Genome Survey Sequences Research Articles

Computational Algorithm for Prediction of miRNA in Plants

The role of bio-informatics and computational biology in life sciences has been growing ever since the emergence of complex and large datasets for understanding the biological processes and expression of traits. Even though, algorithmic approaches are available, individual modules have to be executed for each intermediate result to predict the microRibosomal Nucleic Acid (miRNA). Hence, an attempt was made to develop an integrated model for predicting the miRNA in which all the structures will be generated automatically, once we submit the genomic sequences with varied datasets as an input. A novel algorithm was developed for prediction of miRNA in plants using shell scripting for fast processing of huge amount of data. As a part of the pipeline, software modules for generating RNA secondary structure, RNA structure in Extensible Markup Language(XML) format and RNA structure in pictorial view were developed using shell scripting by imposing various constraints, namely(1) miRNA should be a part of hairpin, (2) miRNA length is approximately 21nt, (3) it should start from 41st position and (4) the length of hairpin of good miRNA is >50 nt. Built-in modules, namely ‘samtools’ and ‘mfold’ were used in the scripting for generating RNA secondary structure in graphical form and in XML format. These modules were executed with the representative tobacco genome survey sequence and able to retrieve the above structures which are considered an input for predicting miRNA, and an output file was generated to display good miRNA sequences from the given structure. This algorithm can be used for predicting miRNA from the genomic sequences from the upcoming tobacco and other plantgenome projects.

Genome-Wide Identification, Localization, and Expression Analysis of Proanthocyanidin-Associated Genes in Brassica.

Proanthocyanidins (PA) is a type of prominent flavonoid compound deposited in seed coats which controls the pigmentation in all Brassica species. Annotation of Brassica juncea genome survey sequences showed 72 PA genes; however, a functional description of these genes, especially how their interactions regulate seed pigmentation, remains elusive. In the present study, we designed 19 primer pairs to screen a bacterial artificial chromosome (BAC) library of B. juncea. A total of 284 BAC clones were identified and sequenced. Alignment of the sequences confirmed that 55 genes were cloned, with every Arabidopsis PA gene having 2–7 homologs in B. juncea. BLAST analysis using the recently released B. rapa or B. napus genome database identified 31 and 58 homologous genes, respectively. Mapping and phylogenetic analysis indicated that 30 B. juncea PA genes are located in the A-genome chromosomes except A04, whereas the remaining 25 genes are mapped to the B-genome chromosomes except B05 and B07. RNA-seq data and Fragments Per Kilobase of a transcript per Million mapped reads (FPKM) analysis showed that most of the PA genes were expressed in the seed coat of B. juncea and B. napus, and that BjuTT3, BjuTT18, BjuANR, BjuTT4-2, BjuTT4-3, BjuTT19-1, and BjuTT19-3 are transcriptionally regulated, and not expressed or downregulated in yellow-seeded testa. Importantly, our study facilitates in better understanding of the molecular mechanism underlying Brassica PA profiles and accumulation, as well as in further characterization of PA genes.

Recurrence of Chromosome Rearrangements and Reuse of DNA Breakpoints in the Evolution of the Triticeae Genomes.

Chromosomal rearrangements (CRs) play important roles in karyotype diversity and speciation. While many CR breakpoints have been characterized at the sequence level in yeast, insects, and primates, little is known about the structure of evolutionary CR breakpoints in plant genomes, which are much more dynamic in genome size and sequence organization. Here, we report identification of breakpoints of a translocation between chromosome arms 4L and 5L of Triticeae, which is fixed in several species, including diploid wheat and rye, by comparative mapping and analysis of the draft genome and chromosome survey sequences of the Triticeae species. The wheat translocation joined the ends of breakpoints downstream of a WD40 gene on 4AL and a gene of the PMEI family on 5AL. A basic helix-loop-helix transcription factor gene in 5AL junction was significantly restructured. Rye and wheat share the same position for the 4L breakpoint, but the 5L breakpoint positions are not identical, although very close in these two species, indicating the recurrence of 4L/5L translocations in the Triticeae. Although barley does not carry the translocation, collinearity across the breakpoints was violated by putative inversions and/or transpositions. Alignment with model grass genomes indicated that the translocation breakpoints coincided with ancient inversion junctions in the Triticeae ancestor. Our results show that the 4L/5L translocation breakpoints represent two CR hotspots reused during Triticeae evolution, and support breakpoint reuse as a widespread mechanism in all eukaryotes. The mechanisms of the recurrent translocation and its role in Triticeae evolution are also discussed.

