Genome-wide association analyses of leaf rust resistance in cultivated emmer wheat.

KEY MESSAGE: Fifteen and eleven loci, with most loci being novel, were identified to associate with seedling and adult resistances, respectively, to the durum-specific races of leaf rust pathogen in cultivated emmer. Leaf rust, caused by Puccinia triticina (Pt), constantly threatens durum (Triticum turgidum ssp. durum) and bread wheat (Triticum aestivum) production worldwide. A Pt race BBBQD detected in California in 2009 poses a potential threat to durum production in North America because resistance source to this race is rare in durum germplasm. To find new resistance sources, we assessed a panel of 180 cultivated emmer wheat (Triticum turgidum ssp. dicoccum) accessions for seedling resistance to BBBQD and for adult resistance to a mixture of durum-specific races BBBQJ, CCMSS, and MCDSS in the field, and genotyped the panel using genotype-by-sequencing (GBS) and the 9 K SNP (Single Nucleotide Polymorphism) Infinium array. The results showed 24 and nine accessions consistently exhibited seedling and adult resistance, respectively, with two accessions providing resistance at both stages. We performed genome-wide association studies using 46,383 GBS and 4,331 9 K SNP markers and identified 15 quantitative trait loci (QTL) for seedling resistance located mostly on chromosomes 2B and 6B, and 11 QTL for adult resistance on 2B, 3B and 6A. Of these QTL, one might be associated with leaf rust resistance (Lr) gene Lr53, and two with the QTL previously reported in durum or hexaploid wheat. The remaining QTL are potentially associated with new Lr genes. Further linkage analysis and gene cloning are necessary to identify the causal genes underlying these QTL. The emmer accessions with high levels of resistance will be useful for developing mapping populations and adapted durum germplasm and varieties with resistance to the durum-specific races.

A rare single nucleotide variant in Pm5e confers powdery mildew resistance in common wheat.

Powdery mildew poses severe threats to wheat production. The most sustainable way to control this disease is through planting resistant cultivars. We report the map-based cloning of the powdery mildew resistance allele Pm5e from a Chinese wheat landrace. We applied a two-step bulked segregant RNA sequencing (BSR-Seq) approach in developing tightly linked or co-segregating markers to Pm5e. The first BSR-Seq used phenotypically contrasting bulks of recombinant inbred lines (RILs) to identify Pm5e-linked markers. The second BSR-Seq utilized bulks of genetic recombinants screened from a fine-mapping population to precisely quantify the associated genomic variation in the mapping interval, and identified the Pm5e candidate genes. The function of Pm5e was validated by transgenic assay, loss-of-function mutants and haplotype association analysis. Pm5e encodes a nucleotide-binding domain leucine-rich-repeat-containing (NLR) protein. A rare nonsynonymous single nucleotide variant (SNV) within the C-terminal leucine rich repeat (LRR) domain is responsible for the gain of powdery mildew resistance function of Pm5e, an allele endemic to wheat landraces of Shaanxi province of China. Results from this study demonstrate the value of landraces in discovering useful genes for modern wheat breeding. The key SNV associated with powdery mildew resistance will be useful for marker-assisted selection of Pm5e in wheat breeding programs.

A rare gain of function mutation in a wheat tandem kinase confers resistance to powdery mildew.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most destructive diseases that pose a great threat to wheat production. Wheat landraces represent a rich source of powdery mildew resistance. Here, we report the map-based cloning of powdery mildew resistance gene Pm24 from Chinese wheat landrace Hulutou. It encodes a tandem kinase protein (TKP) with putative kinase-pseudokinase domains, designated WHEAT TANDEM KINASE 3 (WTK3). The resistance function of Pm24 was validated by transgenic assay, independent mutants, and allelic association analyses. Haplotype analysis revealed that a rare 6-bp natural deletion of lysine-glycine codons, endemic to wheat landraces of Shaanxi Province, China, in the kinase I domain (Kin I) of WTK3 is critical for the resistance function. Transgenic assay of WTK3 chimeric variants revealed that only the specific two amino acid deletion, rather than any of the single or more amino acid deletions, in the Kin I of WTK3 is responsible for gaining the resistance function of WTK3 against the Bgt fungus.

Cloning and characterization of the homoeologous genes for the Rec8-like meiotic cohesin in polyploid wheat.

