Linkage Maps of Lowland and Upland Tetraploid Switchgrass Ecotypes

Switchgrass (Panicum virgatum L.) is a native perennial warm season (C₄) grass that has been identified as a promising species for bioenergy research and production. Consequently, biomass yield and feedstock quality improvements are high priorities for switchgrass research. The objective of this study was to develop a switchgrass genetic linkage map using a full-sib pseudo-testcross mapping population derived from a cross between two heterozygous genotypes selected from the lowland cultivar ‘Alamo’ (AP13) and the upland cultivar ‘Summer’ (VS16). The female parent (AP13) map consists of 515 loci in 18 linkage groups (LGs) and spans 1,733 cM. The male parent (VS16) map arranges 363 loci in 17 LGs and spans 1,508 cM. No obvious cause for the lack of one LG in VS16 could be identified. Comparative analyses between the AP13 and VS16 maps showed that the two major ecotypic classes of switchgrass have highly colinear maps with similar recombination rates, suggesting that chromosomal exchange between the two ecotypes should be able to occur freely. The AP13 and VS16 maps are also highly similar with respect to marker orders and recombination levels to previously published switchgrass maps. The genetic maps will be used to identify quantitative trait loci associated with biomass and quality traits. The AP13 genotype was used for the whole genome-sequencing project and the map will thus also provide a tool for the anchoring of the switchgrass genome assembly.     

Investigation of genomic organization in switchgrass (Panicum virgatum L.) using DNA markers

We report an early investigation into genomic organization and chromosomal transmission in switchgrass based on restriction fragment length polymorphism (RFLP) markers. The segregation of 224 single dose restriction fragments (SDRF) in 85 full-sib progeny of a cross between the genotypes Alamo (AP13) and Summer (VS16) was used to determine linkage associations in each parent. In the seed parent AP13, 11 cosegregation groups were identified by 45 SDRF markers with a cumulative recombination length of 412.4 cM. In the pollen parent VS16, 57 SDRF markers were assigned to 16 cosegregation groups covering a length of 466.5 cM. SDRF markers identified by the same probes and mapping to different cosegregation groups were used to combine the two maps and identify homology groups. Eight homology groups were identified among the nine haploid linkage groups expected in switchgrass. The high incidence of repulsion phase associations indicates that preferential pairing between homologous chromosomes is predominant in switchgrass. Based on marker distribution in the paternal map (VS16), we estimated the recombinational length of switchgrass genome to be 4,617 cM. In order to link 95% of the genome to a marker at a 15-cM distance, a minimum of 459 markers will be required. Using information from the ratio of repulsion to coupling linkages, we infer that switchgrass is an autotetraploid with a high degree of preferential pairing. The information presented in this study establishes a foundation for extending genetic mapping in this crop and constitutes a framework for basic and applied genetic studies.Electronic Supplementary Material Supplementary material is available for this article at     

A genome-wide survey of switchgrass genome structure and organization

The perennial grass, switchgrass (Panicum virgatum L.), is a promising bioenergy crop and the target of whole genome sequencing. We constructed two bacterial artificial chromosome (BAC) libraries from the AP13 clone of switchgrass to gain insight into the genome structure and organization, initiate functional and comparative genomic studies, and assist with genome assembly. Together representing 16 haploid genome equivalents of switchgrass, each library comprises 101,376 clones with average insert sizes of 144 (HindIII-generated) and 110 kb (BstYI-generated). A total of 330,297 high quality BAC-end sequences (BES) were generated, accounting for 263.2 Mbp (16.4%) of the switchgrass genome. Analysis of the BES identified 279,099 known repetitive elements, >50,000 SSRs, and 2,528 novel repeat elements, named switchgrass repetitive elements (SREs). Comparative mapping of 47 full-length BAC sequences and 330K BES revealed high levels of synteny with the grass genomes sorghum, rice, maize, and Brachypodium. Our data indicate that the sorghum genome has retained larger microsyntenous regions with switchgrass besides high gene order conservation with rice. The resources generated in this effort will be useful for a broad range of applications.     

Production of Autopolyploid Lowland Switchgrass Lines Through In Vitro Chromosome Doubling

Switchgrass is considered one of the most promising energy crops. However, breeding of elite switchgrass cultivars is required to meet the challenges of large scale and sustainable biomass production. As a native perennial adapted to North America, switchgrass has lowland and upland ecotypes, where most lowland ecotypes are tetraploid (2n?=?4x?=?36), and most upland ecotypes are predominantly octoploid (2n?=?8x?=?72). Hybridization between lowland and upland switchgrass plants could identify new cultivars with heterosis. However, crossing between tetraploid and octoploid switchgrass is rare in nature. Therefore, in order to break down the cross incompatibility barrier between tetraploid lowland and octoploid upland switchgrass lines, we developed autoployploid switchgrass lines from an anueploid lowland cv. Alamo. In this study, colchicine was used in liquid and solid mediums to chemically induce chromosome doubling in embryogenic calli derived from cv. Alamo. Thirteen autopolyploid switchgrass lines were regenerated from seedlings and identified using flow cytometry. The autoplyploid switchgrass plants exhibited increased stomata aperture and stem size in comparison with the cv. Alamo. The most autooplyploid plants were regenerated from switchgrass calli that were treated with 0.04 % colchicine in liquid medium for 13 days. One autopolyploid switchgrass line, VT8-1, was successfully crossed to the octoploid upland cv. Blackwell. The autoployploid and the derived inter-ecotype hybrids were confirmed by in situ hybridization and molecular marker analysis. Therefore, the results of this study show that an autopolyploid, generated by chemically induced chromosome doubling of lowland cv. Alamo, is cross compatible with upland octoploid switchgrass cultivars. The outcome of this study may have significant applications in switchgrass hybrid breeding.     

