Development and application of a 6.5 million feature affymetrix genechip® for massively parallel discovery of single position polymorphisms in lettuce (Lactuca spp.).

BMC Genomics (Impact Factor: 4.4). 05/2012; 13(1):185. DOI: 10.1186/1471-2164-13-185
Source: PubMed

ABSTRACT BACKGROUND: High-resolution genetic maps are needed in many crops to help characterize the genetic diversity that determines agriculturally important traits. Hybridization to microarrays to detect single feature polymorphisms is a powerful technique for marker discovery and genotyping because of its highly parallel nature. However, microarrays designed for gene expression analysis rarely provide sufficient gene coverage for optimal detection of nucleotide polymorphisms, which limits utility in species with low rates of polymorphism such as lettuce (Lactuca sativa). RESULTS: We developed a 6.5 million feature Affymetrix GeneChip® for efficient polymorphism discovery and genotyping, as well as for analysis of gene expression in lettuce. Probes on the microarray were designed from 26,809 unigenes from cultivated lettuce and an additional 8,819 unigenes from four related species (L. serriola, L. saligna, L. virosa and L. perennis). Where possible, probes were tiled with a 2 bp stagger, alternating on each DNA strand; providing an average of 187 probes covering approximately 600 bp for each of over 35,000 unigenes; resulting in up to 13 fold redundancy in coverage per nucleotide. We developed protocols for hybridization of genomic DNA to the GeneChip® and refined custom algorithms that utilized coverage from multiple, high quality probes to detect single position polymorphisms in 2 bp sliding windows across each unigene. This allowed us to detect greater than 18,000 polymorphisms between the parental lines of our core mapping population, as well as numerous polymorphisms between cultivated lettuce and wild species in the lettuce genepool. Using marker data from our diversity panel comprised of 52 accessions from the five species listed above, we were able to separate accessions by species using both phylogenetic and principal component analyses. Additionally, we estimated the diversity between different types of cultivated lettuce and distinguished morphological types. CONCLUSION: By hybridizing genomic DNA to a custom oligonucleotide array designed for maximum gene coverage, we were able to identify polymorphisms using two approaches for pair-wise.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Minimally processed salad packs often suffer from discolouration on cut leaf edges within a few days after harvest. This limits shelf life of the product and results in high wastage. Recombinant inbred lines (RILs) derived from a cross between lettuce cvs. Saladin and Iceberg were shown to be suitable for genetic analysis of postharvest discolouration traits in lettuce. An intra-specific linkage map based on this population was generated to enable genetic analysis. A total of 424 markers were assigned to 18 linkage groups covering all nine chromosomes. The linkage map has a total length of 1,040 cM with an average marker distance of 2.4 cM within the linkage groups and was anchored to the ultra-dense, transcript-based consensus map. Significant genetic variation in the postharvest traits 'pinking', 'browning' and 'overall discolouration' was detected among the RILs. Seven significant quantitative trait loci (QTL) were identified for postharvest discolouration traits providing markers linked to the QTL that can be used for marker-assisted selection. Phenotypic stability was confirmed for extreme lines possessing the corresponding QTL parental alleles and which had shown transgressive segregation. This study indicates that a desired phenotype with reduced levels of postharvest discolouration can be achieved by breeding using natural variation.
    Theoretical and Applied Genetics 08/2013; · 3.66 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Many cultivars of lettuce (Lactuca sativa L.), the most popular leafy vegetable, are susceptible to downy mildew disease caused by Bremia lactucae. Cultivars Iceberg and Grand Rapids that were released in the 18(th) and 19(th) centuries, respectively, have high levels of quantitative resistance to downy mildew. We developed a population of recombinant inbred lines (RILs) originating from a cross between these two legacy cultivars, constructed a linkage map, and identified two QTLs for resistance on linkage groups 2 (qDM2.1) and 5 (qDM5.1) that determined resistance under field conditions in California and the Netherlands. The same QTLs determined delayed sporulation at the seedling stage in laboratory experiments. Alleles conferring elevated resistance at both QTLs originate from cultivar Iceberg. An additional QTL on linkage group 9 (qDM9.1) was detected through simultaneous analysis of all experiments with mixed-model approach. Alleles for elevated resistance at this locus originate from cultivar Grand Rapids.
    Scientific Reports 01/2013; 3:2875. · 5.08 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Current knowledge of wild Lactuca L. species, their taxonomy, biogeography, gene-pools, germplasm collection quality and quantity, and accession availability is reviewed in this paper. Genetic diversity of Lactuca spp. is characterized at the level of phenotypic and phenological variation, variation in karyology and DNA content, biochemical traits, and protein and molecular polymorphism. The reported variation in reaction to pathogens and pests of wild Lactuca spp. is summarized, including the viral pathogens (Lettuce mosaic virus-LMV, Mirafiori lettuce virus/Lettuce big vein virus-LBV, Beet western yellows virus-BWYV, Tomato spotted wilt virus-TSWV, Cucumber mosaic virus-CMV, Lettuce necrotic stunt virus-LNSV), bacterial pathogens (corky root-Rhizomonas suberifaciens, bacterial leaf spot-Xanthomonas campestris pv. vitians), fungal pathogens (downy mildew-Bremia lactucae, powdery mildew-Golovinomyces cichoracearum, anthracnose-Microdochium panattoniana, stemphylium leaf spot-Stemphylium spp., sclerotinia drop-Sclerotinia spp., verticillium wilt-Verticillium dahliae, fusarium wilt-Fusarium spp., pythium wilt-Pythium tracheiphylum, P. uncinulatum), nematodes (potato cyst nematode-Globodera rostochiensis, root-knot nematode-Meloidogyne spp., incognita, hapla, javanica, enterolobii), insects and mites (the green lettuce aphid-Nasonovia ribisnigri, the green peach aphid-Myzus persicae, the potato aphid-Macrosiphum euphorbiae, leafminer-Liriomyza spp., L. langei). The approaches used to exploit wild Lactuca spp. in lettuce breeding (interspecific hybridization, cell and tissue culture, transformation) are dicussed, and known examples of lettuce cultivars with traits derived from wild Lactuca spp. are described.
    European Journal of Plant Pathology 03/2014; 138(3):597-640. · 1.71 Impact Factor


Available from
May 26, 2014