A genome survey sequence (GSS) analysis and microsatellite marker development for Indian mackerel, Rastrelliger kanagurta, using Ion Torrent technology

A next generation sequencing platform (Ion Torrent PGM™) was used to generate a partial genome survey sequence (GSS) dataset to develop microsatellite markers from R. kanagurta genomic DNA. Data generated included a total of 399,794 sequence reads (81.29Mbp) with 16,209 sequence reads successfully assembled. This produced 327 contigs averaging 677bp in length in addition to the SSR markers. Results based on GSS BLASTx and BLASTn possessed significant similarity (E value<10−6) with available data in public databases, with the majority matching well to previously reported fish sequences. A total of 7891 SSR-containing motif repeats of which 1688 qualified for primer design were generated. After optimization and testing for reproducibility and polymorphism, eight microsatellite markers were deemed suitable for future population genetic analysis in the target species.

Computational identification and characterization of novel microRNA in the mammary gland of dairy goat (Capra hircus).

Many studies have indicated that microRNAs (miRNAs) influence the development of the mammary gland by posttranscriptionally affecting their target genes. The objective of this research was to identify novel miRNAs in the mammary gland of dairy goats with a bioinformatics approach that was based on expressed sequence tag (EST) and genome survey sequence (GSS) analyses. We applied all known major mammals, miRNAs to search against the goat EST and GSS databases for the first time to identify new miRNAs. We, then, validated these newly predicted miRNAs with stem-loop reverse transcription followed by a SYBR Green polymerase chain reaction assay. Finally, 29 mature miRNAs were identified and verified, and of these, 14 were grouped into 13 families based on seed sequence identity and 85 potential target genes of newly verified miRNAs were subsequently predicted, most of which seemed to encode the proteins participating in regulation of metabolism, signal transduction, growth and development. The predicting accuracy of the new miRNAs was 70.37%, which confirmed that the methods used in this study were efficient and reliable. Detailed analyses of the sequence characteristics of the novel miRNAs of the goat mammary gland were performed. In conclusion, these results provide a reference for further identification of miRNAs in animals without a complete genome and thus improve the understanding of miRNAs in the caprine mammary gland.

Genetic diversity in groundnut (Arachis hypogaea L.) genotypes and detection of marker trait associations for plant habit and seed size using genomic and genic SSRs

Groundnut (Arachis hypogaea L.) an important oilseed crop in India is known to have narrow genetic base. Therefore, the assessment of genetic diversity and detection of marker-trait association are important objectives for the genetic improvement of groundnut. The present study involved the development of 192 SSR markers from Arachis genomic survey sequences. From these, seven polymorphic SSRs along with 15 other genomic SSRs, 19 genic SSRs, and three STS markers were used to detect genetic diversity among 44 groundnut genotypes. These polymorphic SSR markers amplified 155 bands (76 genomic and 79 genic), of these 128 bands (67 genomic and 61 genic) were polymorphic. The genomic SSR exhibited 88.1% and genic SSRs displayed 77.2% allelic polymorphism. The polymorphic information content (PIC) of the markers ranged from 0.04 to 0.95. The pair-wise genetic similarity ranged from 24.2 to 90.7% for genomic SSR and 32.9 to 97.9% for genic SSR markers. Cluster analysis based on the pooled data from both genomic and genic SSRs revealed a dendrogram which could distinguish all the genotypes. Further, the AMOVA analysis detected 16.7% genetic variation due to differences in seed size and 13.0% due to plant habit. Based on locus-by-locus AMOVA and Kruskal-Wallis ANOVA and further confirmation by discriminant analysis and general linear model, six markers were found to be associated with plant habit and four markers with seed size.