BACKGROUND: Meiosis is a specialized cell division critical for gamete production in the sexual reproduction of eukaryotes. It ensures genome integrity and generates genetic variability as well. The Rec8-like cohesin is a cohesion protein essential for orderly chromosome segregation in meiotic cell division. The Rec8-like genes and cohesins have been cloned and characterized in diploid models, but not in polyploids. The present study aimed to clone the homoeologous genes (homoeoalleles) for Rec8-like cohesin in polyploid wheat, an important food crop for humans, and to characterize their structure and function under a polyploid condition. RESULTS: We cloned two Rec8-like homoeoalleles from tetraploid wheat (TtRec8-A1 and TtRec8-B1) and one from hexaploid wheat (TaRec8-D1), and performed expression and functional analyses of the homoeoalleles. Also, we identified other two Rec8 homoeoalleles in hexaploid wheat (TaRec8-A1 and TaRec8-B1) and the one in Aegilops tauschii (AetRec8-D1) by referencing the DNA sequences of the Rec8 homoeoalleles cloned in this study. The coding DNA sequences (CDS) of these six Rec8 homoeoalleles are all 1,827 bp in length, encoding 608 amino acids. They differed from each other primarily in introns although single nucleotide polymorphisms were detected in CDS. Substantial difference was observed between the homoeoalleles from the subgenome B (TtRec8-B1 and TaRec8-B1) and those from the subgenomes A and D (TtRec8-A1, TaRec8-A1, and TaRec8-D1). TtRec8-A1 expressed dominantly over TtRec8-B1, but comparably to TaRec8-D1, in polyploid wheat. In addition, we developed the antibody against wheat Rec8 and used the antibody to detect Rec8 cohesin in the Western blotting and subcellular localization analyses. CONCLUSIONS: The Rec8 homoeoalleles from the subgenomes A and D are transcriptionally more active than the one from the subgenome B in polyploid wheat. The structural variation and differential expression of the Rec8 homoeoalleles indicate a unique cross-genome coordination of the homoeologous genes in polyploid wheat, and imply the distinction of the wheat subgenome B from the subgenomes A and D in the origin and evolution.

Toward a better understanding of the genomic region harboring Fusarium head blight resistance QTL Qfhs.ndsu-3AS in durum wheat.

KEY MESSAGE: New molecular markers were developed and mapped to the FHB resistance QTL region in high resolution. Micro-collinearity of the QTL region with rice and Brachypodium was revealed for a better understanding of the genomic region. The wild emmer wheat (Triticum dicoccoides)-derived Fusarium head blight (FHB) resistance quantitative trait locus (QTL) Qfhs.ndsu-3AS previously mapped to the short arm of chromosome 3A (3AS) in a population of recombinant inbred chromosome lines (RICLs). This study aimed to attain a better understanding of the genomic region harboring Qfhs.ndsu-3AS and to improve the utility of the QTL in wheat breeding. Micro-collinearity of the QTL region with rice chromosome 1 and Brachypodium chromosome 2 was identified and used for marker development in saturation mapping. A total of 42 new EST-derived sequence tagged site (STS) and simple sequence repeat (SSR) markers were developed and mapped to the QTL and nearby regions on 3AS. Further comparative analysis revealed a complex collinearity of the 3AS genomic region with their collinear counterparts of rice and Brachypodium. Fine mapping of the QTL region resolved five co-segregating markers (Xwgc1186/Xwgc716/Xwgc1143/Xwgc501/Xwgc1204) into three distinct loci proximal to Xgwm2, a marker previously reported to be closely linked to the QTL. Four other markers (Xwgc1226, Xwgc510, Xwgc1296, and Xwgc1301) mapped farther proximal to the above markers in the QTL region with a higher resolution. Five homozygous recombinants with shortened T. dicoccoides chromosomal segments in the QTL region were recovered by molecular marker analysis and evaluated for FHB resistance. Qfhs.ndsu-3AS was positioned to a 5.2 cM interval flanked by the marker Xwgc501 and Xwgc510. The recombinants containing Qfhs.ndsu-3AS and new markers defining the QTL will facilitate utilization of this resistance source in wheat breeding.

Dynamic evolution of resistance gene analogs in the orthologous genomic regions of powdery mildew resistance gene MlIW170 in Triticum dicoccoides and Aegilops tauschii.

