We developed 21,499 genome-wide insertion–deletion (InDel) markers (2- to 54-bp in silico fragment length polymorphism) by comparing the genomic sequences of four (desi, kabuli and wild C. reticulatum) chickpea [Cicer arietinum (L.)] accessions. InDel markers showing 2- to 6-bp fragment length polymorphism among accessions were abundant (76.8%) in the chickpea genome. The physically mapped 7,643 and 13,856 markers on eight chromosomes and unanchored scaffolds, respectively, were structurally and functionally annotated. The 4,506 coding (23% large-effect frameshift mutations) and regulatory InDel markers were identified from 3,228 genes (representing 11.7% of total 27,571 desi genes), suggesting their functional relevance for trait association/genetic mapping. High amplification (97%) and intra-specific polymorphic (60–83%) potential and wider genetic diversity (15–89%) were detected by genome-wide 6,254 InDel markers among desi, kabuli and wild accessions using even a simpler cost-effective agarose gel-based assay. This signifies added advantages of this user-friendly genetic marker system for manifold large-scale genotyping applications in laboratories with limited infrastructure and resources. Utilizing 6,254 InDel markers-based high-density (inter-marker distance: 0.212 cM) inter-specific genetic linkage map (ICC 4958 × ICC 17160) of chickpea as a reference, three major genomic regions harboring six flowering and maturity time robust QTLs (16.4–27.5% phenotypic variation explained, 8.1–11.5 logarithm of odds) were identified. Integration of genetic and physical maps at these target QTL intervals mapped on three chromosomes delineated five InDel markers-containing candidate genes tightly linked to the QTLs governing flowering and maturity time in chickpea. Taken together, our study demonstrated the practical utility of developing and high-throughput genotyping of such beneficial InDel markers at a genome-wide scale to expedite genomics-assisted breeding applications in chickpea.

Low temperature severely affects plant growth and development. To overcome this constraint, several plant species from regions having a cool season have evolved an adaptive response, called cold acclimation. We have studied this response in olive tree (Olea europaea L.) cv. Picual. Biochemical stress markers and cold-stress symptoms were detected after the first 24 h as sagging leaves. After 5 days, the plants were found to have completely recovered. Control and cold-stressed plants were sequenced by Illumina HiSeq 1000 paired-end technique. We also assembled a new olive transcriptome comprising 157,799 unigenes and found 6,309 unigenes differentially expressed in response to cold. Three types of response that led to cold acclimation were found: short-term transient response, early long-term response, and late long-term response. These subsets of unigenes were related to different biological processes. Early responses involved many cold-stress-responsive genes coding for, among many other things, C-repeat binding factor transcription factors, fatty acid desaturases, wax synthesis, and oligosaccharide metabolism. After long-term exposure to cold, a large proportion of gene down-regulation was found, including photosynthesis and plant growth genes. Up-regulated genes after long-term cold exposure were related to organelle fusion, nucleus organization, and DNA integration, including retrotransposons.

The current gold standard method for methylome analysis is whole-genome bisulfite sequencing (WGBS), but its cost is substantial, especially for the purpose of multi-sample comparison of large methylomes. Shotgun bisulfite sequencing of target-enriched DNA, or targeted methylome sequencing (TMS), can be a flexible, cost-effective alternative to WGBS. However, the current TMS protocol requires a considerable amount of input DNA and hence is hardly applicable to samples of limited quantity. Here we report a method to overcome this limitation by using post-bisulfite adaptor tagging (PBAT), in which adaptor tagging is conducted after bisulfite treatment to circumvent bisulfite-induced loss of intact sequencing templates, thereby enabling TMS of a 100-fold smaller amount of input DNA with far fewer cycles of polymerase chain reaction than in the current protocol. We thus expect that the PBAT-mediated TMS will serve as an invaluable method in epigenomics.