Identification and Characterization of Novel miRNAs in Chlamydomonas reinhardtii by Computational Methods.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs with 18-24 nucleotides in length, which have important roles in posttranscriptional gene regulation. The resemblance of miRNA biogenesis in unicellular green algae and those in plants suggests probable evolutionary conserved pathways. This conservation provides a ground towards prediction of new homologs via computational biology. Here, conserved miRNA genes in Chlamydomonas reinhardtii and plants were examined through homology alignment. Previously known and unique plant miRNAs were BLASTed against expressed sequence tags (ESTs) and genomic survey sequences (GSSs) of C. reinhardtii. All candidate sequences with appropriate fold back structures were screened according to a series of miRNA filtering criteria. Homologous miRNAs (17), belonging to 9 miRNA gene families were predicted. Interestingly and for the first time, a miRNA family of genes was localized to chloroplast. Again and for the first time, here we report identification of C. reinhardtii miRNA orthologs in plants and animals. miRNA target genes were identified based on their sequence complementarities to the respective miRNAs using psRNATarget against C. reinhardtii, Unigene, and DFCI Gene Index (CHRGI). Totally, 152 potential target sites were identified. From the predicted miRNAs, 7 miRNAs had no target sequence in C. reinhardtii protein coding genes. Identifying miRNA and their target transcript(s) would be useful for other research concerned with the function and regulatory mechanisms of C. reinhardtii miRNAs and helps researchers to better understand the nature of its extensive metabolic flexibility and environmental compatibility to survive in distinct environmental niches and nutrient availability.

Identification and characterization of microRNAs and their target genes from Nile tilapia (Oreochromis niloticus).

MicroRNAs (miRNAs) are a class of small single-stranded, endogenous 21-22 nt non-coding RNAs that regulate their target mRNA levels by causing either inactivation or degradation of the mRNAs. In recent years, miRNA genes have been identified from mammals, insects, worms, plants, and viruses. In this research, bioinformatics approaches were used to predict potential miRNAs and their targets in Nile tilapia from the expressed sequence tag (EST) and genomic survey sequence (GSS) database, respectively, based on the conservation of miRNAs in many animal species. A total of 19 potential miRNAs were detected following a range of strict filtering criteria. To test the validity of the bioinformatics method, seven predicted Nile tilapia miRNA genes were selected for further biological validation, and their mature miRNA transcripts were successfully detected by stem-loop RT-PCR experiments. Using these potential miRNAs, we found 56 potential targets in this species. Most of the target mRNAs appear to be involved in development, metabolism, signal transduction, transcription regulation and stress responses. Overall, our findings will provide an important foundation for further research on miRNAs function in the Nile tilapia.

Genomic survey sequencing for development and validation of single-locus SSR markers in peanut (Arachis hypogaea L.).

BackgroundSingle-locus markers have many advantages compared with multi-locus markers in genetic and breeding studies because their alleles can be assigned to particular genomic loci in diversity analyses. However, there is little research on single-locus SSR markers in peanut. Through the de novo assembly of DNA sequencing reads of A. hypogaea, we developed single-locus SSR markers in a genomic survey for better application in genetic and breeding studies of peanut.ResultsIn this study, DNA libraries with four different insert sizes were used for sequencing with 150 bp paired-end reads. Approximately 237 gigabases of clean data containing 1,675,631,984 reads were obtained after filtering. These reads were assembled into 2,102,446 contigs with an N50 length of 1,782 bp, and the contigs were further assembled into 1,176,527 scaffolds with an N50 of 3,920 bp. The total length of the assembled scaffold sequences was 2.0 Gbp, and 134,652 single-locus SSRs were identified from 375,180 SSRs. Among these developed single-locus SSRs, trinucleotide motifs were the most abundant, followed by tetra-, di-, mono-, penta- and hexanucleotide motifs. The most common motif repeats for the various types of single-locus SSRs have a tendency to be A/T rich. A total of 1,790 developed in silico single-locus SSR markers were chosen and used in PCR experiments to confirm amplification patterns. Of them, 1,637 markers that produced single amplicons in twelve inbred lines were considered putative single-locus markers, and 290 (17.7 %) showed polymorphisms. A further F2 population study showed that the segregation ratios of the 97 developed SSR markers, which showed polymorphisms between the parents, were consistent with the Mendelian inheritance law for single loci (1:2:1). Finally, 89 markers were assigned to an A. hypogaea linkage map. A subset of 100 single-locus SSR markers was shown to be highly stable and universal in a collection of 96 peanut accessions. A neighbor-joining tree of this natural population showed that genotypes have obviously correlation with botanical varieties.ConclusionsWe have shown that the detection of single-locus SSR markers from a de novo genomic assembly of a combination of different-insert-size libraries is highly efficient. This is the first report of the development of genome-wide single-locus markers for A. hypogaea, and the markers developed in this study will be useful for gene tagging, sequence scaffold assignment, linkage map construction, diversity analysis, variety identification and association mapping in peanut.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2743-x) contains supplementary material, which is available to authorized users.

