Eric N. Jellen

The genome of Chenopodium quinoa

Chenopodium quinoa (quinoa) is a highly nutritious grain identified as an important crop to improve world food security. Unfortunately, few resources are available to facilitate its genetic improvement. Here we report the assembly of a high-quality, chromosome-scale reference genome sequence for quinoa, which was produced using single-molecule real-time sequencing in combination with optical, chromosome-contact and genetic maps. We also report the sequencing of two diploids from the ancestral gene pools of quinoa, which enables the identification of sub-genomes in quinoa, and reduced-coverage genome sequences for 22 other samples of the allotetraploid goosefoot complex. The genome sequence facilitated the identification of the transcription factor likely to control the production of anti-nutritional triterpenoid saponins found in quinoa seeds, including a mutation that appears to cause alternative splicing and a premature stop codon in sweet quinoa strains. These genomic resources are an important first step towards the genetic improvement of quinoa.

Model SNP development for complex genomes based on hexaploid oat using high-throughput 454 sequencing technology

BackgroundGenetic markers are pivotal to modern genomics research; however, discovery and genotyping of molecular markers in oat has been hindered by the size and complexity of the genome, and by a scarcity of sequence data. The purpose of this study was to generate oat expressed sequence tag (EST) information, develop a bioinformatics pipeline for SNP discovery, and establish a method for rapid, cost-effective, and straightforward genotyping of SNP markers in complex polyploid genomes such as oat.ResultsBased on cDNA libraries of four cultivated oat genotypes, approximately 127,000 contigs were assembled from approximately one million Roche 454 sequence reads. Contigs were filtered through a novel bioinformatics pipeline to eliminate ambiguous polymorphism caused by subgenome homology, and 96 in silico SNPs were selected from 9,448 candidate loci for validation using high-resolution melting (HRM) analysis. Of these, 52 (54%) were polymorphic between parents of the Ogle1040 × TAM O-301 (OT) mapping population, with 48 segregating as single Mendelian loci, and 44 being placed on the existing OT linkage map. Ogle and TAM amplicons from 12 primers were sequenced for SNP validation, revealing complex polymorphism in seven amplicons but general sequence conservation within SNP loci. Whole-amplicon interrogation with HRM revealed insertions, deletions, and heterozygotes in secondary oat germplasm pools, generating multiple alleles at some primer targets. To validate marker utility, 36 SNP assays were used to evaluate the genetic diversity of 34 diverse oat genotypes. Dendrogram clusters corresponded generally to known genome composition and genetic ancestry.ConclusionsThe high-throughput SNP discovery pipeline presented here is a rapid and effective method for identification of polymorphic SNP alleles in the oat genome. The current-generation HRM system is a simple and highly-informative platform for SNP genotyping. These techniques provide a model for SNP discovery and genotyping in other species with complex and poorly-characterized genomes.

SNP Discovery via Genomic Reduction, Barcoding, and 454-Pyrosequencing in Amaranth

The grain amaranths are important pseudo‐cereals native to the New World. During the last decade they have garnered increased international attention for their nutritional quality, tolerance to abiotic stress, and importance as a symbol of indigenous cultures. We report the development of a novel genomic reduction protocol based on restriction‐site conservation and multiplex identifiers (MID) barcodes to discover single nucleotide polymorphisms (SNPs) from a pooled amaranth library of four mapping parents. The incorporation of MID barcodes allowed for DNA sample pooling, sequence deconvolution, and the identification of SNPs for specific mapping populations without additional genotyping. Approximately 1.3 million sequence‐reads with an average read length of 440 bp were collected from a single 454‐pyrosequencing run. Contigs specific to each of the four mapping populations were assembled. The assembled contigs had an average read‐length of 464 bp, and an average read‐depth of 16X. A total of 27,658 SNPs were observed across all populations. The average base coverage at all SNPs was 20X. Thirty‐four of 35 (97%) predicted SNPs were verified via resequencing and the random genomic distribution of the SNPs generated using this approach was shown in Arabidopsis. The method does not require a priori sequence information and should be readily adaptable to other species.

Development, Characterization, and Linkage Mapping of Single Nucleotide Polymorphisms in the Grain Amaranths (Amaranthus sp.)

