Yong Qiang Gu

BatchPrimer3: A high throughput web application for PCR and sequencing primer design

BackgroundMicrosatellite (simple sequence repeat – SSR) and single nucleotide polymorphism (SNP) markers are two types of important genetic markers useful in genetic mapping and genotyping. Often, large-scale genomic research projects require high-throughput computer-assisted primer design. Numerous such web-based or standard-alone programs for PCR primer design are available but vary in quality and functionality. In particular, most programs lack batch primer design capability. Such a high-throughput software tool for designing SSR flanking primers and SNP genotyping primers is increasingly demanded.ResultsA new web primer design program, BatchPrimer3, is developed based on Primer3. BatchPrimer3 adopted the Primer3 core program as a major primer design engine to choose the best primer pairs. A new score-based primer picking module is incorporated into BatchPrimer3 and used to pick position-restricted primers. BatchPrimer3 v1.0 implements several types of primer designs including generic primers, SSR primers together with SSR detection, and SNP genotyping primers (including single-base extension primers, allele-specific primers, and tetra-primers for tetra-primer ARMS PCR), as well as DNA sequencing primers. DNA sequences in FASTA format can be batch read into the program. The basic information of input sequences, as a reference of parameter setting of primer design, can be obtained by pre-analysis of sequences. The input sequences can be pre-processed and masked to exclude and/or include specific regions, or set targets for different primer design purposes as in Primer3Web and primer3Plus. A tab-delimited or Excel-formatted primer output also greatly facilitates the subsequent primer-ordering process. Thousands of primers, including wheat conserved intron-flanking primers, wheat genome-specific SNP genotyping primers, and Brachypodium SSR flanking primers in several genome projects have been designed using the program and validated in several laboratories.ConclusionBatchPrimer3 is a comprehensive web primer design program to develop different types of primers in a high-throughput manner. Additional methods of primer design can be easily integrated into future versions of BatchPrimer3. The program with source code and thousands of PCR and sequencing primers designed for wheat and Brachypodium are accessible at http://wheat.pw.usda.gov/demos/BatchPrimer3/.

Genome comparisons reveal a dominant mechanism of chromosome number reduction in grasses and accelerated genome evolution in Triticeae

Single-nucleotide polymorphism was used in the construction of an expressed sequence tag map of Aegilops tauschii, the diploid source of the wheat D genome. Comparisons of the map with the rice and sorghum genome sequences revealed 50 inversions and translocations; 2, 8, and 40 were assigned respectively to the rice, sorghum, and Ae. tauschii lineages, showing greatly accelerated genome evolution in the large Triticeae genomes. The reduction of the basic chromosome number from 12 to 7 in the Triticeae has taken place by a process during which an entire chromosome is inserted by its telomeres into a break in the centromeric region of another chromosome. The original centromere–telomere polarity of the chromosome arms is maintained in the new chromosome. An intrachromosomal telomere–telomere fusion resulting in a pericentric translocation of a chromosome segment or an entire arm accompanied or preceded the chromosome insertion in some instances. Insertional dysploidy has been recorded in three grass subfamilies and appears to be the dominant mechanism of basic chromosome number reduction in grasses. A total of 64% and 66% of Ae. tauschii genes were syntenic with sorghum and rice genes, respectively. Synteny was reduced in the vicinity of the termini of modern Ae. tauschii chromosomes but not in the vicinity of the ancient termini embedded in the Ae. tauschii chromosomes, suggesting that the dependence of synteny erosion on gene location along the centromere–telomere axis either evolved recently in the Triticeae phylogenetic lineage or its evolution was recently accelerated.

Rapid Genome Evolution Revealed by Comparative Sequence Analysis of Orthologous Regions from Four Triticeae Genomes

Bread wheat (Triticum aestivum) is an allohexaploid species, consisting of three subgenomes (A, B, and D). To study the molecular evolution of these closely related genomes, we compared the sequence of a 307-kb physical contig covering the high molecular weight (HMW)-glutenin locus from the A genome of durum wheat (Triticum turgidum, AABB) with the orthologous regions from the B genome of the same wheat and the D genome of the diploid wheat Aegilops tauschii (Anderson et al., 2003; Kong et al., 2004). Although gene colinearity appears to be retained, four out of six genes including the two paralogous HMW-glutenin genes are disrupted in the orthologous region of the A genome. Mechanisms involved in gene disruption in the A genome include retroelement insertions, sequence deletions, and mutations causing in-frame stop codons in the coding sequences. Comparative sequence analysis also revealed that sequences in the colinear intergenic regions of these different genomes were generally not conserved. The rapid genome evolution in these regions is attributable mainly to the large number of retrotransposon insertions that occurred after the divergence of the three wheat genomes. Our comparative studies indicate that the B genome diverged prior to the separation of the A and D genomes. Furthermore, sequence comparison of two distinct types of allelic variations at the HMW-glutenin loci in the A genomes of different hexaploid wheat cultivars with the A genome locus of durum wheat indicates that hexaploid wheat may have more than one tetraploid ancestor.