Characterization and Transferable Utility of Microsatellite Markers in the Wild and Cultivated Arachis Species.

Microsatellite or simple sequence repeat (SSR) is one of the most widely distributed molecular markers that have been widely utilized to assess genetic diversity and genetic mapping for important traits in plants. However, the understanding of microsatellite characteristics in Arachis species and the currently available amount of high-quality SSR markers remain limited. In this study, we identified 16,435 genome survey sequences SSRs (GSS-SSRs) and 40,199 expressed sequence tag SSRs (EST-SSRs) in Arachis hypogaea and its wild relative species using the publicly available sequence data. The GSS-SSRs had a density of 159.9–239.8 SSRs/Mb for wild Arachis and 1,015.8 SSR/Mb for cultivated Arachis, whereas the EST-SSRs had the density of 173.5–384.4 SSR/Mb and 250.9 SSRs/Mb for wild and cultivated Arachis, respectively. The trinucleotide SSRs were predominant across Arachis species, except that the dinucleotide accounted for most in A. hypogaea GSSs. From Arachis GSS-SSR and EST-SSR sequences, we developed 2,589 novel SSR markers that showed a high polymorphism in six diverse A. hypogaea accessions. A genetic linkage map that contained 540 novel SSR loci and 105 anchor SSR loci was constructed by case of a recombinant inbred lines F6 population. A subset of 82 randomly selected SSR markers were used to screen 39 wild and 22 cultivated Arachis accessions, which revealed a high transferability of the novel SSRs across Arachis species. Our results provided informative clues to investigate microsatellite patterns across A. hypogaea and its wild relative species and potentially facilitate the germplasm evaluation and gene mapping in Arachis species.

Computational prediction of micrornas and their target genes in rainbow trout (Oncorhynchus mykiss)

MicroRNAs (miRNA) are non-coding small RNA molecules consisting of 20–24 nucleotides (nt), which regulate gene expression negatively at the post-transcriptional levels depending on the extent of complementation between miRNA and mRNA in a variety of eukaryotic organisms. They have attracted increasing attention recently. To date, a total of 1637 mature miRNAs from fishes have been deposited in the miRBase database (Release 21) and they are just a small portion of the miRNAs described in fish species. Especially in rainbow trout (Oncorhynchus mykiss) miRNAs, it is far from the numbers of miRNAs found in other fishes and many more miRNA remain to be discovered. In present study, we used known animal miRNAs to systematically search miRNA homologs in available expressed sequence tags (EST), genomic survey sequences (GSS), and transcript sequence assemblies (TSA) of O. mykiss based on the conservation of miRNA sequences. Following the filtering with a combination of stringent criteria, 39 miRNAs belonging to 25 independent families were identified, of which 14 are novel identified miRNAs firstly in O. mykiss. The randomly selected 13 miRNAs were verified by stem-loop RT-PCR indicating that the prediction method that we used to identify the miRNAs was effective. Furthermore, total of 94 target genes were predicted and their probable functions were illustrated. Their target genes are involved in transcription factors, development, metabolism, signaling, immunity, and cell division. The current study has provided an update on O. mykiss miRNAs and their targets, which will be helpful for future research on miRNA-mediated gene regulation in this important fish species.

Genome-wide discovery of single nucleotide polymorphisms (SNPs) and single nucleotide variants (SNVs) in deep-sea mussels: Potential use in population genomics and cross-species application

The present study aimed to generate genome-wide single nucleotide polymorphisms (SNPs) for the deep-sea mussel Bathymodiolus platifrons via a combination of genome survey sequencing and the type IIB endonuclease restriction-site associated DNA (2b-RAD) sequencing, assess the potential use of SNPs in detecting fine-sale population genetic structure and signatures of divergent selection, as well as their cross-species application in other bathymodioline mussels. Genome survey sequencing was conducted for one individual of B. platifrons. De novo assembly resulted in 781,720 sequences with a scaffold N50 of 2.9kb. Using these sequences as a reference, 9307 genome-wide SNPs were identified by 2b-RAD for 28 B. platifrons individuals collected from a seep and a vent population. Among these SNPs, nine outliers showed significant evidence for divergent selection, and their positions in the genes or scaffolds were identified. The FST estimated based on the putative neutral SNPs was low (0.0126) indicating the two B. platifrons populations having a high genetic connectivity. However, the permutation test detected significant differences (P<0.00001), indicating the two populations having clearly detectable genetic differentiation. The Bayesian clustering analyses and principle component analyses (PCA) performed based on either the putative neutral or outlier SNPs also showed that these two populations were genetically differentiated. In addition, 2b-RAD was also conducted to detect 10,199, 6429, and 3811 single nucleotide variants (SNVs) respectively in the bathymodioline mussels Bathymodiolus japonicus, Bathymodiolus aduloides and Idas sp. with different phylogenetic distances from B. platifrons. Overall, our study has demonstrated the feasibility and effectiveness of combining genome survey sequencing and 2b-RAD to rapidly generate genomic resources for use in fine-scale population genetic studies, and various cross-species applications.

