Population structure and linkage disequilibrium in Lupinus albus L. germplasm and its implication for association mapping.

White lupin (Lupinus albus L.) has been around since 300 B.C. and is recognized for its ability to grow on poor soils and application as green manure in addition to seed harvest. The seed has very high levels of protein (33-47 %) and oil (6-13 %). It also has many secondary metabolites that are potentially of nutraceutical value to animals and humans. Despite such a great potential, lupins role in modern agriculture began only in the twentieth century. Although a large collection of Lupinus germplasm accessions is available worldwide, rarely have they been genetically characterized. Additionally, scarce genomic resources in terms of recombinant populations and genome information have been generated for L. albus. With the advancement in association mapping methods, the natural populations have the potential to replace the recombinant populations in gene mapping and marker-trait associations. Therefore, we studied the genetic similarity, population structure and marker-trait association in a USDA germplasm collection for their current and future application in this crop improvement. A total of 122 PI (Plant Inventory) lines were screened with 18 AFLP primer pairs that generated 2,277 fragments. A subset of 892 polymorphic markers with MAF >0.05 (minor allele frequency) were used for association mapping. The cluster analysis failed to group accessions on the basis of their passport information, and a weak structure and low linkage disequilibrium (LD) were observed indicating the usefulness of the collection for association mapping. Moreover, we were also able to identify two markers (a p value of 1.53 × 10(-4) and 2.3 × 10(-4)) that explained 22.69 and 20.5 % of seed weight variation determined using R (LR) (2) . The implications of lack of geographic clustering, population structure, low LD and the ability of AFLP to map seed weight trait using association mapping and the usefulness of the PI collections in breeding programs are discussed.

Generation and analysis of expressed sequence tags (ESTs) for marker development in yam (Dioscorea alata L.).

BACKGROUND: Anthracnose (Colletotrichum gloeosporioides) is a major limiting factor in the production of yam (Dioscorea spp.) worldwide. Availability of high quality sequence information is necessary for designing molecular markers associated with resistance. However, very limited sequence information pertaining to yam is available at public genome databases. Therefore, this collaborative project was developed for genetic improvement and germplasm characterization of yams using molecular markers. The current investigation is focused on studying gene expression, by large scale generation of ESTs, from one susceptible (TDa 95-0310) and two resistant yam genotypes (TDa 87-01091, TDa 95-0328) challenged with the fungus. Total RNA was isolated from young leaves of resistant and susceptible genotypes and cDNA libraries were sequenced using Roche 454 technology. RESULTS: A total of 44,757 EST sequences were generated from the cDNA libraries of the resistant and susceptible genotypes. Greater than 56% of ESTs were annotated using MapMan Mercator tool and Blast2GO search tools. Gene annotations were used to characterize the transcriptome in yam and also perform a differential gene expression analysis between the resistant and susceptible EST datasets. Mining for SSRs in the ESTs revealed 1702 unique sequences containing SSRs and 1705 SSR markers were designed using those sequences. CONCLUSION: We have developed a comprehensive annotated transcriptome data set in yam to enrich the EST information in public databases. cDNA libraries were constructed from anthracnose fungus challenged leaf tissues for transcriptome characterization, and differential gene expression analysis. Thus, it helped in identifying unique transcripts in each library for disease resistance. These EST resources provide the basis for future microarray development, marker validation, genetic linkage mapping and QTL analysis in Dioscorea species.

Optimization of PCR conditions to amplify microsatellite loci in the bunchgrass lizard (Sceloporus slevini) genomic DNA.

BACKGROUND: Microsatellites, also called Simple Sequence Repeats (SSRs), repetitions of nucleotide motifs of 1-5 bases, are currently the markers of choice due to their abundant distribution in the genomes, and suitability for high-throughput analysis. A total of five different primer pairs were optimized for polymerase chain reaction (PCR) to amplify microsatellite loci in total genomic DNA of bunchgrass lizards (Sceloporus slevini) collected from three sites in southeastern Arizona; the Sonoita Plain, Chiricahua Mountains and Huachuca Mountains. FINDINGS: The primers used for current investigation were originally designed for the Eastern Fence Lizard (Sceloporus undulatus). Five primer pairs were selected based on annealing temperatures for optimizing the PCR conditions to amplify with bunchgrass lizards. Different concentrations of DNA and annealing temperature were optimized. While keeping other reagents constant, a DNA concentration, 37.5 ng in the final reaction volume and PCR conditions of an initial denaturation of 94°C for five minutes, an annealing temperature of 55°C and final extension of 72°C for four minutes gave the best amplification for all the primer pairs. CONCLUSIONS: Modifying the standard protocol for annealing temperatures and final extension time increases the success of cross amplification of specific microsatellite loci in the bunchgrass lizard. A loading volume of 5 ul DNA at a concentration of 10 ng/ul and a 2% agarose for gel electrophoresis were observed the best for cross amplification of selected five primer pairs on bunch grass lizard. TRIAL REGISTRATION: The research was conducted with Arizona Game and Fish Department scientific collecting permits SP565256, SP657407 & SP749119 to Dr. Christian A d'Orgeix.