Double Digest RADseq: An Inexpensive Method for De Novo SNP Discovery and Genotyping in Model and Non-Model Species

The ability to efficiently and accurately determine genotypes is a keystone technology in modern genetics, crucial to studies ranging from clinical diagnostics, to genotype-phenotype association, to reconstruction of ancestry and the detection of selection. To date, high capacity, low cost genotyping has been largely achieved via “SNP chip” microarray-based platforms which require substantial prior knowledge of both genome sequence and variability, and once designed are suitable only for those targeted variable nucleotide sites. This method introduces substantial ascertainment bias and inherently precludes detection of rare or population-specific variants, a major source of information for both population history and genotype-phenotype association. Recent developments in reduced-representation genome sequencing experiments on massively parallel sequencers (commonly referred to as RAD-tag or RADseq) have brought direct sequencing to the problem of population genotyping, but increased cost and procedural and analytical complexity have limited their widespread adoption. Here, we describe a complete laboratory protocol, including a custom combinatorial indexing method, and accompanying software tools to facilitate genotyping across large numbers (hundreds or more) of individuals for a range of markers (hundreds to hundreds of thousands). Our method requires no prior genomic knowledge and achieves per-site and per-individual costs below that of current SNP chip technology, while requiring similar hands-on time investment, comparable amounts of input DNA, and downstream analysis times on the order of hours. Finally, we provide empirical results from the application of this method to both genotyping in a laboratory cross and in wild populations. Because of its flexibility, this modified RADseq approach promises to be applicable to a diversity of biological questions in a wide range of organisms

A perspective on the measurement of time in plant disease epidemiology

The growth and development of plant pathogens and their hosts generally respond strongly to the temperature of their environment. However, most studies of plant pathology record pathogen/host measurements against physical time (e.g. hours or days) rather than thermal time (e.g. degree-days or degree-hours). This confounds the comparison of epidemiological measurements across experiments and limits the value of the scientific literature

Development of a RAD-Seq Based DNA Polymorphism Identification Software, AgroMarker Finder, and Its Application in Rice Marker-Assisted Breeding

Abstract Rapid and accurate genome-wide marker detection is essential to the marker-assisted breeding and functional genomics studies. In this work, we developed an integrated software, AgroMarker Finder (AMF: http://erp.novelbio.com/AMF), for providing graphical user interface (GUI) to facilitate the recently developed restriction-site associated DNA (RAD) sequencing data analysis in rice. By application of AMF, a total of 90,743 high-quality markers (82,878 SNPs and 7,865 InDels) were detected between rice varieties JP69 and Jiaoyuan5A. The density of the identified markers is 0.2 per Kb for SNP markers, and 0.02 per Kb for InDel markers. Sequencing validation revealed that the accuracy of genome-wide marker detection by AMF is 93%. In addition, a validated subset of 82 SNPs and 31 InDels were found to be closely linked to 117 important agronomic trait genes, providing a basis for subsequent marker-assisted selection (MAS) and variety identification. Furthermore, we selected 12 markers from 31 validated InDel markers to identify seed authenticity of variety Jiaoyuanyou69, and we also identified 10 markers closely linked to the fragrant gene BADH2 to minimize linkage drag for Wuxiang075 (BADH2 donor)/Jiachang1 recombinants selection. Therefore, this software provides an efficient approach for marker identification from RAD-seq data, and it would be a valuable tool for plant MAS and variety protection

Genetic dissection of fruit quality traits in the octoploid cultivated strawberry highlights the role of homoeo-QTL in their control

Fruit quality traits are major breeding targets in the Rosaceae. Several of the major Rosaceae species are current or ancient polyploids. To dissect the inheritance of fruit quality traits in polyploid fleshy fruit species, we used a cultivated strawberry segregating population comprising a 213 full-sibling F1 progeny from a cross between the variety ‘Capitola’ and the genotype ‘CF1116’. We previously developed the most comprehensive strawberry linkage map, which displays seven homoeology groups (HG), including each four homoeology linkage groups (Genetics 179:2045–2060, 2008). The map was used to identify quantitative trait loci (QTL) for 19 fruit traits related to fruit development, texture, colour, anthocyanin, sugar and organic acid contents. Analyses were carried out over two or three successive years on field-grown plants. QTL were detected for all the analysed traits. Because strawberry is an octopolyploid species, QTL controlling a given trait and located at orthologous positions on different homoeologous linkage groups within one HG are considered as homoeo-QTL. We found that, for various traits, about one-fourth of QTL were putative homoeo-QTL and were localised on two linkage groups. Several homoeo-QTL could be detected the same year, suggesting that several copies of the gene underlying the QTL are functional. The detection of some other homoeo-QTL was year-dependent. Therefore, changes in allelic expression could take place in response to environmental changes. We believe that, in strawberry as in other polyploid fruit species, the mechanisms unravelled in the present study may play a crucial role in the variations of fruit quality