Computational identification and functional annotation of microRNAs and their targets from expressed sequence tags (ESTs) and genome survey sequences (GSSs) of coffee (Coffea arabica L.)

MicroRNAs (miRNAs) are conserved, small non-coding endogenous RNAs, which are typically 21–24 nucleotides in length and play a pivotal role in post transcriptional gene regulation either by translational repression or cleavage. The regulation of target genes by miRNA has a central role in plant growth and development as well as in stress condition; however, only a few reports on coffee miRNA functions have been published. In the present study, we report the computational identification of miRNAs and their targets from expressed sequence tags (ESTs) and genome survey sequences (GSSs) of Coffea arabica as well as functionally annotated the target genes based on Gene ontology terms (GO). By following stringent filtering criteria, a total of 20 new potential miRNAs belonging to 13 different miRNA families (miR393, miR390, miR397, miR482, miR2118, miR414, miR1879, miR1134, miR1110, miR533, miR5809, miR426 and miR5212) were identified through homology search. The psRNATarget server predicted 142 potential target genes for 17 miRNAs and their probable functions were illustrated. Most of the predicted target genes encoded transcription factors and genes involved in plant growth and development, signal transduction, metabolism, defense and stress responses. The result from this study will shed light on understanding the functions of miRNAs in C. arabica.

Genome Survey Sequencing for the Characterization of the Genetic Background of Rosa roxburghii Tratt and Leaf Ascorbate Metabolism Genes.

Rosa roxburghii Tratt is an important commercial horticultural crop in China that is recognized for its nutritional and medicinal values. In spite of the economic significance, genomic information on this rose species is currently unavailable. In the present research, a genome survey of R. roxburghii was carried out using next-generation sequencing (NGS) technologies. Total 30.29 Gb sequence data was obtained by HiSeq 2500 sequencing and an estimated genome size of R. roxburghii was 480.97 Mb, in which the guanine plus cytosine (GC) content was calculated to be 38.63%. All of these reads were technically assembled and a total of 627,554 contigs with a N50 length of 1.484 kb and furthermore 335,902 scaffolds with a total length of 409.36 Mb were obtained. Transposable elements (TE) sequence of 90.84 Mb which comprised 29.20% of the genome, and 167,859 simple sequence repeats (SSRs) were identified from the scaffolds. Among these, the mono-(66.30%), di-(25.67%), and tri-(6.64%) nucleotide repeats contributed to nearly 99% of the SSRs, and sequence motifs AG/CT (28.81%) and GAA/TTC (14.76%) were the most abundant among the dinucleotide and trinucleotide repeat motifs, respectively. Genome analysis predicted a total of 22,721 genes which have an average length of 2311.52 bp, an average exon length of 228.15 bp, and average intron length of 401.18 bp. Eleven genes putatively involved in ascorbate metabolism were identified and its expression in R. roxburghii leaves was validated by quantitative real-time PCR (qRT-PCR). This is the first report of genome-wide characterization of this rose species.

Identification and Characterization of Novel microRNAs and Their Targets in Cucumis melo L.: An in silico Approach

Introduction: MicroRNAs are small non protein coding entities that play crucial regulatory role in plants as well as animals by altering their gene expression either by wrecking or blocking the process of translation of the homologous mRNAs. It is an accepted fact that the plant miRNAs are conserved in nature and hence comparative genomics approach can be employed to identify novel miRNAs from the expressed sequence tags (ESTs) and genome survey sequences (GSSs).Methods: In the present study, ESTs and GSSs of Cucumis melo were subjected to BLASTn against non redundant miRNAs from miRBase. BLASTX was performed to remove non coding sequences. The remaining sequences were further subjected to MFOLD to predict the secondary structure and MFEI values. psRNATarget server was employed to predict the targets for newly identified miRNAs which were functionally annotated using GO and KEGG. MEGA version 6 was used to generate phylogenetic tree.Results: The present study identified 15 novel miRNAs from Cucumis melo ESTs and GSSs having average MFEI value of 0.803. A total of 178 targets were predicted. GO analysis revealed a maximum of 8 and 3 miRNAs involved in response to abiotic and biotic stress, respectively while 1 miRNA was found to be involved in both the type of stresses.Conclusion: The identified targets were observed to have a role in plant growth and development, metabolism, senescence, disease resistance and various other stress responses. The findings will further contribute towards understanding of the miRNAs function and their regulatory mechanisms in melon.

