A Standardized RNA Isolation Protocol for Yam (Dioscorea alata L) cDNA Library Construction

For the purpose of constructing yam cDNA libraries, attempts to isolate high quality RNA using several previously reported protocols were unsuccessful. Therefore a protocol was standardized for yam total RNA isolation by using guanidium buffer at the Department of Biology, Virginia State University. The RNA isolated using this standardized protocol was high in quality and led to successful good quality cDNA library construction and identification of functional ESTs in yam

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

<p>Abstract</p> <p>Background</p> <p>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 (<it>Sceloporus slevini</it>) collected from three sites in southeastern Arizona; the Sonoita Plain, Chiricahua Mountains and Huachuca Mountains.</p> <p>Findings</p> <p>The primers used for current investigation were originally designed for the Eastern Fence Lizard (<it>Sceloporus undulatus</it>). 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.</p> <p>Conclusions</p> <p>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.</p> <p>Trial Registration</p> <p>The research was conducted with Arizona Game and Fish Department scientific collecting permits SP565256, SP657407 & SP749119 to Dr. Christian A d'Orgeix.</p

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

<p>Abstract</p> <p>Background</p> <p>Anthracnose (<it>Colletotrichum </it><it>gloeosporioides</it>) is a major limiting factor in the production of yam (<it>Dioscorea </it>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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 <it>Dioscorea </it>species.</p

Alfalfa and flax sprouts supplementation enriches the content of bioactive compounds and lowers the cholesterol in hen egg

Abstract The effect of dietary supplementation with flax and alfalfa sprouts (40 g/d) on bioactive compounds and cholesterol contents of hen's egg was examined. Thirty White Leghorn hens, 26 weeks of age, were fed, for 66 days, three diets that included control (standard diet – C), standard diet + alfalfa sprouts (A), and standard diet + flax sprouts (F). Productive performance of hens was recorded daily. The cholesterol content of plasma and yolk, and the presence of bioactive compounds in the egg, were also analysed. Supplementation of flax and alfalfa sprouts reduced plasma and egg cholesterol probably due to the synergy between different compounds of the sprouts (polyunsaturated fatty acids - PUFAs, lignans, isoflavones and sterols). Eggs from A and F groups also had higher contents of n-3 PUFA, vitamins (α-tocopherol, α-, γ-tocotrienol, retinol), carotenes (β-carotene, lutein, zeaxanthin) and phytoestrogens (daidzein, equol, isolariciresinol) than eggs from the C group

ANTI-NUTRIENTS AND BIOACTIVE COMPOUNDS IN DROUGHT RESPONSIVE NATIVE TEPARY BEANS

ABSTRACT: Phytonutrients are gaining importance due to their contribution towards healthy nutrition to eliminate the risk of cancer and heart diseases. Food legumes were one of the richest sources of these compounds. Tepary bean is drought tolerant food legume native to USA preferred by various native and international communities and not explored to date for its seed quality traits. Twelve drought responsive cultivars were evaluated to know the content of antinutrients and antioxidants and their interaction with seed coat color (cultivar). The cultivars with brown and black colored seed coats showing high antioxidant activity compared to those with white color. The cultivars showed significant (p&lt;0.0001) variation for total phenolic content (0-6.59 mg of tannic acid equivalent/g), condensed tannins (0-12.54 % leucocyanad in equivalent), total tannins (0-6.48 mg of tannic acid equivalent/g) and antioxidant activity (0.56-19.72µmol/Trolox Equivalent/ g).Significant variation among the seed components and the cultivars was observed with high contents in seed coat followed by whole ground soaked seed, bean without seed coat and whole ground dry seed. The cultivars TB 4,7,18, 24, 29 were observed with high contents of phenols, tannins and antioxidant activity. The cultivars with high phenolic content are positively associated with high antioxidant activity and drought tolerance. The contents were high in seed coat compared to bean and whole seed due to accumulation of pigments and phenolic compounds in the seed coat and testa. The significant variation observed among cultivars indicated the genetic control of the trait to utilize in breeding new tepary varieties for these traits to produce healthy food grain by studying the nature of inheritance and heritability