The growth habit of lateral shoots (also termed “branching habit”) is an important descriptive and agronomic character of peanut. Yet, both the inheritance of branching habit and the genetic mechanism that controls it in this crop remain unclear. In addition, the low degree of polymorphism among cultivated peanut varieties hinders fine-mapping of this and other traits in non-homozygous genetic structures. Here, we combined high-throughput sequencing with a well-defined genetic system to study these issues in peanut. Initially, segregating F2 populations derived from a reciprocal cross between very closely related Virginia-type peanut cultivars with spreading and bunch growth habits were examined. The spreading/bunch trait was shown to be controlled by a single gene with no cytoplasmic effect. That gene was named Bunch1 and was significantly correlated with pod yield per plant, time to maturation and the ratio of “dead-end” pods. Subsequently, bulked segregant analysis was performed on 52 completely bunch, and 47 completely spreading F3 families. In order to facilitate the process of SNP detection and candidate-gene analysis, the transcriptome was used instead of genomic DNA. Young leaves were sampled and bulked. Reads from Illumina sequencing were aligned against the peanut reference transcriptome and the diploid genomes. Inter-varietal SNPs were detected, scored and quality-filtered. Thirty-four candidate SNPs were found to have a bulk frequency ratio value >10 and 6 of those SNPs were found to be located in the genomic region of linkage group B5. Three best hits from that over-represented region were further analyzed in the segregating population. The trait locus was found to be located in a ~1.1 Mbp segment between markers M875 (B5:145,553,897; 1.9 cM) and M255 (B5:146,649,943; 2.25 cM). The method was validated using a population of recombinant inbreed lines of the same cross and a new DNA SNP-array. This study demonstrates the relatively straight-forward utilization of bulk segregant analysis for trait fine-mapping in the low polymeric and heterozygous germplasm of cultivated peanut and provides a baseline for candidate gene discovery and map-based cloning of Bunch1.

Real-time qPCR is currently the most sensitive technique available for the detection of lowlevel mRNA expression. For more reliable and precise gene expression analyses, real-time PCR data for a sequence of interest must be normalized against that of a control gene, which is uniformly expressed in various tissues and during different phases of development. So far, suitable internal controls for gene expression studies in peanut have not been identified. We assessed the expression of 10 frequently used housekeeping genes, specifically ubq10, gapdh, hel1, yls8, 14-3-3, 60s, ubc, ef-1a, act7, and adh3. Using the algorithms available through the GeNorm and NormFinder programs, the stability of their expression was estimated in a set of five diverse peanut tissue samples derived from a Virginiatype peanut cultivar (Shulamit). Collectively, the gene with the most stable expression across all of the examined tissues and both programs was adh3, followed by 60s and yls8, which had minimal estimated intra-and inter-tissue variation. The stability of two stable reference genes (adh3 and yls8) compared with two less stable (14-3-3 and ubq10) reference genes was validated in unpooled tissue samples from five peanut kernel developmental stages. Finally, the effect of the use of one or more reference genes on the observed relative expression levels of an important seed oil metabolism gene, diacylglycerol acyltransferase 1 (Dgat1), during kernel development was demonstrated. Based on findings, the suggestion is that adh3, or a combination of this gene with 60s and yls8 should be considered for use in quantitative mRNA expression analyses in Arachis, particularly in studies involving seed development; whereas ubq10 and gapdh should be avoided.

The stearoyl-acyl carrier protein (ACP) desaturase (SAD) is a nuclear-encoded, plastid-localized soluble desaturase that catalyzes the conversion of stearoyl-ACP to oleoyl-ACP and plays a key role in the determination of the properties of the majority of cellular glycerolipids. Sad genes from a variety of plant species have been cloned and characterized. However, in peanut (Arachis hypogaea), an important edible and oilseed crop, these genes have not yet been characterized. By searching peanut expressed sequence tag (EST) and parallel sequencing (454) libraries, we have identified three members of the ahSad gene family. Among them, only one gene, ahSad3, was exclusively expressed during seed development and in a manner fully corresponding to oil accumulation. Both ahSad3 homeologous genes (ahSad3A and ahSad3B) were recovered from the allotetraploid peanut, and their mRNA expression levels were characterized. The open reading frames for ahSad3A and ahSad3B are 98% identical and consist of 1,158 bp, encoding a 386-full-amino-acid protein, with one intron in the coding sequence. Comparisons of the sequences of these two homeologous genes revealed seven singlenucleotide polymorphisms and one triplet insertion in the coding region. Southern blot analysis indicated that there are only two copies of the ahSad3 gene in the peanut genome. Homeolog-specific gene expression analysis showed that both ahSad3 homeologs are expressed in developing seeds, but gene expression is significantly biased toward the B genome. Our results point to ahSad3 as a possible target gene for manipulation of fatty acid saturation in A. hypogaea.

In the in-shell peanut market, consumers favour the bright yellow shells characteristic of peanuts grown in sandy soils. Expanding cultivation of commercial varieties into areas with heavier soils has led to a less desirable brown tint, and reduced crop marketability. To overcome this, we evaluated a collection of 97 genotypes for shell colour when grown in sandy or semi-red soils to identify genotypes that have bright shells when grown in heavier soils. Residual maximum likelihood analyses of spectrophotometer-based parameters indicated significant genotype · soil interaction effects for all of the examined colour variables: brightness, red and yellow. In the heavier soil, there was also a significant correlation between maturity and the red variable. The pods of several early-maturing genotypes, mainly from the fastigiata group, had similar or better scores in the heavier soil than in the sandy soil. We did not observe any significant correlations between colour variables and other important pod traits, aside from maturity and shell thickness, highlighting the potential for introducing desirable pod colour into local cultivars to be grown in heavy soils.