Conserved simple sequence repeats for the Limnanthaceae (Brassicales)

Abstract

The Limnanthaceae (Order Brassicales) is a family of 18 taxa of Limnanthes (meadowfoam) native to California, Oregon, and British Columbia. Cultivated meadowfoam ( L. alba Benth.), a recently domesticated plant, has been the focus of research and development as an industrial oilseed for three decades. The goal of the present research was to develop several hundred simple sequence repeat (SSR) markers for genetic mapping, molecular breeding, and genomics research in wild and cultivated meadowfoam taxa. We developed 389 SSR markers for cultivated meadowfoam by isolating and sequencing 1,596 clones from L. alba genomic DNA libraries enriched for AG(n) or AC(n) repeats, identifying one or more unique SSRs in 696 clone sequences, and designing and testing primers for 624 unique SSRs. The SSR markers were screened for cross- taxa utility and polymorphisms among ten of 17 taxa in the Limnanthaceae; 373 of these markers were polymorphic and 106 amplified loci from every taxon. Cross-taxa amplification percentages ranged from 37.3% in L. douglasii ssp. rosea (145/389) to 85.6% in L. montana (333/389). The SSR markers amplified 4,160 unique bands from 14 genotypes sampled from ten taxa (10.7 unique bands per SSR marker), of which 972 were genotype-specific. Mean and maximum haplotype heterozygosities were 0.71 and 0.90, respectively, among six L. alba genotypes and 0.63 and 0.93, respectively, among 14 genotypes (ten taxa). The SSR markers supply a critical mass of high-throughput DNA markers for biological and agricultural research across the Limnanthaceae and open the way to the development of a genetic linkage map for meadowfoam ( x = 5).

Glucosinolates in the new oilseed crop meadowfoam: natural variation in Section Inflexae of Limnanthes, a new glucosinolate in L. floccosa, and QTL analysis in L. alba

“…versicolor accession PI 374801 (Knapp and Crane 1997). DNA extracted from young leaves of 160 BC 1 plants was genotyped with 57 SSR markers (Kishore et al. 2004) that were polymorphic in the parents.…”

Genetic diversity in the USDA Limnanthes germplasm collection assessed by simple sequence repeats

“…The amplification of multiple loci in Limnanthes using SSR markers was also observed in a study by Kishore et al (2004), in which 1-28 unique bands were amplified for each SSR marker, with 56% of the primers amplifying multiple loci. Here, where samples were bulked, the level of allelic richness of 9.5 alleles per SSR primer set is comparable to the average number of alleles found in the survey of individual genotypes (10.7 per marker) in Kishore et al (2004). In other studies of SSRs in higher plants, where specific allelic status was unknown because of polyploidy, bulked samples found comparable levels of allelic richness.…”

“…A total of 140 SSLPs were identified using 15 SSR markers, ranging from 144 to 437 bp in size. Eleven of the fifteen SSR primer pairs amplified products within 10 bp of the expected size ranges reported by Kishore et al (2004). The remaining four primers ampli fied products within 50 bp of the expected ranges ( Table 2).…”

Conservation genetics of Butte County meadowfoam (Limnanthes floccosa ssp. californica Arroyo), an endangered vernal pool endemic

“…To develop informative SSR markers we screened 32 highly polymorphic primers with high rates of heterozygosity in test populations of L. alba (Kishore et al 2004). Of these we were able to adapt nine polymorphic markers suitable for genotyping with L. floccosa ssp.…”

“…Several sites (including DRCB-N, DRCB-W, SCH-W, SCH-E, Airport sites and BR; see Table 1 for site codes) had degraded leaf tissue (yellow or dried material) and were extracted with the Qiagen DNeasy kit using the manufacturer's protocol for frozen leaf tissue. PCR reactions were performed according to the methods in Kishore et al (2004), and using fluorescently labeled forward primers. PCR products were sized using an ABI 3100 16-capillary DNA analyzer and ABI GeneMapper 3.0 software (Applied Biosystems, Cupertino, CA).…”