Identification of candidate genes responsible for chasmogamy in wheat.

BACKGROUND: The flowering biology of wheat plants favours self-pollination which causes obstacles in wheat hybrid breeding. Wheat flowers can be divided into two groups, the first one is characterized by flowering and pollination within closed flowers (cleistogamy), while the second one possesses the ability to open flowers during processes mentioned above (chasmogamy). The swelling of lodicules is involved in the flowering of cereals and among others their morphology, calcium and potassium content differentiate between cleistogamic and non-cleistogamous flowers. A better understanding of the chasmogamy mechanism can lead to the development of tools for selection of plants with the desired outcrossing rate. To learn more, the sequencing of transcriptomes (RNA-Seq) and Representational Difference Analysis products (RDA-Seq) were performed to investigate the global transcriptomes of wheat lodicules in two highly chasmogamous (HCH, Piko and Poezja) and two low chasmogamous (LCH, Euforia and KWS Dacanto) varieties at two developmental stages-pre-flowering and early flowering. RESULTS: The differentially expressed genes were enriched in five, main pathways: "metabolism", "organismal systems", "genetic information processing", "cellular processes" and "environmental information processing", respectively. Important genes with opposite patterns of regulation between the HCH and LCH lines have been associated with the lodicule development i.e. expression levels of MADS16 and MADS58 genes may be responsible for quantitative differences in chasmogamy level in wheat. CONCLUSIONS: We conclude that the results provide a new insight into lodicules involvement in the wheat flowering process. This study generated important genomic information to support the exploitation of the chasmogamy in wheat hybrid breeding programs.

Identification of Rf Genes in Hexaploid Wheat (Triticumaestivum L.) by RNA-Seq and Paralog Analyses.

Among the natural mechanisms used for wheat hybrid breeding, the most desirable is the system combining the cytoplasmic male sterility (cms) of the female parent with the fertility-restoring genes (Rf) of the male parent. The objective of this study was to identify Rf candidate genes in the wheat genome on the basis of transcriptome sequencing (RNA-seq) and paralog analysis data. Total RNA was isolated from the anthers of two fertility-restorer (Primépi and Patras) and two non-restorer (Astoria and Grana) varieties at the tetrad and late uninucleate microspore stages. Of 36,912 differentially expressed genes (DEGs), 21 encoding domains in known fertility-restoring proteins were selected. To enrich the pool of Rf candidates, 52 paralogs (PAGs) of the 21 selected DEGs were included in the analyses. The expression profiles of most of the DEGs and PAGs determined bioinformatically were as expected (i.e., they were overexpressed in at least one fertility-restorer variety). However, these results were only partially consistent with the quantitative real-time PCR data. The DEG and PAG promoters included cis-regulatory elements common among PPR-encoding genes. On the basis of the obtained results, we designated seven genes as Rf candidate genes, six of which were identified for the first time in this study.

Evaluation of genetic structure in European wheat cultivars and advanced breeding lines using high-density genotyping-by-sequencing approach.

BACKGROUND: The genetic diversity and gene pool characteristics must be clarified for efficient genome-wide association studies, genomic selection, and hybrid breeding. The aim of this study was to evaluate the genetic structure of 509 wheat accessions representing registered varieties and advanced breeding lines via the high-density genotyping-by-sequencing approach. RESULTS: More than 30% of 13,499 SNP markers representing 2162 clusters were mapped to genes, whereas 22.50% of 26,369 silicoDArT markers overlapped with coding sequences and were linked in 3527 blocks. Regarding hexaploidy, perfect sequence matches following BLAST searches were not sufficient for the unequivocal mapping to unique loci. Moreover, allelic variations in homeologous loci interfered with heterozygosity calculations for some markers. Analyses of the major genetic changes over the last 27 years revealed the selection pressure on orthologs of the gibberellin biosynthesis-related GA2 gene and the senescence-associated SAG12 gene. A core collection representing the wheat population was generated for preserving germplasm and optimizing breeding programs. CONCLUSIONS: Our results confirmed considerable differences among wheat subgenomes A, B and D, with D characterized by the lowest diversity but the highest LD. They revealed genomic regions that have been targeted by breeding.

