Douglas Senalik

Using next-generation sequencing approaches to isolate simple sequence repeat (SSR) loci in the plant sciences

The application of next-generation sequencing (NGS) technologies for the development of simple sequence repeat (SSR) or microsatellite loci for genetic research in the botanical sciences is described. Microsatellite markers are one of the most informative and versatile DNA-based markers used in plant genetic research, but their development has traditionally been a difficult and costly process. NGS technologies allow the efficient identification of large numbers of microsatellites at a fraction of the cost and effort of traditional approaches. The major advantage of NGS methods is their ability to produce large amounts of sequence data from which to isolate and develop numerous genome-wide and gene-based microsatellite loci. The two major NGS technologies with emergent application in SSR isolation are 454 and Illumina. A review is provided of several recent studies demonstrating the efficient use of 454 and Illumina technologies for the discovery of microsatellites in plants. Additionally, important aspects during NGS isolation and development of microsatellites are discussed, including the use of computational tools and high-throughput genotyping methods. A data set of microsatellite loci in the plastome and mitochondriome of cranberry (Vaccinium macrocarpon Ait.) is provided to illustrate a successful application of 454 sequencing for SSR discovery. In the future, NGS technologies will massively increase the number of SSRs and other genetic markers available to conduct genetic research in understudied but economically important crops such as cranberry.

De novo assembly and characterization of the carrot transcriptome reveals novel genes, new markers, and genetic diversity

BackgroundAmong next generation sequence technologies, platforms such as Illumina and SOLiD produce short reads but with higher coverage and lower cost per sequenced nucleotide than 454 or Sanger. A challenge now is to develop efficient strategies to use short-read length platforms for de novo assembly and marker development. The scope of this study was to develop a de novo assembly of carrot ESTs from multiple genotypes using the Illumina platform, and to identify polymorphisms.ResultsA de novo assembly of transcriptome sequence from four genetic backgrounds produced 58,751 contigs and singletons. Over 50% of these assembled sequences were annotated allowing detection of transposable elements and new carrot anthocyanin genes. Presence of multiple genetic backgrounds in our assembly allowed the identification of 114 computationally polymorphic SSRs, and 20,058 SNPs at a depth of coverage of 20× or more. Polymorphisms were predominantly between inbred lines except for the cultivated x wild RIL pool which had high intra-sample polymorphism. About 90% and 88% of tested SSR and SNP primers amplified a product, of which 70% and 46%, respectively, were of the expected size. Out of verified SSR and SNP markers 84% and 82% were polymorphic. About 25% of SNPs genotyped were polymorphic in two diverse mapping populations.ConclusionsThis study confirmed the potential of short read platforms for de novo EST assembly and identification of genetic polymorphisms in carrot. In addition we produced the first large-scale transcriptome of carrot, a species lacking genomic resources.

De novo assembly of the carrot mitochondrial genome using next generation sequencing of whole genomic DNA provides first evidence of DNA transfer into an angiosperm plastid genome

BackgroundSequence analysis of organelle genomes has revealed important aspects of plant cell evolution. The scope of this study was to develop an approach for de novo assembly of the carrot mitochondrial genome using next generation sequence data from total genomic DNA.ResultsSequencing data from a carrot 454 whole genome library were used to develop a de novo assembly of the mitochondrial genome. Development of a new bioinformatic tool allowed visualizing contig connections and elucidation of the de novo assembly. Southern hybridization demonstrated recombination across two large repeats. Genome annotation allowed identification of 44 protein coding genes, three rRNA and 17 tRNA. Identification of the plastid genome sequence allowed organelle genome comparison. Mitochondrial intergenic sequence analysis allowed detection of a fragment of DNA specific to the carrot plastid genome. PCR amplification and sequence analysis across different Apiaceae species revealed consistent conservation of this fragment in the mitochondrial genomes and an insertion in Daucus plastid genomes, giving evidence of a mitochondrial to plastid transfer of DNA. Sequence similarity with a retrotransposon element suggests a possibility that a transposon-like event transferred this sequence into the plastid genome.ConclusionsThis study confirmed that whole genome sequencing is a practical approach for de novo assembly of higher plant mitochondrial genomes. In addition, a new aspect of intercompartmental genome interaction was reported providing the first evidence for DNA transfer into an angiosperm plastid genome. The approach used here could be used more broadly to sequence and assemble mitochondrial genomes of diverse species. This information will allow us to better understand intercompartmental interactions and cell evolution.