KEY MESSAGE: Rapid evolution of powdery mildew resistance gene MlIW170 orthologous genomic regions in wheat subgenomes. Wheat is one of the most important staple grain crops in the world and also an excellent model for plant ploidy evolution research with different ploidy levels from diploid to hexaploid. Powdery mildew disease caused by Blumeria graminis f.sp. tritici can result in significant loss in both grain yield and quality in wheat. In this study, the wheat powdery mildew resistance gene MlIW170 locus located at the Triticum dicoccoides chromosome 2B short arm was further characterized by constructing and sequencing a BAC-based physical map contig covering a 0.3 cM genetic distance region (880 kb) and developing additional markers to delineate the resistance gene within a 0.16 cM genetic interval (372 kb). Comparative analyses of the T. dicoccoides 2BS region with the orthologous Aegilops tauschii 2DS region showed great gene colinearity, including the structure organization of both types of RGA1/2-like and RPS2-like resistance genes. Comparative analyses with the orthologous regions from Brachypodium and rice genomes revealed considerable dynamic evolutionary changes that have re-shaped this MlIW170 region in the wheat genome, resulting in a high number of non-syntenic genes including resistance-related genes. This result might reflect the rapid evolution in R-gene regions. Phylogenetic analysis on these resistance-related gene sequences indicated the duplication of these genes in the MlIW170 region, occurred before the separation of the wheat B and D genomes.

Haplotype variation of Glu-D1 locus and the origin of Glu-D1d allele conferring superior end-use qualities in common wheat.

In higher plants, seed storage proteins (SSPs) are frequently expressed from complex gene families, and allelic variation of SSP genes often affects the quality traits of crops. In common wheat, the Glu-D1 locus, encoding 1Dx and 1Dy SSPs, has multiple alleles. The Glu-D1d allele frequently confers superior end-use qualities to commercial wheat varieties. Here, we studied the haplotype structure of Glu-D1 genomic region and the origin of Glu-D1d. Using seven diagnostic DNA markers, 12 Glu-D1 haplotypes were detected among common wheat, European spelt wheat (T. spelta, a primitive hexaploid relative of common wheat), and Aegilops tauschii (the D genome donor of hexaploid wheat). By comparatively analyzing Glu-D1 haplotypes and their associated 1Dx and 1Dy genes, we deduce that the haplotype carrying Glu-D1d was likely differentiated in the ancestral hexaploid wheat around 10,000 years ago, and was subsequently transmitted to domesticated common wheat and T. spelta. A group of relatively ancient Glu-D1 haplotypes was discovered in Ae. tauschii, which may serve for the evolution of other haplotypes. Moreover, a number of new Glu-D1d variants were found in T. spelta. The main steps in Glu-D1d differentiation are proposed. The implications of our work for enhancing the utility of Glu-D1d in wheat quality improvement and studying the SSP alleles in other crop species are discussed.

Genetic association of OPR genes with resistance to Hessian fly in hexaploid wheat.

BACKGROUND: Hessian fly (Mayetiola destructor) is one of the most destructive pests of wheat. The genes encoding 12-oxo-phytodienoic acid reductase (OPR) and lipoxygenase (LOX) play critical roles in insect resistance pathways in higher plants, but little is known about genes controlling resistance to Hessian fly in wheat. RESULTS: In this study, 154 F6:8 recombinant inbred lines (RILs) generated from a cross between two cultivars, 'Jagger' and '2174' of hexaploid wheat (2n = 6 × =42; AABBDD), were used to map genes associated with resistance to Hessian fly. Two QTLs were identified. The first one was a major QTL on chromosome 1A (QHf.osu-1A), which explained 70% of the total phenotypic variation. The resistant allele at this locus in cultivar 2174 could be orthologous to one or more of the previously mapped resistance genes (H9, H10, H11, H16, and H17) in tetraploid wheat. The second QTL was a minor QTL on chromosome 2A (QHf.osu-2A), which accounted for 18% of the total phenotypic variation. The resistant allele at this locus in 2174 is collinear to an Yr17-containing-fragment translocated from chromosome 2N of Triticum ventricosum (2n = 4 × =28; DDNN) in Jagger. Genetic mapping results showed that two OPR genes, TaOPR1-A and TaOPR2-A, were tightly associated with QHf.osu-1A and QHf.osu-2A, respectively. Another OPR gene and three LOX genes were mapped but not associated with Hessian fly resistance in the segregating population. CONCLUSIONS: This study has located two major QTLs/genes in bread wheat that can be directly used in wheat breeding programs and has also provided insights for the genetic association and disassociation of Hessian fly resistance with OPR and LOX genes in wheat.

A radiation hybrid map of chromosome 1D reveals synteny conservation at a wheat speciation locus.