Representational Difference Analysis of Transcripts Involved in Jervine Biosynthesis.

Veratrum-type steroidal alkaloids (VSA) are the major bioactive ingredients that strongly determine the pharmacological activities of Veratrum nigrum. Biosynthesis of VSA at the molecular and genetic levels is not well understood. Next-generation sequencing of representational difference analysis (RDA) products after elicitation and precursor feeding was applied to identify candidate genes involved in VSA biosynthesis. A total of 12,048 contigs with a median length of 280 bases were received in three RDA libraries obtained after application of methyl jasmonate, squalene and cholesterol. The comparative analysis of annotated sequences was effective in identifying candidate genes. GABAT2 transaminase and hydroxylases active at C-22, C-26, C-11, and C-16 positions in late stages of jervine biosynthesis were selected. Moreover, genes coding pyrroline-5-carboxylate reductase and enzymes from the short-chain dehydrogenases/reductases family (SDR) associated with the reduction reactions of the VSA biosynthesis process were proposed. The data collected contribute to better understanding of jervine biosynthesis and may accelerate implementation of biotechnological methods of VSA biosynthesis.

Identification of candidate genes involved in steroidal alkaloids biosynthesis in organ-specific transcriptomes of Veratrum nigrum L.

Veratrum nigrum is protected plant of Melanthiaceae family, able to synthetize unique steroidal alkaloids important for pharmacy. Transcriptomes from leaves, stems and rhizomes of in vitro maintained V. nigrum plants were sequenced and annotated for genes and markers discovery. Sequencing of samples derived from the different organs resulted in a total of 108,511 contigs with a mean length of 596 bp. Transcripts derived from leaf and stalk were annotated at 28%, and 38% in Nr nucleotide database, respectively. The sequencing revealed 949 unigenes related with lipid metabolism, including 73 transcripts involved in steroids and genus-specific steroid alkaloids biosynthesis. Additionally, 3203 candidate SSRs markers we identified in unigenes with average density of one SSR locus every 6.2 kb sequence. Unraveling of biochemical machinery of the pathway responsible for steroidal alkaloids will open possibility to design and optimize biotechnological process. The transcriptomic data provide valuable resources for biochemical, molecular genetics, comparative transcriptomics, functional genomics, ecological and evolutionary studies of V. nigrum.

Changes in fenugreek transcriptome induced by methyl jasmonate and steroid precursors revealed by RNA-Seq.

Changes in fenugreek transcriptome related to enhanced production of steroids were induced by methyl jasmonate, cholesterol and squalene, and recorded using RNA-seq. A total of 112,850 unigenes were obtained after de novo assembling of next generation sequencing data, and used for functional annotations. In steroidal saponins pathway, transcripts involved in mevalonate, terpenoid backbone and plant sterol synthesis were annotated. Overexpression of several transcripts from phytosterol biosynthesis pathway was confirmed by quantitative RT-PCR. In diosgenin biosynthesis pathway, fatty acid ω-hydroxylase (CYP86A2) and steroid 22-alpha-hydroxylase (CYP90B1) genes were annotated in all induced transcriptomes. Moreover, direct sequencing confirmed increased levels of CYP90B1, unspecific monooxygenase and 26-hydroxylase genes in plants with elevated level of diosgenin. New unigenes corresponding to enzymes involved in biosynthesis of diosgenin from cycloartenol via cholesterol were obtained and the role of CYP72A family in steroidal saponin biosynthesis was proposed. Additional support for biosynthetic pathway from cycloartenol to diosgenin was provided.

Next-generation sequencing of representational difference analysis products for identification of genes involved in diosgenin biosynthesis in fenugreek (Trigonella foenum-graecum).