A high-quality carrot genome assembly provides new insights into carotenoid accumulation and asterid genome evolution

We report a high-quality chromosome-scale assembly and analysis of the carrot (Daucus carota) genome, the first sequenced genome to include a comparative evolutionary analysis among members of the euasterid II clade. We characterized two new polyploidization events, both occurring after the divergence of carrot from members of the Asterales order, clarifying the evolutionary scenario before and after radiation of the two main asterid clades. Large- and small-scale lineage-specific duplications have contributed to the expansion of gene families, including those with roles in flowering time, defense response, flavor, and pigment accumulation. We identified a candidate gene, DCAR_032551, that conditions carotenoid accumulation (Y) in carrot taproot and is coexpressed with several isoprenoid biosynthetic genes. The primary mechanism regulating carotenoid accumulation in carrot taproot is not at the biosynthetic level. We hypothesize that DCAR_032551 regulates upstream photosystem development and functional processes, including photomorphogenesis and root de-etiolation.

Microsatellite isolation and marker development in carrot - genomic distribution, linkage mapping, genetic diversity analysis and marker transferability across Apiaceae

BackgroundThe Apiaceae family includes several vegetable and spice crop species among which carrot is the most economically important member, with ~21 million tons produced yearly worldwide. Despite its importance, molecular resources in this species are relatively underdeveloped. The availability of informative, polymorphic, and robust PCR-based markers, such as microsatellites (or SSRs), will facilitate genetics and breeding of carrot and other Apiaceae, including integration of linkage maps, tagging of phenotypic traits and assisting positional gene cloning. Thus, with the purpose of isolating carrot microsatellites, two different strategies were used; a hybridization-based library enrichment for SSRs, and bioinformatic mining of SSRs in BAC-end sequence and EST sequence databases. This work reports on the development of 300 carrot SSR markers and their characterization at various levels.ResultsEvaluation of microsatellites isolated from both DNA sources in subsets of 7 carrot F2 mapping populations revealed that SSRs from the hybridization-based method were longer, had more repeat units and were more polymorphic than SSRs isolated by sequence search. Overall, 196 SSRs (65.1%) were polymorphic in at least one mapping population, and the percentage of polymophic SSRs across F2 populations ranged from 17.8 to 24.7. Polymorphic markers in one family were evaluated in the entire F2, allowing the genetic mapping of 55 SSRs (38 codominant) onto the carrot reference map. The SSR loci were distributed throughout all 9 carrot linkage groups (LGs), with 2 to 9 SSRs/LG. In addition, SSR evaluations in carrot-related taxa indicated that a significant fraction of the carrot SSRs transfer successfully across Apiaceae, with heterologous amplification success rate decreasing with the target-species evolutionary distance from carrot. SSR diversity evaluated in a collection of 65 D. carota accessions revealed a high level of polymorphism for these selected loci, with an average of 19 alleles/locus and 0.84 expected heterozygosity.ConclusionsThe addition of 55 SSRs to the carrot map, together with marker characterizations in six other mapping populations, will facilitate future comparative mapping studies and integration of carrot maps. The markers developed herein will be a valuable resource for assisting breeding, genetic, diversity, and genomic studies of carrot and other Apiaceae.

Genetic structure and domestication of carrot (Daucus carota subsp. sativus) (Apiaceae)

UNLABELLED PREMISE OF THE STUDY Analyses of genetic structure and phylogenetic relationships illuminate the origin and domestication of modern crops. Despite being an important worldwide vegetable, the genetic structure and domestication of carrot (Daucus carota) is poorly understood. We provide the first such study using a large data set of molecular markers and accessions that are widely dispersed around the world. • METHODS Sequencing data from the carrot transcriptome were used to develop 4000 single nucleotide polymorphisms (SNPs). Eighty-four genotypes, including a geographically well-distributed subset of wild and cultivated carrots, were genotyped using the KASPar assay. • KEY RESULTS Analysis of allelic diversity of SNP data revealed no reduction of genetic diversity in cultivated vs. wild accessions. Structure and phylogenetic analysis indicated a clear separation between wild and cultivated accessions as well as between eastern and western cultivated carrot. Among the wild carrots, those from Central Asia were genetically most similar to cultivated accessions. Furthermore, we found that wild carrots from North America were most closely related to European wild accessions. • CONCLUSIONS Comparing the genetic diversity of wild and cultivated accessions suggested the absence of a genetic bottleneck during carrot domestication. In conjunction with historical documents, our results suggest an origin of domesticated carrot in Central Asia. Wild carrots from North America were likely introduced as weeds with European colonization. These results provide answers to long-debated questions of carrot evolution and domestication and inform germplasm curators and breeders on genetic substructure of carrot genetic resources.