Breeding new varieties with low seed glucosinolate (GS) concentrations has long been a prime target in Brassica napus. In this study, a novel association mapping methodology termed ‘associative transcriptomics’ (AT) was applied to a panel of 101 B. napus lines to define genetic regions and also candidate genes controlling total seed GS contents. Over 100,000 informative single-nucleotide polymorphisms (SNPs) and gene expression markers (GEMs) were developed for AT analysis, which led to the identification of 10 SNP and 7 GEM association peaks. Within these peaks, 26 genes were inferred to be involved in GS biosynthesis. A weighted gene co-expression network analysis provided additional 40 candidate genes. The transcript abundance in leaves of two candidate genes, BnaA.GTR2a located on chromosome A2 and BnaC.HAG3b on C9, was correlated with seed GS content, explaining 18.8 and 16.8% of phenotypic variation, respectively. Resequencing of genomic regions revealed six new SNPs in BnaA.GTR2a and four insertions or deletions in BnaC.HAG3b. These deletion polymorphisms were then successfully converted into polymerase chain reaction–based diagnostic markers that can, due to high linkage disequilibrium observed in these regions of the genome, be used for marker-assisted breeding for low seed GS lines.

Despite their biological importance, a significant number of genes for secondary metabolite biosynthesis (SMB) remain undetected due largely to the fact that they are highly diverse and are not expressed under a variety of cultivation conditions. Several software tools including SMURF and antiSMASH have been developed to predict fungal SMB gene clusters by finding core genes encoding polyketide synthase, nonribosomal peptide synthetase and dimethylallyltryptophan synthase as well as several others typically present in the cluster. In this work, we have devised a novel comparative genomics method to identify SMB gene clusters that is independent of motif information of the known SMB genes. The method detects SMB gene clusters by searching for a similar order of genes and their presence in nonsyntenic blocks. With this method, we were able to identify many known SMB gene clusters with the core genes in the genomic sequences of 10 filamentous fungi. Furthermore, we have also detected SMB gene clusters without core genes, including the kojic acid biosynthesis gene cluster of Aspergillus oryzae. By varying the detection parameters of the method, a significant difference in the sequence characteristics was detected between the genes residing inside the clusters and those outside the clusters.

Analysis of microbiota in various biological and environmental samples under a variety of conditions has recently become more practical due to remarkable advances in next-generation sequencing. Changes leading to specific biological states including some of the more complex diseases can now be characterized with relative ease. It is known that gut microbiota is involved in the pathogenesis of inflammatory bowel disease (IBD), mainly Crohn's disease and ulcerative colitis, exhibiting symptoms in the gastrointestinal tract. Recent studies also showed increased frequency of oral manifestations among IBD patients, indicating aberrations in the oral microbiota. Based on these observations, we analyzed the composition of salivary microbiota of 35 IBD patients by 454 pyrosequencing of the bacterial 16S rRNA gene and compared it with that of 24 healthy controls (HCs). The results showed that Bacteroidetes was significantly increased with a concurrent decrease in Proteobacteria in the salivary microbiota of IBD patients. The dominant genera, Streptococcus, Prevotella, Neisseria, Haemophilus, Veillonella, and Gemella, were found to largely contribute to dysbiosis (dysbacteriosis) observed in the salivary microbiota of IBD patients. Analysis of immunological biomarkers in the saliva of IBD patients showed elevated levels of many inflammatory cytokines and immunoglobulin A, and a lower lysozyme level. A strong correlation was shown between lysozyme and IL-1β levels and the relative abundance of Streptococcus, Prevotella, Haemophilus and Veillonella. Our data demonstrate that dysbiosis of salivary microbiota is associated with inflammatory responses in IBD patients, suggesting that it is possibly linked to dysbiosis of their gut microbiota.

Mouse embryonic stem (ES) cells are prototypical stem cells that remain undifferentiated in culture for long periods, yet maintain the ability to differentiate into essentially all cell types. Previously, we have reported that ES cells oscillate between two distinct states, which can be distinguished by the transient expression of Zscan4 genes originally identified for its specific expression in mouse two-cell stage embryos. Here, we report that the nascent protein synthesis is globally repressed in the Zscan4-positive state of ES cells, which is mediated by the transient expression of newly identified eukaryotic translation initiation factor 1A (Eif1a)-like genes. Eif1a-like genes, clustered on Chromosome 12, show the high sequence similarity to the Eifa1 and consist of 10 genes (Eif1al1–Eif1al10) and 9 pseudogenes (Eif1al-ps1–Eif1al-ps9). The analysis of the expressed sequence tag database showed that Eif1a-like genes are expressed mostly in the two-cell stage mouse embryos. Microarray analyses and quantitative real-time polymerase chain reaction analyses show that Eif1a-like genes are expressed specifically in the Zscan4-positive state of ES cells. These results indicate a novel mechanism to repress protein synthesis by Eif1a-like genes and a unique mode of protein synthesis regulation in ES cells, which undergo a transient and reversible repression of global protein synthesis in the Zscan4-positive state.