A unigene set for European beech (Fagus sylvatica L.) and its use to decipher the molecular mechanisms involved in dormancy regulation

Systematic sequencing is the method of choice for generating genomic resources for molecular marker development and candidate gene identification in nonmodel species. We generated 47 357 Sanger ESTs and 2.2M Roche‐454 reads from five cDNA libraries for European beech (Fagus sylvatica L.). This tree species of high ecological and economic value in Europe is among the most representative trees of deciduous broadleaf forests. The sequences generated were assembled into 21 057 contigs with MIRA software. Functional annotations were obtained for 85% of these contigs, from the proteomes of four plant species, Swissprot accessions and the Gene Ontology database. We were able to identify 28 079 in silico SNPs for future marker development. Moreover, RNAseq and qPCR approaches identified genes and gene networks regulated differentially between two critical phenological stages preceding vegetative bud burst (the quiescent and swelling buds stages). According to climatic model‐based projection, some European beech populations may be endangered, particularly at the southern and eastern edges of the European distribution range, which are strongly affected by current climate change. This first genomic resource for the genus Fagus should facilitate the identification of key genes for beech adaptation and management strategies for preserving beech adaptability.     

High‐density linkage map reveals QTL underlying growth traits in AP13×VS16 biparental population of switchgrass

Switchgrass (Panicum virgatum L.), a native warm‐season perennial grass, is being considered as a feedstock for biofuel production in the United States. To expedite its genetic improvement and enhance genetic gain per selection cycle, application of marker‐assisted selection is indispensable. A high‐density linkage map was constructed in a pseudo‐F₁ testcross mapping population of AP13×VS16, consisting of 349 progenies. A total of 8,757 single nucleotide polymorphism (SNP) markers generated through genotype‐by‐sequencing (GBS) were used to construct the linkage map. The total map length spans up to 2,540.2 cM with the marker density of one marker in every 0.25–0.34 cM. Spring green‐up (SG), days to flowering (FL), and the vegetative growth period (VP) data were analyzed and used for quantitative trait loci (QTL) mapping. The population showed significant variations and exhibited transgressive segregation for SG, FL, and VP. QTL analyses were performed using trait mean of each year and location along with BLUP (best linear unbiased prediction) values of the traits. A total of 35, 37, and 34 QTL for SG, FL, and VP, respectively, were identified. Phenotypic variability explained by each QTL ranged from 11.29% to 27.85%. The additive genetic effects of individual QTL ranged from ?1.81 to 2.40, ?6.12 to 7.58, and ?16.01 to 6.38 for SG, FL, and VP, respectively. Comparing major QTL regions in the switchgrass genome, 20 candidate genes were identified which were reported to be involved in growth‐, development‐, and flowering‐related traits in switchgrass.     

Next‐generation DNA barcoding: using next‐generation sequencing to enhance and accelerate DNA barcode capture from single specimens

DNA barcoding is an efficient method to identify specimens and to detect undescribed/cryptic species. Sanger sequencing of individual specimens is the standard approach in generating large‐scale DNA barcode libraries and identifying unknowns. However, the Sanger sequencing technology is, in some respects, inferior to next‐generation sequencers, which are capable of producing millions of sequence reads simultaneously. Additionally, direct Sanger sequencing of DNA barcode amplicons, as practiced in most DNA barcoding procedures, is hampered by the need for relatively high‐target amplicon yield, coamplification of nuclear mitochondrial pseudogenes, confusion with sequences from intracellular endosymbiotic bacteria (e.g. Wolbachia) and instances of intraindividual variability (i.e. heteroplasmy). Any of these situations can lead to failed Sanger sequencing attempts or ambiguity of the generated DNA barcodes. Here, we demonstrate the potential application of next‐generation sequencing platforms for parallel acquisition of DNA barcode sequences from hundreds of specimens simultaneously. To facilitate retrieval of sequences obtained from individual specimens, we tag individual specimens during PCR amplification using unique 10‐mer oligonucleotides attached to DNA barcoding PCR primers. We employ 454 pyrosequencing to recover full‐length DNA barcodes of 190 specimens using 12.5% capacity of a 454 sequencing run (i.e. two lanes of a 16 lane run). We obtained an average of 143 sequence reads for each individual specimen. The sequences produced are full‐length DNA barcodes for all but one of the included specimens. In a subset of samples, we also detected Wolbachia, nontarget species, and heteroplasmic sequences. Next‐generation sequencing is of great value because of its protocol simplicity, greatly reduced cost per barcode read, faster throughout and added information content.     

