Translation of continuous artificial selection on phenotype into genotype during rice breeding programs.

Understanding genetic diversity among local populations is a primary goal of modern crop breeding programs. Here, we demonstrated the genetic relationships of rice varieties in Hokkaido, Japan, one of the northern limits of rice cultivation around the world. Furthermore, artificial selection during rice breeding programs has been characterized using genome sequences. We utilized 8,565 single nucleotide polymorphisms and insertion/deletion markers distributed across the genome in genotype-by-sequencing for genetic diversity analyses. Phylogenetics, genetic population structure, and principal component analysis showed that a total of 110 varieties were classified into four distinct clusters according to different populations geographically and historically. Furthermore, the genome sequences of 19 rice varieties along with historic representations in Hokkaido, nucleotide diversity and FST values in each cluster revealed that artificial selection of elite phenotypes focused on chromosomal regions. These results clearly demonstrated the history of the selections on agronomic traits as genome sequences among current rice varieties from Hokkaido.

Population structure of Nepali spring wheat (Triticum aestivum L.) germplasm.

BACKGROUND: Appropriate information about genetic diversity and population structure of germplasm improves the efficiency of plant breeding. The low productivity of Nepali bread wheat (Triticum aestivum L.) is a major concern particularly since Nepal is ranked the 4th most vulnerable nation globally to climate change. The genetic diversity and population structure of Nepali spring wheat have not been reported. This study aims to improve the exploitation of more diverse and under-utilized genetic resources to contribute to current and future breeding efforts for global food security. RESULTS: We used genotyping-by-sequencing (GBS) to characterize a panel of 318 spring wheat accessions from Nepal including 166 landraces, 115 CIMMYT advanced lines, and 34 Nepali released varieties. We identified 95 K high-quality SNPs. The greatest genetic diversity was observed among the landraces, followed by CIMMYT lines, and released varieties. Though we expected only 3 groupings corresponding to these 3 seed origins, the population structure revealed two large, distinct subpopulations along with two smaller and scattered subpopulations in between, with significant admixture. This result was confirmed by principal component analysis (PCA) and UPGMA distance-based clustering. The pattern of LD decay differed between subpopulations, ranging from 60 to 150 Kb. We discuss the possibility that germplasm explorations during the 1970s-1990s may have mistakenly collected exotic germplasm instead of local landraces and/or collected materials that had already cross-hybridized since exotic germplasm was introduced starting in the 1950s. CONCLUSION: We suggest that only a subset of wheat "landraces" in Nepal are authentic which this study has identified. Targeting these authentic landraces may accelerate local breeding programs to improve the food security of this climate-vulnerable nation. Overall, this study provides a novel understanding of the genetic diversity of wheat in Nepal and this may contribute to global wheat breeding initiatives.

Genotype-by-Sequencing Analysis of Mutations and Recombination in Pepper Progeny of Gamma-Irradiated Gametophytes.

The irradiation of dry seeds is the most widely-used irradiation method for improving seed-propagated crops; however, the irradiation of other tissues also has useful effects. The irradiation of plant reproductive organs, rather than seeds, for mutation breeding has advantages, such as producing non-chimeric progeny. However, the mutation frequency and spectrum produced using this method have not been analyzed on a genome-wide level. We performed a genotype-by-sequencing analysis to determine the frequencies of single-base substitutions and small (1-2 bp) insertions and deletions in hot pepper (Capsicum annuum L.) plants derived from crosses using gamma-irradiated female or male gametophytes. The progeny of irradiated gametophytes showed similar or higher DNA mutation frequencies, which were dependent on the irradiation dose and irradiated tissue, and less biased single base substitutions than progeny of irradiated seeds. These characteristics were expected to be beneficial for development of mutation population with a high frequency of small DNA mutations and performing reverse-genetics-based mutation screening. We also examined the possible use of this irradiation method in manipulating the meiotic recombination frequency; however, no statistically significant increase was detected. Our results provide useful information for further research and breeding using irradiated gametophytes.

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).

Comparative Analysis of Genotyping by Sequencing and Whole-Genome Sequencing Methods in Diversity Studies of Olea europaea L.

Olive, Olea europaea L., is a tree of great economic and cultural importance in the Mediterranean basin. Thousands of cultivars have been described, of which around 1200 are conserved in the different olive germplasm banks. The genetic characterisation of these cultivars can be performed in different ways. Whole-genome sequencing (WGS) provides more information than the reduced representation methods such as genotype by sequencing (GBS), but at a much higher cost. This may change as the cost of sequencing continues to drop, but, currently, genotyping hundreds of cultivars using WGS is not a realistic goal for most research groups. Our aim is to systematically compare both methodologies applied to olive genotyping and summarise any possible recommendations for the geneticists and molecular breeders of the olive scientific community. In this work, we used a selection of 24 cultivars from an olive core collection from the World Olive Germplasm Collection of the Andalusian Institute of Agricultural and Fisheries Research and Training (WOGBC), which represent the most of the cultivars present in cultivated fields over the world. Our results show that both methodologies deliver similar results in the context of phylogenetic analysis and popular population genetic analysis methods such as clustering. Furthermore, WGS and GBS datasets from different experiments can be merged in a single dataset to perform these analytical methodologies with proper filtering. We also tested the influence of the different olive reference genomes in this type of analysis, finding that they have almost no effect when estimating genetic relationships. This work represents the first comparative study between both sequencing techniques in olive. Our results demonstrate that the use of GBS is a perfectly viable option for replacing WGS and reducing research costs when the goal of the experiment is to characterise the genetic relationship between different accessions. Besides this, we show that it is possible to combine variants from GBS and WGS datasets, allowing the reuse of publicly available data.