NAC [no apical meristem (NAM), Arabidopsis thaliana transcription activation factor [ATAF1/2] and cup-shaped cotyledon (CUC2)] proteins belong to one of the largest plant-specific transcription factor (TF) families and play important roles in plant development processes, response to biotic and abiotic cues and hormone signalling. Our genome-wide analysis identified 110 StNAC genes in potato encoding for 136 proteins, including 14 membrane-bound TFs. The physical map positions of StNAC genes on 12 potato chromosomes were non-random, and 40 genes were found to be distributed in 16 clusters. The StNAC proteins were phylogenetically clustered into 12 subgroups. Phylogenetic analysis of StNACs along with their Arabidopsis and rice counterparts divided these proteins into 18 subgroups. Our comparative analysis has also identified 36 putative TNAC proteins, which appear to be restricted to Solanaceae family. In silico expression analysis, using Illumina RNA-seq transcriptome data, revealed tissue-specific, biotic, abiotic stress and hormone-responsive expression profile of StNAC genes. Several StNAC genes, including StNAC072 and StNAC101that are orthologs of known stress-responsive Arabidopsis RESPONSIVE TO DEHYDRATION 26 (RD26) were identified as highly abiotic stress responsive. Quantitative real-time polymerase chain reaction analysis largely corroborated the expression profile of StNAC genes as revealed by the RNA-seq data. Taken together, this analysis indicates towards putative functions of several StNAC TFs, which will provide blue-print for their functional characterization and utilization in potato improvement.

The availability of well-validated informative co-dominant microsatellite markers and saturated genetic linkage map has been limited in foxtail millet (Setaria italica L.). In view of this, we conducted a genome-wide analysis and identified 28 342 microsatellite repeat-motifs spanning 405.3 Mb of foxtail millet genome. The trinucleotide repeats (∼48%) was prevalent when compared with dinucleotide repeats (∼46%). Of the 28 342 microsatellites, 21 294 (∼75%) primer pairs were successfully designed, and a total of 15 573 markers were physically mapped on 9 chromosomes of foxtail millet. About 159 markers were validated successfully in 8 accessions of Setaria sp. with ∼67% polymorphic potential. The high percentage (89.3%) of cross-genera transferability across millet and non-millet species with higher transferability percentage in bioenergy grasses (∼79%, Switchgrass and ∼93%, Pearl millet) signifies their importance in studying the bioenergy grasses. In silico comparative mapping of 15 573 foxtail millet microsatellite markers against the mapping data of sorghum (16.9%), maize (14.5%) and rice (6.4%) indicated syntenic relationships among the chromosomes of foxtail millet and target species. The results, thus, demonstrate the immense applicability of developed microsatellite markers in germplasm characterization, phylogenetics, construction of genetic linkage map for gene/quantitative trait loci discovery, comparative mapping in foxtail millet, including other millets and bioenergy grass species.

The cultivated strawberry (Fragaria× ananassa) is an octoploid (2n = 8x = 56) of the Rosaceae family whose genomic architecture is still controversial. Several recent studies support the AAA′A′BBB′B′ model, but its complexity has hindered genetic and genomic analysis of this important crop. To overcome this difficulty and to assist genome-wide analysis of F. × ananassa, we constructed an integrated linkage map by organizing a total of 4474 of simple sequence repeat (SSR) markers collected from published Fragaria sequences, including 3746 SSR markers [Fragaria vesca expressed sequence tag (EST)-derived SSR markers] derived from F. vesca ESTs, 603 markers (F. × ananassa EST-derived SSR markers) from F. × ananassa ESTs, and 125 markers (F. × ananassa transcriptome-derived SSR markers) from F. × ananassa transcripts. Along with the previously published SSR markers, these markers were mapped onto five parent-specific linkage maps derived from three mapping populations, which were then assembled into an integrated linkage map. The constructed map consists of 1856 loci in 28 linkage groups (LGs) that total 2364.1 cM in length. Macrosynteny at the chromosome level was observed between the LGs of F. × ananassa and the genome of F. vesca. Variety distinction on 129 F. × ananassa lines was demonstrated using 45 selected SSR markers.