Modified protocols for rapid carrot genomic DNA extraction and AFLP™ analysis using silver stain or radioisotopes

AFLP™ is one of the most frequently used techniques for identification of molecular markers. We have modified the procedures for genomic DNA extraction, AFLP product generation and silver staining in order to speed up analyses and screen large numbers of plant samples. Using this protocol, we were able to achieve an 82% reduction of costs without compromising the reliability and quality of data gathered.

Path analysis suggests phytoene accumulation is the key step limiting the carotenoid pathway in white carrot roots

Two F2 carrot (Daucus carota L.) populations (orange rooted Brasilia x very dark orange rooted High Carotene Mass - HCM cross and the dark orange rooted cultivated variety B493 x white rooted wild carrot Queen Annes Lace - QAL cross) with very unrelated genetic backgrounds were used to investigate intrinsic factors limiting carotenoid accumulation in carrots by applying phenotypic correlation and path analysis to study the relationships between major root carotenes, root color and several other morphological traits. Most of the correlations between traits were close and agreed in sign between the two populations. Root weight had a moderate to highly significant positive correlation with leaf length, root length and top and middle root diameter. Although phenotypic correlations failed to identify the order of the substrates and products in the carotenoid pathway the correct order of substrates and products (phytoene ® zeta-carotene ® lycopene) was identified in the causal diagram of beta-carotene for the Brasilia x HCM population. Path analysis of beta-carotene synthesis in the B493 x QAL population suggested that selection for root carotenes had little effect on plant morphological traits. Causal model of beta-carotene and lycopene in the B493 x QAL population suggested that phytoene synthesis is the key step limiting the carotenoid pathway in white carrots. Path analysis, first presented by Sewall Wright to study quantitative traits, appears to be a powerful statistical approach for the identification of key compounds in complex pathways.

Comparative FISH mapping of Daucus species (Apiaceae family).

The cytogenetic characterization of the carrot genome (Daucus carota L., 2n = 18) has been limited so far, partly because of its somatic chromosome morphology and scant of chromosome markers. Here, we integrate the carrot linkage groups with pachytene chromosomes by fluorescent in situ hybridization (FISH) mapping genetically anchored bacterial artificial chromosomes (BACs). We isolated a satellite repeat from the centromeric regions of the carrot chromosomes, which facilitated the study of the pachytene-based karyotype and demonstrated that heterochromatic domains were mainly confined to the pericentromeric regions of each chromosome. Chromosome-specific BACs were used to: (1) physically locate genetically unanchored DNA sequences, (2) reveal relationships between genetic and physical distances, and (3) address chromosome evolution in Daucus. Most carrot BACs generated distinct FISH signals in 22-chromosome Daucus species, providing evidence for syntenic chromosome segments and rearrangements among them. These results provide a foundation for further cytogenetic characterization and chromosome evolution studies in Daucus.

Phylogenomics of the carrot genus (Daucus, Apiaceae).

UNLABELLED • PREMISE OF THE STUDY We explored the utility of multiple nuclear orthologs for the taxonomic resolution of wild and cultivated carrot, Daucus species.• METHODS We studied the phylogeny of 92 accessions of 13 species and two subspecies of Daucus and 15 accessions of related genera (107 accessions total) with DNA sequences of 94 nuclear orthologs. Reiterative analyses examined data of both alleles using ambiguity codes or a single allele with the highest coverage, trimmed vs. untrimmed homopolymers; pure exonic vs. pure intronic data; the use of all 94 markers vs. a reduced subset of markers; and analysis of a concatenated data set vs. a coalescent (species tree) approach.• KEY RESULTS Our maximum parsimony and maximum likelihood trees were highly resolved, with 100% bootstrap support for most of the external and many of the internal clades. They resolved multiple accessions of many different species as monophyletic with strong support, but failed to support other species. The single allele analysis gave slightly better topological resolution; trimming homopolymers failed to increase taxonomic resolution; the exonic data had a smaller proportion of parsimony-informative characters. Similar results demonstrating the same dominant topology can be obtained with many fewer markers. A Bayesian concordance analysis provided an overall similar phylogeny, but the coalescent analysis provided drastic changes in topology to all the above.• CONCLUSIONS Our research highlights some difficult species groups in Daucus and misidentifications in germplasm collections. It highlights a useful subset of markers and approaches for future studies of dominant topologies in Daucus.