John McCallum

A Unique Set of 11,008 Onion Expressed Sequence Tags Reveals Expressed Sequence and Genomic Differences between the Monocot Orders Asparagales and Poales

Enormous genomic resources have been developed for plants in the monocot order Poales; however, it is not clear how representative the Poales are for the monocots as a whole. The Asparagales are a monophyletic order sister to the lineage carrying the Poales and possess economically important plants such as asparagus, garlic, and onion. To assess the genomic differences between the Asparagales and Poales, we generated 11,008 unique ESTs from a normalized cDNA library of onion. Sequence analyses of these ESTs revealed microsatellite markers, single nucleotide polymorphisms, and homologs of transposable elements. Mean nucleotide similarity between rice and the Asparagales was 78% across coding regions. Expressed sequence and genomic comparisons revealed strong differences between the Asparagales and Poales for codon usage and mean GC content, GC distribution, and relative GC content at each codon position, indicating that genomic characteristics are not uniform across the monocots. The Asparagales were more similar to eudicots than to the Poales for these genomic characteristics.

A low-density genetic map of onion reveals a role for tandem duplication in the evolution of an extremely large diploid genome

Abstract The bulb onion, Allium cepa L., is a diploid (2n=2x=16) plant with a huge nuclear genome. Previous genetic and cytogenetic analyses have not supported a polyploid origin for onion. We developed a low-density genetic map of morphological markers, randomly amplified polymorphic DNAs (RAPD), and restriction fragment length polymorphisms (RFLP) as a tool for onion improvement and to study the genome organization of onion. A mapping population of 58 F3 families was produced from a single F1 plant from the cross of two partially inbred lines (Brigham Yellow Globe 15-23 and Alisa Craig 43). Segregations were established for restoration of male fertility in sterile cytoplasm, complementary light-red bulb color, 14 RAPDs, 110 RFLPs revealed by 90 anonymous cDNA clones, and 2 RFLPs revealed by a cDNA clone of alliinase, the enzyme responsible for the characteristic Allium flavors. Duplicated RFLP loci were detected by 21% of the clones, of which 53% were unlinked (>30 cM), 5% loosely linked (10–30 cM), and 42% tightly linked (<10 cM). This duplication frequency is less than that reported for paleopolyploids but higher than for diploid species. We observed 40% dominant RFLPs, the highest yet reported among plants. Among duplicated RFLP loci, 19% segregated as two loci each with two codominant alleles, 52% segregated as one locus with codominant alleles and one locus with only a dominant fragment, and 29% segregated as two loci with only dominant fragments. We sequenced cDNAs detecting duplicated RFLPs; 63% showed homology to known gene families (e.g., chlorophyll binding proteins, ubiquitin, or RuBISCO), and 37% were unique clones showing significant homology to known genes of low-copy number or no homology to database sequences. Duplicated RFLPs showing linkage could be due to retroviral-like sequences in adjacent coding regions or intrachromosomal, as opposed to whole genome, duplications. Previous cytological analyses and this genetic map support intrachromosomal duplication as a mechanism contributing to the huge onion genome.

A linkage map of the pea (Pisum sativum L.) genome containing cloned sequences of known function and expressed sequence tags (ESTs)

Abstract A linkage map of the pea (Pisum sativum L.) genome is presented which is based on F2 plants produced by crossing the marrowfat cultivar ‘Primo’ and the blue-pea breeding line ‘OSU442-15’. This linkage map consists of 209 markers and covers 1330 cM (Kosambi units) and includes RFLP, RAPD and AFLP markers. By mapping a number of anchor loci, the ‘Primo’בOSU442-15’ map has been related to other pea linkage maps. A feature of the map is the incorporation of 29 loci representing genes of known function, obtained from other laboratories. The map also contains RFLP loci detected using sequence-characterized cDNA clones developed in our laboratory. The putative identities of 38 of these cDNA clones were assigned by examining public-sequence databases for protein or nucleotide-sequence similarities. The conversion of sequence-characterized pea cDNAs into PCR-amplifiable and polymorphic sequence-tagged sites (STSs) was investigated using 18 pairs of primers designed for single-copy sequences. Eleven polymorphic STSs were developed.

FLOWERING LOCUS T genes control onion bulb formation and flowering

Onion (Allium cepa L.) is a biennial crop that in temperate regions is planted in the spring and, after a juvenile stage, forms a bulb in response to the lengthening photoperiod of late spring/summer. The bulb then overwinters and in the next season it flowers and sets seed. FLOWERING LOCUS T (FT) encodes a mobile signaling protein involved in regulating flowering, as well as other aspects of plant development. Here we show that in onions, different FT genes regulate flowering and bulb formation. Flowering is promoted by vernalization and correlates with the upregulation of AcFT2, whereas bulb formation is regulated by two antagonistic FT-like genes. AcFT1 promotes bulb formation, while AcFT4 prevents AcFT1 upregulation and inhibits bulbing in transgenic onions. Long-day photoperiods lead to the downregulation of AcFT4 and the upregulation of AcFT1, and this promotes bulbing. The observation that FT proteins can repress and promote different developmental transitions highlights the evolutionary versatility of FT.

