Genes affecting novel seed constituents in Limnanthes alba Benth: transcriptome analysis of developing embryos and a new genetic map of meadowfoam.

The seed oil of meadowfoam, a new crop in the Limnanthaceae family, is highly enriched in very long chain fatty acids that are desaturated at the Δ5 position. The unusual oil is desirable for cosmetics and innovative industrial applications and the seed meal remaining after oil extraction contains glucolimnanthin, a methoxylated benzylglucosinolate whose degradation products are herbicidal and anti-microbial. Here we describe EST analysis of the developing seed transcriptome that identified major genes involved in biosynthesis and assembly of the seed oil and in glucosinolate metabolic pathways. mRNAs encoding acyl-CoA Δ5 desaturase were notably abundant. The library was searched for simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). Fifty-four new SSR markers and eight candidate gene markers were developed and combined with previously developed SSRs to construct a new genetic map for Limnanthes alba. Mapped genes in the lipid biosynthetic pathway encode 3-ketoacyl-CoA synthase (KCS), Δ5 desaturase (Δ5DS), lysophosphatidylacyl-acyl transferase (LPAT), and acyl-CoA diacylglycerol acyl transferase (DGAT). Mapped genes in glucosinolate biosynthetic and degradation pathways encode CYP79A, myrosinase (TGG), and epithiospecifier modifier protein (ESM). The resources developed in this study will further the domestication and improvement of meadowfoam as an oilseed crop.

SSRs and INDELs mined from the sunflower EST database: abundance, polymorphisms, and cross-taxa utility.

Simple sequence repeats (SSRs) are abundant and frequently highly polymorphic in transcribed sequences and widely targeted for marker development in eukaryotes. Sunflower (Helianthus annuus) transcript assemblies were built and mined to identify SSRs and insertions-deletions (INDELs) for marker development, comparative mapping, and other genomics applications in sunflower. We describe the spectrum and frequency of SSRs identified in the sunflower EST database, a catalog of 16,643 EST-SSRs, a collection of 484 EST-SSR and 43 EST-INDEL markers developed from common sunflower ESTs, polymorphisms of the markers among the parents of several intraspecific and interspecific mapping populations, and the transferability of the markers to closely and distantly related species in the Compositae. Of 17,904 unigenes in the transcript assembly, 1,956 (10.9%) harbored one or more SSRs with repeat counts of n > or = 5. EST-SSR markers were 1.6-fold more polymorphic among exotic than elite genotypes and 0.7-fold less polymorphic than non-genic SSR markers. Of 466 EST-SSR or INDEL markers screened for cross-species amplification and polymorphisms, 413 (88.6%) amplified alleles from one or more wild species (H. argophyllus, H. tuberosus, H. anomalus, H. paradoxus, and H. deserticola), whereas 69 (14.8%) amplified alleles from safflower (Carthamus tinctorius) and 67 (14.4%) amplified alleles from lettuce (Lactuca sativa); hence, only a fraction were transferable to distantly related genera in the Compositae, whereas most were transferable to wild relatives of H. annuus. Several thousand additional SSRs were identified in the EST database and supply a wealth of templates for EST-SSR marker development in sunflower.

PCR-multiplexes for a genome-wide framework of simple sequence repeat marker loci in cultivated sunflower.

Simple sequence repeat (SSR) and other DNA sequence-tagged site markers can be genotyped more rapidly and cost efficiently by simultaneously amplifying multiple loci (multiplex PCR). The development of PCR-multiplexes for a nearly genome-wide framework of 78 SSR marker loci in cultivated sunflower ( Helianthus annuus L.) is described herein. The most outstanding single-locus SSR markers in the public collection (300 out of 1,089) were identified and screened for polymorphisms among 24 elite inbred lines, preparatory to selecting SSR markers for testing in multiplex PCRs. The selected SSR markers produced robust PCR products, amplified a single locus each, were polymorphic among elite inbred lines (minimum, mean and maximum heterozygosities were 0.08, 0.53 and 0.85, respectively), and supply a dense genome-wide framework of predominantly or completely codominant, single-locus DNA markers for molecular breeding and genomics research in sunflower. Thirteen six-locus multiplex PCRs were developed for 78 SSR marker loci strategically positioned throughout the sunflower genome (three to five per linkage group) by identifying compatible SSR primer combinations and optimizing multiplex PCR protocols. The multiplexed SSR markers, when coupled with 17 complementary SSR marker loci, create a 'standard genotyping' set ideal for first-pass scans of the genome, as are often needed when screening bulked-segregant DNA samples or mapping phenotypic trait loci. The minimum, mean and maximum heterozygosities of the multiplexed SSR markers were 0.38, 0.62 and 0.83, respectively. The PCR-multiplexes increase genotyping throughput, reduce reagent costs, and are ideal for repetitive genotyping applications where common sets of SSR marker loci are required or advantageous.