MAIN CONCLUSION: Representational difference analysis of cDNA was performed and differential products were sequenced and annotated. Candidate genes involved in biosynthesis of diosgenin in fenugreek were identified. Detailed mechanism of diosgenin synthesis was proposed. Fenugreek (Trigonella foenum-graecum L.) is a valuable medicinal and crop plant. It belongs to Fabaceae family and has a unique potential to synthesize valuable steroidal saponins, e.g., diosgenin. Elicitation (methyl jasmonate) and precursor feeding (cholesterol and squalene) were used to enhance the content of sterols and steroidal sapogenins in in vitro grown plants for representational difference analysis of cDNA (cDNA-RDA). To identify candidate genes involved in diosgenin biosynthesis, differential, factor-specific libraries were subject to the next-generation sequencing. Approximately 9.9 million reads were obtained, trimmed, and assembled into 31,491 unigenes with an average length of 291 bp. Then, functional annotation and gene ontogeny enrichment analysis was performed by aligning all-unigenes with public databases. Within the transcripts related to sterol and steroidal saponin biosynthesis, we discovered novel candidate genes of diosgenin biosynthesis and validated their expression using quantitative RT-PCR analysis. Based on these findings, we supported the idea that diosgenin is biosynthesized from cycloartenol via cholesterol. This is the first report on the next-generation sequencing of cDNA-RDA products. Analysis of the transcriptomes enriched in low copy sequences contributed substantially to our understanding of the biochemical pathways of steroid synthesis in fenugreek.

Characterization of the genome, proteome, and structure of yersiniophage ϕR1-37.

The bacteriophage vB_YecM-ϕR1-37 (ϕR1-37) is a lytic yersiniophage that can propagate naturally in different Yersinia species carrying the correct lipopolysaccharide receptor. This large-tailed phage has deoxyuridine (dU) instead of thymidine in its DNA. In this study, we determined the genomic sequence of phage ϕR1-37, mapped parts of the phage transcriptome, characterized the phage particle proteome, and characterized the virion structure by cryo-electron microscopy and image reconstruction. The 262,391-bp genome of ϕR1-37 is one of the largest sequenced phage genomes, and it contains 367 putative open reading frames (ORFs) and 5 tRNA genes. Mass-spectrometric analysis identified 69 phage particle structural proteins with the genes scattered throughout the genome. A total of 269 of the ORFs (73%) lack homologues in sequence databases. Based on terminator and promoter sequences identified from the intergenic regions, the phage genome was predicted to consist of 40 to 60 transcriptional units. Image reconstruction revealed that the ϕR1-37 capsid consists of hexameric capsomers arranged on a T=27 lattice similar to the bacteriophage ϕKZ. The tail of ϕR1-37 has a contractile sheath. We conclude that phage ϕR1-37 is a representative of a novel phage type that carries the dU-containing genome in a ϕKZ-like head.

Rapid differentiation of mixed influenza A/H1N1 virus infections with seasonal and pandemic variants by multitemperature single-stranded conformational polymorphism analysis.

Mixed infections of a single host with different variants of influenza A virus are the main source of reassortants which may have unpredictable properties when they establish themselves in the human population. In this report we describe a method for rapid detection of mixed influenza virus infections with the seasonal A/H1N1 human strain and the pandemic A/H1N1/v strain which emerged in 2009 in Mexico and the United States. The influenza virus A/H1N1 variants were characterized by the multitemperature single-stranded conformational polymorphism (MSSCP) method. The MSSCP gel patterns of hemagglutinin gene fragments of pandemic A/H1N1/v and different seasonal A/H1N1 strains were easily distinguishable 2 h after completion of reverse transcription-PCR (RT-PCR). Using the MSSCP-based genotyping approach, coinfections with seasonal and pandemic variants of the A/H1N1 subtype were identified in 4 out of 23 primary samples obtained from patients that presented with influenza-like symptoms to hospitals across Poland during the 2009-2010 epidemic season. Pandemic influenza virus strain presence was confirmed in all these primary samples by real-time RT-PCR. The sensitivity level of the MSSCP-based minor genetic variant detection was 0.1%, as determined on a mixture of DNA fragments obtained from amplification of the hemagglutinin gene of seasonal and pandemic strains. The high sensitivity of the method suggests its applicability for characterization of new viral variants long before they become dominant.