Genetic mapping of a major gene affecting onion bulb fructan content.

The non-structural dry matter content of onion bulbs consists principally of fructose, glucose, sucrose and fructans. The objective of this study was to understand the genetic basis for the wide variation observed in the relative amounts of these carbohydrates. Bulb carbohydrate composition was evaluated in progeny from crosses between high dry matter storage onion varieties and sweet, low dry matter varieties. When samples were analysed on a dry weight basis, reducing sugar and fructan content exhibited high negative correlations and bimodal segregation suggestive of the action of a major gene. A polymorphic SSR marker, ACM235, was identified which exhibited strong disequilibrium with bulb fructan content in F2:3 families from the ‘W202A’ × ‘Texas Grano 438’ mapping population evaluated in two environments. This marker was mapped to chromosome 8 in the interspecific population ‘Allium cepa × A. roylei’. Mapping in the ‘Colossal Grano PVP’ × ‘Early Longkeeper P12’ F2 population showed that a dominant major gene conditioning high-fructan content lay in the same genomic region. QTL analysis of total bulb fructan content in the intraspecific mapping population ‘BYG15-23’ × ‘AC43’ using a complete molecular marker map revealed only one significant QTL in the same chromosomal region. This locus, provisionally named Frc, may account for the major phenotypic differences in bulb carbohydrate content between storage and sweet onion varieties.

Linkage mapping of quantitative trait loci controlling seed weight in pea (Pisum sativum L.).

Quantitative trait loci (QTLs) affecting seed weight in pea (Pisum sativum L.) were mapped using two populations, a field-grown F2 progeny of a cross between two cultivated types (‘Primo’ and ‘OSU442-15’) and glasshouse-grown single-seed-descent recombinant inbred lines (RILs) from a wide cross between a P. sativum ssp. sativum line (‘Slow’) and a P. sativum ssp. humile accession (‘JI1794’). Linkage maps for these crosses consisted of 199 and 235 markers, respectively. QTLs for seed weight in the ‘Primo’ x ‘OSU442-15’ cross were identified by interval mapping, bulked segregant analysis, and selective genotyping. Four QTLs were identified in this cross, demonstrating linkage to four intervals on three linkage groups. QTLs for seed weight in the ‘JI1794’ x ‘Slow’ cross were identified by single-marker analyses. Linkage were demonstrated to four intervals on three linkage groups plus three unlinked loci. In the two crosses, only one common genomic region was identified as containing seed-weight QTLs. Seed-weight QTLs mapped to the same region of linkage group III in both crosses. Conserved linkage relationships were demonstrated for pea, mungbean (Vigna radiata L.), and cowpea (V. unguiculata L.) genomic regions containing seed-weight QTLs by mapping RFLP loci from the Vigna maps in the ‘Primo’ x ‘OSU442-15’ and ‘JI1794’ x ‘Slow’ crosses.

Expressed sequence markers for genetic analysis of bulb onion (Allium cepa L.)

Abstract  Sequencing of cDNA clones previously screened for ability to reveal RFLPs in bulb onion has been completed and a further 128 ESTs from 111 clones have been deposited in public databases. A putative function was assigned to 66% (84/128) of ESTs by BLASTX searches against public databases and FASTA comparisons were used to determine similarity among clones, including those which detected linked RFLP loci. Cleavage amplified polymorphisms (CAPs) and single-stranded conformation polymorphisms (SSCP) were evaluated as strategies for converting onion expressed sequence tags (ESTs) into PCR-based assays for gene mapping. We screened 14 ESTs with 8 to 12 restriction enzymes and detected two CAPs, which mapped in the ’Brigham Yellow Globe’ (BYG15–23)×’Ailsa Craig’ (AC43) mapping population. A wider survey of CAPs for ESTs among eight bulb onion populations with six frequently cutting restriction enzymes detected variation, but too little to be practical for routine gene mapping. By contrast, non-radioactive SSCP of amplicons from 3´ UTRs of ESTs was found to detect useful levels of variation within bulb onion germplasm. In addition to SSCPs, homo- and hetero-duplex polymorphisms (duplex polymorphisms) were also frequently observed on the same gels. Of a total of 31 ESTs surveyed, 26 exhibited SSCP/duplex variation among bulb onion populations. SSCP/duplex polymorphisms in 11 ESTs were mapped in the ’BYG15–23’×’AC43’ family and, of these, ten were linked to an RFLP locus revealed by the original cDNA. The SSCP/duplex assays of five additional ESTs showed Mendelian segregations in the ’Colossal Grano’×’Pukekohe Longkeeper’ (P12) F2 population. Two of these markers were linked, as predicted from linkage of their corresponding RFLPs in the ’BYG15–23’×’AC43’ family. Ninety two percent (12/13) of EST PCR products that amplified in Allium roylei exhibited marked differences in SSCP patterns from bulb onion. ESTs for invertase and sucrose-sucrose fructosyltransferase were mapped by SSCP and an ATP sulfurylase gene cloned by RT-PCR revealed SSCP/ duplex polymorphism within bulb onion. These results demonstrate that SSCP/duplex is an efficient and economical technique for exploiting onion EST information for gene mapping in onion.