The grain amaranths (Amaranthus sp.) are important pseudo‐cereals native to the New World. During the last decade they have garnered increased international attention for their nutritional quality, tolerance to abiotic stress, and importance as a symbol of indigenous cultures. We describe the development of the first single nucleotide polymorphism (SNP) assays for amaranth. In addition, we report the characterization of the first complete genetic linkage map in the genus. The SNP assays are based on KASPar genotyping chemistry and were detected using the Fluidigm dynamic array platform. A diversity screen of 41 accessions of the cultivated amaranth species and their putative ancestor species (Amaranth hybridus L.) showed that the minor allele frequency (MAF) of these markers ranged from 0.05 to 0.5 with an average MAF of 0.27 per SNP locus. One hundred and forty‐one of the SNP loci were considered highly polymorphic (MAF ≥ 0.3). Linkage mapping placed all 411 markers into 16 linkage groups, presumably corresponding to each of the 16 amaranth haploid chromosomes. The map spans 1288 cM with an average marker density of 3.1 cM per marker. The work reported here represents the initial first steps toward the genetic dissection of agronomically important characteristics in amaranth.

SNP Discovery and Chromosome Anchoring Provide the First Physically-Anchored Hexaploid Oat Map and Reveal Synteny with Model Species

A physically anchored consensus map is foundational to modern genomics research; however, construction of such a map in oat (Avena sativa L., 2n = 6x = 42) has been hindered by the size and complexity of the genome, the scarcity of robust molecular markers, and the lack of aneuploid stocks. Resources developed in this study include a modified SNP discovery method for complex genomes, a diverse set of oat SNP markers, and a novel chromosome-deficient SNP anchoring strategy. These resources were applied to build the first complete, physically-anchored consensus map of hexaploid oat. Approximately 11,000 high-confidence in silico SNPs were discovered based on nine million inter-varietal sequence reads of genomic and cDNA origin. GoldenGate genotyping of 3,072 SNP assays yielded 1,311 robust markers, of which 985 were mapped in 390 recombinant-inbred lines from six bi-parental mapping populations ranging in size from 49 to 97 progeny. The consensus map included 985 SNPs and 68 previously-published markers, resolving 21 linkage groups with a total map distance of 1,838.8 cM. Consensus linkage groups were assigned to 21 chromosomes using SNP deletion analysis of chromosome-deficient monosomic hybrid stocks. Alignments with sequenced genomes of rice and Brachypodium provide evidence for extensive conservation of genomic regions, and renewed encouragement for orthology-based genomic discovery in this important hexaploid species. These results also provide a framework for high-resolution genetic analysis in oat, and a model for marker development and map construction in other species with complex genomes and limited resources.

Assessment of genetic diversity in the USDA and CIP-FAO international nursery collections of quinoa (Chenopodium quinoa Willd.) using microsatellite markers

Quinoa ( Chenopodium quinoa Willd.) is a staple food crop for millions of impoverished rural inhabitants of Andean South America where it has been cultivated for millennia. Interest in quinoa, due largely to its superior nutritional characteristics, is fuelling a growing export market and has led to an increased focus on genetic research and the development of quinoa breeding programmes throughout South America. The success of these breeding programmes will rely heavily on the development of core germplasm collections and germplasm conservation. We report the development of a set of fluorescence-tagged microsatellite molecular markers that can be used to characterize genetic diversity within quinoa germplasm and we use this set of 36 microsatellites markers to genetically characterize the diversity of 121 accessions of C. quinoa held in the USDA germplasm bank, 22 accessions from the CIP-FAO international nursery collection and eight accessions representing parents from genetic mapping populations. A total of 420 alleles were detected among the quinoa accessions with an average of 11 alleles detected per microsatellite locus. Genetic heterogeneity was observed in 32% of the quinoa accessions at a given locus and suggests that many of these accessions represent heterogeneous seed lots or landraces. Both unweighted pair-group method with arithmetic averages (UPGMA) and principle components analysis (PCA) analyses partitioned the quinoa accessions into two main clusters. The first major cluster consisted of accessions from the Andean highlands of Peru, Bolivia, Ecuador, Argentina and extreme northeastern Chile. The other main cluster contained accessions from both the lowlands of Chile and a set of USDA accessions with no known passport data, collected by Emigdio Ballon. Using the patterns of genetic diversity detected within the C. quinoa accessions we discuss hypotheses regarding quinoas centre of diversity, including highland and lowland ecotype clustering patterns, origin of lowland varieties, origin of domestication, and diversity levels in the USDA and CIP-FAO collections.