Structural analysis of the α subunit of Na(+)/K(+) ATPase genes in invertebrates.

The Na(+)/K(+) ATPase is a ubiquitous pump coordinating the transport of Na(+) and K(+) across the membrane of cells and its role is fundamental to cellular functions. It is heteromer in eukaryotes including two or three subunits (α, β and γ which is specific to the vertebrates). The catalytic functions of the enzyme have been attributed to the α subunit. Several complete α protein sequences are available, but only few gene structures were characterized. We identified the genomic sequences coding the α-subunit of the Na(+)/K(+) ATPase, from the whole-genome shotgun contigs (WGS), NCBI Genomes (chromosome), Genomic Survey Sequences (GSS) and High Throughput Genomic Sequences (HTGS) databases across distinct phyla. One copy of the α subunit gene was found in Annelida, Arthropoda, Cnidaria, Echinodermata, Hemichordata, Mollusca, Placozoa, Porifera, Platyhelminthes, Urochordata, but the nematodes seem to possess 2 to 4 copies. The number of introns varied from 0 (Platyhelminthes) to 26 (Porifera); and their localization and length are also highly variable. Molecular phylogenies (Maximum Likelihood and Maximum Parsimony methods) showed some clusters constituted by (Chordata/(Echinodermata/Hemichordata)) or (Plathelminthes/(Annelida/Mollusca)) and a basal position for Porifera. These structural analyses increase our knowledge about the evolutionary events of the α subunit genes in the invertebrates.

Genome-wide mining, characterization, and development of microsatellite markers in Marsupenaeus japonicus by genome survey sequencing

The kuruma prawn, Marsupenaeus japonicus, is one of the most cultivated and consumed species of shrimp. However, very few molecular genetic/genomic resources are publically available for it. Thus, the characterization and distribution of simple sequence repeats (SSRs) remains ambiguous and the use of SSR markers in genomic studies and marker-assisted selection is limited. The goal of this study is to characterize and develop genome-wide SSR markers in M. japonicus by genome survey sequencing for application in comparative genomics and breeding. A total of 326 945 perfect SSRs were identified, among which dinucleotide repeats were the most frequent class (44.08%), followed by mononucleotides (29.67%), trinucleotides (18.96%), tetranucleotides (5.66%), hexanucleotides (1.07%), and pentanucleotides (0.56%). In total, 151 541 SSR loci primers were successfully designed. A subset of 30 SSR primer pairs were synthesized and tested in 42 individuals from a wild population, of which 27 loci (90.0%) were successfully amplified with specific products and 24 (80.0%) were polymorphic. For the amplified polymorphic loci, the alleles ranged from 5 to 17 (with an average of 9.63), and the average PIC value was 0.796. A total of 58 256 SSR-containing sequences had significant Gene Ontology annotation; these are good functional molecular marker candidates for association studies and comparative genomic analysis. The newly identified SSRs significantly contribute to the M. japonicus genomic resources and will facilitate a number of genetic and genomic studies, including high density linkage mapping, genome-wide association analysis, marker-aided selection, comparative genomics analysis, population genetics, and evolution.

Identification and Evaluation of Single-Nucleotide Polymorphisms in Allotetraploid Peanut (Arachis hypogaea L.) Based on Amplicon Sequencing Combined with High Resolution Melting (HRM) Analysis.

