Moreover, the majority of the STS markers can be considered as "universal markers" because 81% of these STS markers amplified successfully across 20 related grass species. These results permit us to consider the use of these markers in synteny studies.Communicated by G. Wenzel     

Catalytic mechanism of ancestral L-lysine oxidase assigned by sequence data mining

A large number of protein sequences are registered in public databases such as PubMed. Functionally uncharacterized enzymes are included in these databases, some of which likely have potential for industrial applications. However, assignment of the enzymes remained difficult tasks for now. In this study, we assigned a total of 28 original sequences to uncharacterized enzymes in the FAD-dependent oxidase family expressed in some species of bacteria including Chryseobacterium, Flavobacterium, and Pedobactor. Progenitor sequence of the assigned 28 sequences was generated by ancestral sequence reconstruction, and the generated sequence exhibited L-lysine oxidase activity; thus, we named the enzyme AncLLysO. Crystal structures of ligand-free and ligand-bound forms of AncLLysO were determined, indicating that the enzyme recognizes L-Lys by hydrogen bond formation with R76 and E383. The binding of L-Lys to AncLLysO induced dynamic structural change at a plug loop formed by residues 251 to 254. Biochemical assays of AncLLysO variants revealed the functional importance of these substrate recognition residues and the plug loop. R76A and E383D variants were also observed to lose their activity, and the k_cat/K_m value of G251P and Y253A mutations were approximately 800- to 1800-fold lower than that of AncLLysO, despite the indirect interaction of the substrates with the mutated residues. Taken together, our data demonstrate that combinational approaches to sequence classification from database and ancestral sequence reconstruction may be effective not only to find new enzymes using databases of unknown sequences but also to elucidate their functions.