The species cytoplasm specific (scs) genes affect nuclear-cytoplasmic interactions in interspecific hybrids. A radiation hybrid (RH) mapping population of 188 individuals was employed to refine the location of the scs (ae) locus on Triticum aestivum chromosome 1D. "Wheat Zapper," a comparative genomics tool, was used to predict synteny between wheat chromosome 1D, Oryza sativa, Brachypodium distachyon, and Sorghum bicolor. A total of 57 markers were developed based on synteny or literature and genotyped to produce a RH map spanning 205.2 cR. A test-cross methodology was devised for phenotyping of RH progenies, and through forward genetic, the scs (ae) locus was pinpointed to a 1.1 Mb-segment containing eight genes. Further, the high resolution provided by RH mapping, combined with chromosome-wise synteny analysis, located the ancestral point of fusion between the telomeric and centromeric repeats of two paleochromosomes that originated chromosome 1D. Also, it indicated that the centromere of this chromosome is likely the result of a neocentromerization event, rather than the conservation of an ancestral centromere as previously believed. Interestingly, location of scs locus in the vicinity of paleofusion is not associated with the expected disruption of synteny, but rather with a good degree of conservation across grass species. Indeed, these observations advocate the evolutionary importance of this locus as suggested by "Maan's scs hypothesis."

Complete chloroplast genome sequences of Mongolia medicine Artemisia frigida and phylogenetic relationships with other plants.

BACKGROUND: Artemisia frigida Willd. is an important Mongolian traditional medicinal plant with pharmacological functions of stanch and detumescence. However, there is little sequence and genomic information available for Artemisia frigida, which makes phylogenetic identification, evolutionary studies, and genetic improvement of its value very difficult. We report the complete chloroplast genome sequence of Artemisia frigida based on 454 pyrosequencing. METHODOLOGY/PRINCIPAL FINDINGS: The complete chloroplast genome of Artemisia frigida is 151,076 bp including a large single copy (LSC) region of 82,740 bp, a small single copy (SSC) region of 18,394 bp and a pair of inverted repeats (IRs) of 24,971 bp. The genome contains 114 unique genes and 18 duplicated genes. The chloroplast genome of Artemisia frigida contains a small 3.4 kb inversion within a large 23 kb inversion in the LSC region, a unique feature in Asteraceae. The gene order in the SSC region of Artemisia frigida is inverted compared with the other 6 Asteraceae species with the chloroplast genomes sequenced. This inversion is likely caused by an intramolecular recombination event only occurred in Artemisia frigida. The existence of rich SSR loci in the Artemisia frigida chloroplast genome provides a rare opportunity to study population genetics of this Mongolian medicinal plant. Phylogenetic analysis demonstrates a sister relationship between Artemisia frigida and four other species in Asteraceae, including Ageratina adenophora, Helianthus annuus, Guizotia abyssinica and Lactuca sativa, based on 61 protein-coding sequences. Furthermore, Artemisia frigida was placed in the tribe Anthemideae in the subfamily Asteroideae (Asteraceae) based on ndhF and trnL-F sequence comparisons. CONCLUSION: The chloroplast genome sequence of Artemisia frigida was assembled and analyzed in this study, representing the first plastid genome sequenced in the Anthemideae tribe. This complete chloroplast genome sequence will be useful for molecular ecology and molecular phylogeny studies within Artemisia species and also within the Asteraceae family.

Exploring the diploid wheat ancestral A genome through sequence comparison at the high-molecular-weight glutenin locus region.

The polyploid nature of hexaploid wheat (T. aestivum, AABBDD) often represents a great challenge in various aspects of research including genetic mapping, map-based cloning of important genes, and sequencing and accurately assembly of its genome. To explore the utility of ancestral diploid species of polyploid wheat, sequence variation of T. urartu (A(u)A(u)) was analyzed by comparing its 277-kb large genomic region carrying the important Glu-1 locus with the homologous regions from the A genomes of the diploid T. monococcum (A(m)A(m)), tetraploid T. turgidum (AABB), and hexaploid T. aestivum (AABBDD). Our results revealed that in addition to a high degree of the gene collinearity, nested retroelement structures were also considerably conserved among the A(u) genome and the A genomes in polyploid wheats, suggesting that the majority of the repetitive sequences in the A genomes of polyploid wheats originated from the diploid A(u) genome. The difference in the compared region between A(u) and A is mainly caused by four differential TE insertion and two deletion events between these genomes. The estimated divergence time of A genomes calculated on nucleotide substitution rate in both shared TEs and collinear genes further supports the closer evolutionary relationship of A to A(u) than to A(m). The structure conservation in the repetitive regions promoted us to develop repeat junction markers based on the A(u) sequence for mapping the A genome in hexaploid wheat. Eighty percent of these repeat junction markers were successfully mapped to the corresponding region in hexaploid wheat, suggesting that T. urartu could serve as a useful resource for developing molecular markers for genetic and breeding studies in hexaploid wheat.