The easy road to genome‐wide medium density SNP screening in a non‐model species: development and application of a 10 K SNP‐chip for the house sparrow (Passer domesticus)

With the advent of next generation sequencing, new avenues have opened to study genomics in wild populations of non‐model species. Here, we describe a successful approach to a genome‐wide medium density Single Nucleotide Polymorphism (SNP) panel in a non‐model species, the house sparrow (Passer domesticus), through the development of a 10 K Illumina iSelect HD BeadChip. Genomic DNA and cDNA derived from six individuals were sequenced on a 454 GS FLX system and generated a total of 1.2 million sequences, in which SNPs were detected. As no reference genome exists for the house sparrow, we used the zebra finch (Taeniopygia guttata) reference genome to determine the most likely position of each SNP. The 10 000 SNPs on the SNP‐chip were selected to be distributed evenly across 31 chromosomes, giving on average one SNP per 100 000 bp. The SNP‐chip was screened across 1968 individual house sparrows from four island populations. Of the original 10 000 SNPs, 7413 were found to be variable, and 99% of these SNPs were successfully called in at least 93% of all individuals. We used the SNP‐chip to demonstrate the ability of such genome‐wide marker data to detect population sub‐division, and compared these results to similar analyses using microsatellites. The SNP‐chip will be used to map Quantitative Trait Loci (QTL) for fitness‐related phenotypic traits in natural populations.     

Tissue expression and bioinformatics analysis of the vitellogenin gene of Asian arowana (Scleropages formosus)

The RACE technique was used to clone the full‐length vitellogenin (VTG) cDNA sequence of Asian arowana (Scleropages formosus). The full‐length sequence was 5,550 bp with an open reading frame of 5,238 bp, encoding 1,745 amino acids, and 5′ and 3′ UTRs (untranslated regions) of 45 bp and 267 bp, respectively. Phylogenetic analysis showed that S. formosus and silver arowana (Osteoglossum bicirrhosum) share a close evolutionary relationship (bootstrap 100%). The quantitative real‐time PCR results showed that vtg expression was significantly higher in liver and gonads of male and female fish compared with its expression in the other tissues tested (p < 0.01). The relative expression levels of vtg in liver, gland, kidney, heart, head kidney, and brain of female fish were significantly higher than in the corresponding tissues of male fish (p < 0.05).     

ANALYSIS OF EXPRESSED SEQUENCE TAGS FROM THE GREEN ALGA ULVA LINZA (CHLOROPHYTA)1

There is a general lack of genomic information available for chlorophyte seaweed genera such as Ulva, and in particular there is no information concerning the genes that contribute to adhesion and cell wall biosynthesis for this organism. Partial sequencing of cDNA libraries to generate expressed sequence tags (ESTs) is an effective means of gene discovery and characterization of expression patterns. In this study, a cDNA library was created from sporulating tissue of Ulva linza L. Initially, 650 ESTs were randomly selected from a cDNA library and sequenced from their 5′ ends to obtain an indication of the level of redundancy of the library (21%). The library was normalized to enrich for rarer sequences, and a further 1920 ESTs were sequenced. These sequences were subjected to contig assembly that resulted in a unigene set of approximately 1104 ESTs. Forty‐eight percent of these sequences exhibited significant similarity to sequences in the databases. Phylogenetic comparisons are made between selected sequences with similarity in the databases to proteins involved in aspects of extracellular matrix/cell wall assembly and adhesion.     

The Quantification of Representative Sequences pipeline for amplicon sequencing: case study on within‐population ITS1 sequence variation in a microparasite infecting Daphnia

Next generation sequencing (NGS) platforms are replacing traditional molecular biology protocols like cloning and Sanger sequencing. However, accuracy of NGS platforms has rarely been measured when quantifying relative frequencies of genotypes or taxa within populations. Here we developed a new bioinformatic pipeline (QRS) that pools similar sequence variants and estimates their frequencies in NGS data sets from populations or communities. We tested whether the estimated frequency of representative sequences, generated by 454 amplicon sequencing, differs significantly from that obtained by Sanger sequencing of cloned PCR products. This was performed by analysing sequence variation of the highly variable first internal transcribed spacer (ITS1) of the ichthyosporean Caullerya mesnili, a microparasite of cladocerans of the genus Daphnia. This analysis also serves as a case example of the usage of this pipeline to study within‐population variation. Additionally, a public Illumina data set was used to validate the pipeline on community‐level data. Overall, there was a good correspondence in absolute frequencies of C. mesnili ITS1 sequences obtained from Sanger and 454 platforms. Furthermore, analyses of molecular variance (amova ) revealed that population structure of C. mesnili differs across lakes and years independently of the sequencing platform. Our results support not only the usefulness of amplicon sequencing data for studies of within‐population structure but also the successful application of the QRS pipeline on Illumina‐generated data. The QRS pipeline is freely available together with its documentation under GNU Public Licence version 3 at http://code.google.com/p/quantification-representative-sequences .