Digital PCR for Genotype Quantification: A Case Study in a Pasta Production Chain

Digital polymerase chain reaction (dPCR) is a breakthrough technology based on the partitioning of the analytical sample and detection of individual end-point amplifications into the separate compartments. Among the numerous applications of this technology, its suitability in mutation detection is relevant and characterized by unprecedented levels of precision. The actual applicability of this analytical technique to quantify the presence of a specific plant genotype, in both raw materials and transformed products, by exploiting a point polymorphism has been evaluated. As proof of concept, an Italian premium pasta production chain was considered and a dPCR assay based on a durum wheat target variety private point mutation was designed and evaluated in supply-chain samples. From the results obtained, the assay can be applied to confirm the presence of a target variety and to quantify it in raw materials and transformed products, such as commercial grain lots and pasta. The performance, costs, and applicability of the assay has been compared to analytical alternatives, namely simple sequence repeats (SSRs) and genotype-by-sequencing based on Diversity Arrays Technology sequencing (DArTseqTM).

Identification of quantitative trait loci (QTL) controlling resistance to pea weevil (Bruchus pisorum) in a high-density integrated DArTseq SNP-based genetic map of pea

Pea weevil (Bruchus pisorum) is a damaging insect pest affecting pea (Pisum sativum) production worldwide. No resistant cultivars are available, although some levels of incomplete resistance have been identified in Pisum germplasm. To decipher the genetic control underlying the resistance previously identify in P. sativum ssp. syriacum, a recombinant inbred line (RIL F8:9) population was developed. The RIL was genotyped through Diversity Arrays Technology PL’s DArTseq platform and screened under field conditions for weevil seed infestation and larval development along 5 environments. A newly integrated genetic linkage map was generated with a subset of 6,540 markers, assembled into seven linkage groups, equivalent to the number of haploid pea chromosomes. An accumulated distance of 2,503 cM was covered with an average density of 2.61 markers cM−1. The linkage map allowed the identification of three QTLs associated to reduced seed infestation along LGs I, II and IV. In addition, a QTL for reduced larval development was also identified in LGIV. Expression of these QTLs varied with the environment, being particularly interesting QTL BpSI.III that was detected in most of the environments studied. This high-saturated pea genetic map has also allowed the identification of seven potential candidate genes co-located with QTLs for marker-assisted selection, providing an opportunity for breeders to generate effective and sustainable strategies for weevil control.

Verification of taxonomic relationships within the genus Secale (Poaceae: Pooideae: Triticeae) based on multiple molecular methods

This study aimed to verify the taxonomic relationships within the genus Secale. The plant material included 16 wild rye accessions from four species. Two approaches were applied: 1) whole genome scanning using three molecular marker systems: diversity arrays technology sequencing, simple sequence repeats and sequence-specific amplification polymorphism; and 2) characterisation based on polymorphisms within the sequences of two genes involved in benzoxazinoid biosynthesis: ScBx1 and ScBx5. Bayesian and neighbour-joining clustering and principal coordinate analysis were applied to illustrate relationships among species and accessions of Secale based on genetic distance (GD) matrices. Pearson’s correlation analysis between GD matrices was conducted. Clustering of Secale accessions revealed that S. sylvestre samples were the most divergent. The remaining accessions formed two clusters. One of them comprised S. strictum accessions while the second cluster consisted of subspecies of S. cereale, the species S. vavilovii and S. strictum subsp. ciliatoglume.