A SNP Genotyping Array for Hexaploid Oat

Recognizing a need in cultivated hexaploid oat (Avena sativa L.) for a reliable set of reference single nucleotide polymorphisms (SNPs), we have developed a 6000 (6K) BeadChip design containing 257 Infinium I and 5486 Infinium II designs corresponding to 5743 SNPs. Of those, 4975 SNPs yielded successful assays after array manufacturing. These SNPs were discovered based on a variety of bioinformatics pipelines in complementary DNA (cDNA) and genomic DNA originating from 20 or more diverse oat cultivars. The array was validated in 1100 samples from six recombinant inbred line (RIL) mapping populations and sets of diverse oat cultivars and breeding lines, and provided approximately 3500 discernible Mendelian polymorphisms. Here, we present an annotation of these SNPs, including methods of discovery, gene identification and orthology, population‐genetic characteristics, and tentative positions on an oat consensus map. We also evaluate a new cluster‐based method of calling SNPs. The SNP design sequences are made publicly available, and the full SNP genotyping platform is available for commercial purchase from an independent third party.

Simple sequence repeat marker development and genetic mapping in quinoa (Chenopodium quinoa Willd.)

Quinoa is a regionally important grain crop in the Andean region of South America. Recently quinoa has gained international attention for its high nutritional value and tolerances of extreme abiotic stresses. DNA markers and linkage maps are important tools for germplasm conservation and crop improvement programmes. Here we report the development of 216 new polymorphic SSR (simple sequence repeats) markers from libraries enriched for GA, CAA and AAT repeats, as well as 6 SSR markers developed from bacterial artificial chromosome-end sequences (BES-SSRs). Heterozygosity (H) values of the SSR markers ranges from 0.12 to 0.90, with an average value of 0.57. A linkage map was constructed for a newly developed recombinant inbred lines (RIL) population using these SSR markers. Additional markers, including amplified fragment length polymorphisms (AFLPs), two 11S seed storage protein loci, and the nucleolar organizing region (NOR), were also placed on the linkage map. The linkage map presented here is the first SSR-based map in quinoa and contains 275 markers, including 200 SSR. The map consists of 38 linkage groups (LGs) covering 913 cM. Segregation distortion was observed in the mapping population for several marker loci, indicating possible chromosomal regions associated with selection or gametophytic lethality. As this map is based primarily on simple and easily-transferable SSR markers, it will be particularly valuable for research in laboratories in Andean regions of South America.

Assignment of RFLP linkage groups to chromosomes using monosomic F1 analysis in hexaploid oat

Abstract The availability of molecular genetic maps in oat (Avena spp.) and improved identification of chromosomes by C-banding are two recent developments that have made locating linkage groups to chromosomes possible in cultivated hexaploid oat, 2n=6x=42. Monosomic series derived from Avena byzantina C. Koch cv Kanota and from Avena sativa L. cv Sun II were used as maternal plants in crosses with the parents, Kanota-1 and Ogle-C, of the oat RFLP mapping population. Monosomic F1 plants were identified by root-tip cell chromosome counts. For marker analysis, DNAs of eight F2 plants from a monosomic F1 were combined to provide a larger source of DNA that mimicked that of the monosomic F1 plant. Absence of maternal alleles in monosomic F1s served to associate linkage groups with individual chromosomes. Twenty two linkage groups were associated with 16 chromosomes. In seven instances, linkage groups that were independent of each other in recombination analyses were associated with the same chromosome. Five linkage groups were shown to be associated with translocation differences among oat lines. Additionally, the results better-characterized the oat monosomic series through the detection of duplicates and translocation differences among the various monosomic lines. The F1 monosomic series represents a powerful cytogenetic tool with the potential to greatly improve understanding of the oat genome.

A consensus map in cultivated hexaploid oat reveals conserved grass synteny with substantial subgenome rearrangement

We constructed a hexaploid oat consensus map from 12 populations representing 19 parents. The map represents the most common physical chromosome arrangements in oat. Deviations from the consensus map may indicate physical rearrangements. Large chromosomal translocations vary among different varieties. There is regional synteny with rice but considerable subgenome rearrangement.