The cultivated peanut (Arachis hypogaea L.) is an allotetraploid (AABB) species derived from the A-genome (Arachis duranensis) and B-genome (Arachis ipaensis) progenitors. Presence of two versions of a DNA sequence based on the two progenitor genomes poses a serious technical and analytical problem during single nucleotide polymorphism (SNP) marker identification and analysis. In this context, we have analyzed 200 amplicons derived from expressed sequence tags (ESTs) and genome survey sequences (GSS) to identify SNPs in a panel of genotypes consisting of 12 cultivated peanut varieties and two diploid progenitors representing the ancestral genomes. A total of 18 EST-SNPs and 44 genomic-SNPs were identified in 12 peanut varieties by aligning the sequence of A. hypogaea with diploid progenitors. The average frequency of sequence polymorphism was higher for genomic-SNPs than the EST-SNPs with one genomic-SNP every 1011 bp as compared to one EST-SNP every 2557 bp. In order to estimate the potential and further applicability of these identified SNPs, 96 peanut varieties were genotyped using high resolution melting (HRM) method. Polymorphism information content (PIC) values for EST-SNPs ranged between 0.021 and 0.413 with a mean of 0.172 in the set of peanut varieties, while genomic-SNPs ranged between 0.080 and 0.478 with a mean of 0.249. Total 33 SNPs were used for polymorphism detection among the parents and 10 selected lines from mapping population Y13Zh (Zhenzhuhei × Yueyou13). Of the total 33 SNPs, nine SNPs showed polymorphism in the mapping population Y13Zh, and seven SNPs were successfully mapped into five linkage groups. Our results showed that SNPs can be identified in allotetraploid peanut with high accuracy through amplicon sequencing and HRM assay. The identified SNPs were very informative and can be used for different genetic and breeding applications in peanut.

Physical Mapping of Bread Wheat Chromosome 5A: An Integrated Approach.

The huge size, redundancy, and highly repetitive nature of the bread wheat [Triticum aestivum (L.)] genome, makes it among the most difficult species to be sequenced. To overcome these limitations, a strategy based on the separation of individual chromosomes or chromosome arms and the subsequent production of physical maps was established within the frame of the International Wheat Genome Sequence Consortium (IWGSC). A total of 95,812 bacterial artificial chromosome (BAC) clones of short-arm chromosome 5A (5AS) and long-arm chromosome 5A (5AL) arm-specific BAC libraries were fingerprinted and assembled into contigs by complementary analytical approaches based on the FingerPrinted Contig (FPC) and Linear Topological Contig (LTC) tools. Combined anchoring approaches based on polymerase chain reaction (PCR) marker screening, microarray, and sequence homology searches applied to several genomic tools (i.e., genetic maps, deletion bin map, neighbor maps, BAC end sequences (BESs), genome zipper, and chromosome survey sequences) allowed the development of a high-quality physical map with an anchored physical coverage of 75% for 5AS and 53% for 5AL with high portions (64 and 48%, respectively) of contigs ordered along the chromosome. In the genome of grasses, Brachypodium [Brachypodium distachyon (L.) Beauv.], rice (Oryza sativa L.), and sorghum [Sorghum bicolor (L.) Moench] homologs of genes on wheat chromosome 5A were separated into syntenic blocks on different chromosomes as a result of translocations and inversions during evolution. The physical map presented represents an essential resource for fine genetic mapping and map-based cloning of agronomically relevant traits and a reference for the 5A sequencing projects.

Phylogeny and photosynthesis of the grass tribe Paniceae.

The grass tribe Paniceae includes important food, forage, and bioenergy crops such as switchgrass, napiergrass, various millet species, and economically important weeds. Paniceae are also valuable for answering scientific and evolutionary questions about C4 photosynthetic evolution, drought tolerance, and spikelet variation. However, the phylogeny of the tribe remains incompletely resolved. Forty-five taxa were selected from across the tribe Paniceae and outgroups for genome survey sequencing (GSS). These data were used to build a phylogenetic tree of the Paniceae based on 102 markers (78 chloroplast, 22 mitochondrial, 2 nrDNA). Ancestral state reconstruction analyses were also performed within the Paniceae using both the traditional and two subtype classification systems to test hypotheses of C4 subtype evolution. The phylogenetic tree resolves many areas of the Paniceae with high support and provides insight into the origin and number of C4 evolution events within the tribe. The recovered phylogeny and ancestral state reconstructions support between four and seven independent origins of C4 photosynthesis within the tribe and indicate which species are potentially the closest C3 sister taxa of each of these events. Although the sequence of evolutionary events that produced multiple C4 subtypes within the Paniceae remains undetermined, the results presented here are consistent with only a subset of currently proposed models. The species used in this study constitute a panel of C3 and C4 grasses that are suitable for further studies on C4 photosynthesis, bioenergy, food and forage crops, and various developmental features of the Paniceae.