Fine mapping of the Bsr1 barley stripe mosaic virus resistance gene in the model grass Brachypodium distachyon.

The ND18 strain of Barley stripe mosaic virus (BSMV) infects several lines of Brachypodium distachyon, a recently developed model system for genomics research in cereals. Among the inbred lines tested, Bd3-1 is highly resistant at 20 to 25 °C, whereas Bd21 is susceptible and infection results in an intense mosaic phenotype accompanied by high levels of replicating virus. We generated an F(6:7) recombinant inbred line (RIL) population from a cross between Bd3-1 and Bd21 and used the RILs, and an F(2) population of a second Bd21 × Bd3-1 cross to evaluate the inheritance of resistance. The results indicate that resistance segregates as expected for a single dominant gene, which we have designated Barley stripe mosaic virus resistance 1 (Bsr1). We constructed a genetic linkage map of the RIL population using SNP markers to map this gene to within 705 Kb of the distal end of the top of chromosome 3. Additional CAPS and Indel markers were used to fine map Bsr1 to a 23 Kb interval containing five putative genes. Our study demonstrates the power of using RILs to rapidly map the genetic determinants of BSMV resistance in Brachypodium. Moreover, the RILs and their associated genetic map, when combined with the complete genomic sequence of Brachypodium, provide new resources for genetic analyses of many other traits.

Conserved globulin gene across eight grass genomes identify fundamental units of the loci encoding seed storage proteins.

The wheat high molecular weight (HMW) glutenins are important seed storage proteins that determine bread-making quality in hexaploid wheat (Triticum aestivum). In this study, detailed comparative sequence analyses of large orthologous HMW glutenin genomic regions from eight grass species, representing a wide evolutionary history of grass genomes, reveal a number of lineage-specific sequence changes. These lineage-specific changes, which resulted in duplications, insertions, and deletions of genes, are the major forces disrupting gene colinearity among grass genomes. Our results indicate that the presence of the HMW glutenin gene in Triticeae genomes was caused by lineage-specific duplication of a globulin gene. This tandem duplication event is shared by Brachypodium and Triticeae genomes, but is absent in rice, maize, and sorghum, suggesting the duplication occurred after Brachypodium and Triticeae genomes diverged from the other grasses ~35 Ma ago. Aside from their physical location in tandem, the sequence similarity, expression pattern, and conserved cis-acting elements responsible for endosperm-specific expression further support the paralogous relationship between the HMW glutenin and globulin genes. While the duplicated copy in Brachypodium has apparently become nonfunctional, the duplicated copy in wheat has evolved to become the HMW glutenin gene by gaining a central prolamin repetitive domain.

Rapid development of PCR-based genome-specific repetitive DNA junction markers in wheat.

In hexaploid wheat (Triticum aestivum L.) (AABBDD, C=17 000 Mb), repeat DNA accounts for approximately 90% of the genome, of which transposable elements (TEs) constitute 60%-80%. Despite the dynamic evolution of TEs, our previous study indicated that the majority of TEs are conserved and collinear between the homologous wheat genomes, based on identical insertion patterns. In this study, we exploited the unique and abundant TE insertion junction regions identified from diploid Aegilops tauschii to develop genome-specific repeat DNA junction markers (RJM) for use in hexaploid wheat. In this study, both BAC end and random shotgun sequences were used to search for RJM. Of the 300 RJM primer pairs tested, 269 (90%) amplified single bands from diploid Ae. tauschii. Of these 269 primer pairs, 260 (97%) amplified hexaploid wheat and 9 (3%) amplified Ae. tauschii only. Among the RJM primers that amplified hexaploid wheat, 88% were successfully assigned to individual chromosomes of the hexaploid D genome. Among the 38 RJM primers mapped on chromosome 6D, 31 (82%) were unambiguously mapped to delineated bins of the chromosome using various wheat deletion lines. Our results suggest that the unique RJM derived from the diploid D genome could facilitate genetic, physical, and radiation mapping of the hexaploid wheat D genome.

Saturation and comparative mapping of the genomic region harboring Hessian fly resistance gene H26 in wheat.