OrthoVenn: a web server for genome wide comparison and annotation of orthologous clusters across multiple species

Genome wide analysis of orthologous clusters is an important component of comparative genomics studies. Identifying the overlap among orthologous clusters can enable us to elucidate the function and evolution of proteins across multiple species. Here, we report a web platform named OrthoVenn that is useful for genome wide comparisons and visualization of orthologous clusters. OrthoVenn provides coverage of vertebrates, metazoa, protists, fungi, plants and bacteria for the comparison of orthologous clusters and also supports uploading of customized protein sequences from user-defined species. An interactive Venn diagram, summary counts, and functional summaries of the disjunction and intersection of clusters shared between species are displayed as part of the OrthoVenn result. OrthoVenn also includes in-depth views of the clusters using various sequence analysis tools. Furthermore, OrthoVenn identifies orthologous clusters of single copy genes and allows for a customized search of clusters of specific genes through key words or BLAST. OrthoVenn is an efficient and user-friendly web server freely accessible at http://probes.pw.usda.gov/OrthoVenn or http://aegilops.wheat.ucdavis.edu/OrthoVenn.

Types and Rates of Sequence Evolution at the High-Molecular-Weight Glutenin Locus in Hexaploid Wheat and Its Ancestral Genomes

The Glu-1 locus, encoding the high-molecular-weight glutenin protein subunits, controls bread-making quality in hexaploid wheat (Triticum aestivum) and represents a recently evolved region unique to Triticeae genomes. To understand the molecular evolution of this locus region, three orthologous Glu-1 regions from the three subgenomes of a single hexaploid wheat species were sequenced, totaling 729 kb of sequence. Comparing each Glu-1 region with its corresponding homologous region from the D genome of diploid wheat, Aegilops tauschii, and the A and B genomes of tetraploid wheat, Triticum turgidum, revealed that, in addition to the conservation of microsynteny in the genic regions, sequences in the intergenic regions, composed of blocks of nested retroelements, are also generally conserved, although a few nonshared retroelements that differentiate the homologous Glu-1 regions were detected in each pair of the A and D genomes. Analysis of the indel frequency and the rate of nucleotide substitution, which represent the most frequent types of sequence changes in the Glu-1 regions, demonstrated that the two A genomes are significantly more divergent than the two B genomes, further supporting the hypothesis that hexaploid wheat may have more than one tetraploid ancestor.

Dynamics of the evolution of orthologous and paralogous portions of a complex locus region in two genomes of allopolyploid wheat

Two overlapping bacterial artificial chromosome (BAC) clones from the B genome of the tetraploid wheat Triticum turgidum were identified, each of which contains one of the two high-molecular-weight (HMW) glutenin genes, comprising the complex Glu-B1 locus. The complete sequence (285 506 bp of DNA) of this chromosomal region was determined. The two paralogous x-type (Glu-1-1) and y-type (Glu-1-2) HMW-glutenin genes of the complex Glu-B1 locus were found to be separated by ca. 168 000 bp instead of the 51 000 bp separation previously reported for the orthologous Glu-D1 locus of Aegilops tauschii, the D-genome donor of hexaploid wheat. This difference in intergene spacing is due almost entirely to be the insertion of clusters of nested retrotransposons. Otherwise, the orientation and order of the HMW glutenins and adjacent genes were identical in the two genomes. A comparison of these orthologous regions indicates modes and patterns of sequence divergence, with implications for the overall Triticeae genome structure and evolution. A duplicate globulin gene, found 5′ of each HMW-glutenin gene, assists to tentatively define the original duplication event leading to the paralogous x- and y-type HMW-glutenin genes. The intergenic regions of the two loci are composed of different patterns and classes of retrotransposons, indicating that insertion times of these retroelements were after the divergence of the two wheat genomes. In addition, a putative receptor kinase gene near the y-type HMW-glutenin gene at the Glu-B1 locus is likely active as it matches recently reported ESTs from germinating barley endosperm. The presence of four genes represented only in the Triticeae endosperm ESTs suggests an endosperm-specific chromosome domain.

Genomic organization of the complex α-gliadin gene loci in wheat

To better understand the molecular evolution of the large α-gliadin gene family, a half-million bacterial artificial chromosome (BAC) library clones from tetraploid durum wheat, Triticum turgidum ssp. durum (2n=4x=28, genome AB), were screened for large genomic segments carrying the α-gliadin genes of the Gli-2 loci on the group 6 homoeologous chromosomes. The resulting 220 positive BAC clones—each containing between one and four copies of α-gliadin sequences—were fingerprinted for contig assembly to produce contiguous chromosomal regions covering the Gli-2 loci. While contigs consisting of as many as 21 BAC clones and containing up to 17 α-gliadin genes were formed, many BAC clones remained as singletons. The accuracy of the order of BAC clones in the contigs was verified by Southern hybridization analysis of the BAC fingerprints using an α-gliadin probe. These results indicate that α-gliadin genes are not evenly dispersed in the Gli-2 locus regions. Hybridization of these BACs with probes for long terminal repeat retrotransposons was used to determine the abundance and distribution of repetitive DNA in this region. Sequencing of BAC ends indicated that 70% of the sequences were significantly similar to different classes of retrotransposons, suggesting that these elements are abundant in this region. Several mechanisms underlying the dynamic evolution of the Gli-2 loci are discussed.