A Toolkit for bulk PCR-based marker design from next-generation sequence data: application for development of a framework linkage map in bulb onion (Allium cepa L.)

BackgroundAlthough modern sequencing technologies permit the ready detection of numerous DNA sequence variants in any organisms, converting such information to PCR-based genetic markers is hampered by a lack of simple, scalable tools. Onion is an example of an under-researched crop with a complex, heterozygous genome where genome-based research has previously been hindered by limited sequence resources and genetic markers.ResultsWe report the development of generic tools for large-scale web-based PCR-based marker design in the Galaxy bioinformatics framework, and their application for development of next-generation genetics resources in a wide cross of bulb onion (Allium cepa L.). Transcriptome sequence resources were developed for the homozygous doubled-haploid bulb onion line ‘CUDH2150’ and the genetically distant Indian landrace ‘Nasik Red’, using 454™ sequencing of normalised cDNA libraries of leaf and shoot. Read mapping of ‘Nasik Red’ reads onto ‘CUDH2150’ assemblies revealed 16836 indel and SNP polymorphisms that were mined for portable PCR-based marker development. Tools for detection of restriction polymorphisms and primer set design were developed in BioPython and adapted for use in the Galaxy workflow environment, enabling large-scale and targeted assay design. Using PCR-based markers designed with these tools, a framework genetic linkage map of over 800cM spanning all chromosomes was developed in a subset of 93 F2 progeny from a very large F2 family developed from the ‘Nasik Red’ x ‘CUDH2150’ inter-cross. The utility of tools and genetic resources developed was tested by designing markers to transcription factor-like polymorphic sequences. Bin mapping these markers using a subset of 10 progeny confirmed the ability to place markers within 10 cM bins, enabling increased efficiency in marker assignment and targeted map refinement. The major genetic loci conditioning red bulb colour (R) and fructan content (Frc) were located on this map by QTL analysis.ConclusionsThe generic tools developed for the Galaxy environment enable rapid development of sets of PCR assays targeting sequence variants identified from Illumina and 454 sequence data. They enable non-specialist users to validate and exploit large volumes of next-generation sequence data using basic equipment.

AlliumMap-A comparative genomics resource for cultivated Allium vegetables

BackgroundVegetables of the genus Allium are widely consumed but remain poorly understood genetically. Genetic mapping has been conducted in intraspecific crosses of onion (Allium cepa L.), A. fistulosum and interspecific crosses between A. roylei and these two species, but it has not been possible to access genetic maps and underlying data from these studies easily.DescriptionAn online comparative genomics database, AlliumMap, has been developed based on the GMOD CMap tool at http://alliumgenetics.org. It has been populated with curated data linking genetic maps with underlying markers and sequence data from multiple studies. It includes data from multiple onion mapping populations as well as the most closely related species A. roylei and A. fistulosum. Further onion EST-derived markers were evaluated in the A. cepa x A. roylei interspecific population, enabling merging of the AFLP-based maps. In addition, data concerning markers assigned in multiple studies to the Allium physical map using A. cepa-A. fistulosum alien monosomic addition lines have been compiled. The compiled data reveal extensive synteny between onion and A. fistulosum.ConclusionsThe database provides the first online resource providing genetic map and marker data from multiple Allium species and populations. The additional markers placed on the interspecific Allium map confirm the value of A. roylei as a valuable bridge between the genetics of onion and A. fistulosum and as a means to conduct efficient mapping of expressed sequence markers in Allium. The data presented suggest that comparative approaches will be valuable for genetic and genomic studies of onion and A. fistulosum. This online resource will provide a valuable means to integrate genetic and sequence-based explorations of Allium genomes.

Complex formation between recombinant ATP sulfurylase and APS reductase of Allium cepa (L.)

Recombinant ATP sulfurylase (AcATPS1) and adenosine‐5′‐phosphosulfate reductase (AcAPR1) from Allium cepa have been used to determine if these enzymes form protein–protein complexes in vitro. Using a solid phase binding assay, AcAPR1 was shown to interact with AcATPS1. The AcAPR1 enzyme was also expressed in E. coli as the N‐terminal reductase domain (AcAPR1‐N) and the C‐terminal glutaredoxin domain (AcAPR1‐C), but neither of these truncated proteins interacted with AcATPS1. The solid‐phase interactions were confirmed by immune‐precipitation, where anti‐AcATPS1 IgG precipitated the full‐length AcAPR1 protein, but not AcAPR1‐N and AcAPR1‐C. Finally, using the ligand binding assay, full‐length AcATPS1 has been shown to bind to membrane‐localised full‐length AcAPR1. The significance of an interaction between chloroplastidic ATPS and APR in A. cepa is evaluated with respect to the control of the reductive assimilation of sulfate.