Resistance gene H26, derived from Aegilops tauschii Coss., is one of the most effective R genes against the Hessian fly [Mayetiola destructor (Say)], an important pest of wheat (Triticum aestivum L.). Using a limited number of PCR-based molecular markers a previous study mapped H26 to the wheat chromosomal deletion bin 3DL3-0.81-1.00. The objectives of this study were to saturate the chromosomal region harboring H26 with newly developed PCR-based markers and to investigate the collinearity of this wheat chromosomal region with rice (Oryza sativa L.) and Brachypodium distachyon genome. A population of 96 F(2) individuals segregating at the H26 gene locus was used for saturation mapping. All wheat ESTs assigned to the deletion bin 3DL3-0.81-1.00 were used to design STS (sequence tagged site) primers. The wheat ESTs mapped near H26 were further used to BLAST rice and B. distachyon genomic sequences for comparative mapping. To date, 26 newly developed STS markers have been mapped to the chromosomal region spanning the H26 locus. Two of them were mapped 1.0 cM away from the H26 locus. Comparative analysis identified genomic regions on rice chromosome 1 and Brachypodium Super contig 13 which are collinear with the genomic region spanning the H26 locus within the distal region of 3DL. The newly developed STS markers closely linked to H26 will be useful for mapped-based cloning of H26 and marker-assisted selection of this gene in wheat breeding. The results will also enhance understanding of this chromosomal region which contains several other Hessian fly resistance genes.

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

BACKGROUND: Microsatellite (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. RESULTS: A 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. CONCLUSION: BatchPrimer3 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/.

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 omega-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.

Application of optical tweezers in the research of molecular interaction between lymphocyte function associated antigen-1 and its monoclonal antibody.

Lymphocyte function associated antigen-1 (CD11a/CD18, LFA-1) plays an important role in the structure of the immunological synapse and is required for efficient lysis of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. To study the activation mode of LFA-1 on the NK cell surface, optical tweezers were used in the work. As an emerging technology, optical tweezers are widely used to manipulate microscopic objects and measure the forces of molecular interactions in the field of biological research. In our study, a new platform was constructed to study the single molecular behavior of receptor on cell surface using optical tweezers. Based on the platform, the interaction between an NK cell and a polystyrene microsphere coated with anti-LFA-1 antibody was observed. The result confirmed that the adhesion forces between an NK cell and a polystyrene bead were time-dependent. According to our findings, we propose that anti-LFA-1 antibody may cause the clustering of LFA-1 on NK cell surface.

New insights into Oryza genome evolution: high gene colinearity and differential retrotransposon amplification.

An approximately 247-kb genomic region from FF genome of wild rice Oryza brachyantha, possessing the smallest Oryza genome, was compared to the orthologous approximately 450-kb region from AA genome, O. sativa L. ssp. japonica. 37 of 38 genes in the orthologous regions are shared between japonica and O. brachyantha. Analyses of nucleotide substitution in coding regions suggest the two genomes diverged approximately 10 million years ago. Comparisons of transposable elements (TEs) reveal that the density of DNA TEs in O. brachyantha is comparable to O. sativa; however, the density of RNA TEs is dramatically lower. The genomic fraction of RNA TEs in japonica is two times greater than in O. brachyantha. Differences, particularly in RNA TEs, in this region and in BAC end sequences from five wild and two cultivated Oryza species explain major genome size differences between sativa and brachyantha. Gene expression analyses of three ObDREB1 genes in the sequenced region indicate orthologous genes retain similar expression patterns following cold stress. Our results demonstrate that size and number of RNA TEs play a major role in genomic differentiation and evolution in Oryza. Additionally, distantly related O. brachyantha shares colinearity with O. sativa, offering opportunities to use comparative genomics to explore the genetic diversity of wild species to improve cultivated rice.

GenoProfiler: batch processing of high-throughput capillary fingerprinting data.

UNLABELLED: High-throughput content fingerprinting techniques employing capillary electrophoresis place new demands on the editing of fingerprint files for the downstream contig assembly program, FPC. A cross-platform software application, GenoProfiler, was developed for automated editing of sized fingerprinting profiles generated by the ABI Genetic Analyzers. The batch-processing module extracts the sized fragment information directly from the ABI raw trace files, or from data files exported from GeneMapper or other size calling software, removes the background noise and undesired fragments, and generates fragment size files compatible with the FPC software. AVAILABILITY: http://wheat.pw.usda.gov/PhysicalMapping/