Rapid evolution and complex structural organization in genomic regions harboring multiple prolamin genes in the polyploid wheat genome.

Genes encoding wheat prolamins belong to complicated multi-gene families in the wheat genome. To understand the structural complexity of storage protein loci, we sequenced and analyzed orthologous regions containing both gliadin and LMW-glutenin genes from the A and B genomes of a tetraploid wheat species, Triticum turgidum ssp. durum. Despite their physical proximity to one another, the gliadin genes and LMW-glutenin genes are organized quite differently. The gliadin genes are found to be more clustered than the LMW-glutenin genes which are separated from each other by much larger distances. The separation of the LMW-glutenin genes is the result of both the insertion of large blocks of repetitive DNA owing to the rapid amplification of retrotransposons and the presence of genetic loci interspersed between them. Sequence comparisons of the orthologous regions reveal that gene movement could be one of the major factors contributing to the violation of microcolinearity between the homoeologous A and B genomes in wheat. The rapid sequence rearrangements and differential insertion of repetitive DNA has caused the gene islands to be not conserved in compared regions. In addition, we demonstrated that the i-type LMW-glutenin originated from a deletion of 33-bps in the 5′ coding region of the m-type gene. Our results show that multiple rounds of segmental duplication of prolamin genes have driven the amplification of the ω-gliadin genes in the region; such segmental duplication could greatly increase the repetitive DNA content in the genome depending on the amount of repetitive DNA present in the original duplicate region.

Gene Space Dynamics During the Evolution of Aegilops tauschii, Brachypodium distachyon, Oryza sativa, and Sorghum bicolor Genomes

Nine different regions totaling 9.7 Mb of the 4.02 Gb Aegilops tauschii genome were sequenced using the Sanger sequencing technology and compared with orthologous Brachypodium distachyon, Oryza sativa (rice), and Sorghum bicolor (sorghum) genomic sequences. The ancestral gene content in these regions was inferred and used to estimate gene deletion and gene duplication rates along each branch of the phylogenetic tree relating the four species. The total gene number in the extant Ae. tauschii genome was estimated to be 36,371. The gene deletion and gene duplication rates and total gene numbers in the four genomes were used to estimate the total gene number in each node of the phylogenetic tree. The common ancestor of the Brachypodieae and Triticeae lineages was estimated to have had 28,558 genes, and the common ancestor of the Panicoideae, Ehrhartoideae, and Pooideae subfamilies was estimated to have had 27,152 or 28,350 genes, depending on the ancestral gene scenario. Relative to the Brachypodieae and Triticeae common ancestor, the gene number was reduced in B. distachyon by 3,026 genes and increased in Ae. tauschii by 7,813 genes. The sum of gene deletion and gene duplication rates, which reflects the rate of gene synteny loss, was correlated with the rate of structural chromosome rearrangements and was highest in the Ae. tauschii lineage and lowest in the rice lineage. The high rate of gene space evolution in the Ae. tauschii lineage accounts for the fact that, contrary to the expectations, the level of synteny between the phylogenetically more related Ae. tauschii and B. distachyon genomes is similar to the level of synteny between the Ae. tauschii genome and the genomes of the less related rice and sorghum. The ratio of gene duplication to gene deletion rates in these four grass species closely parallels both the total number of genes in a species and the overall genome size. Because the overall genome size is to a large extent a function of the repeated sequence content in a genome, we suggest that the amount and activity of repeated sequences are important factors determining the number of genes in a genome.

PIECE: a database for plant gene structure comparison and evolution

Gene families often show degrees of differences in terms of exon–intron structures depending on their distinct evolutionary histories. Comparative analysis of gene structures is important for understanding their evolutionary and functional relationships within plant species. Here, we present a comparative genomics database named PIECE (http://wheat.pw.usda.gov/piece) for Plant Intron and Exon Comparison and Evolution studies. The database contains all the annotated genes extracted from 25 sequenced plant genomes. These genes were classified based on Pfam motifs. Phylogenetic trees were pre-constructed for each gene category. PIECE provides a user-friendly interface for different types of searches and a graphical viewer for displaying a gene structure pattern diagram linked to the resulting bootstrapped dendrogram for each gene family. The gene structure evolution of orthologous gene groups was determined using the GLOOME, Exalign and GECA software programs that can be accessed within the database. PIECE also provides a web server version of the software, GSDraw, for drawing schematic diagrams of gene structures. PIECE is a powerful tool for comparing gene sequences and provides valuable insights into the evolution of gene structure in plant genomes.