Development of an SSR marker-based genetic linkage map and identification of a QTL associated with flowering time in Eustoma.

Lisianthus (Eustoma grandiflorum) is an important floricultural crop cultivated worldwide. Despite its commercial importance, few DNA markers are available for molecular genetic research. In this study, we constructed a genetic linkage map and to detect quantitative trait loci (QTLs) for important agronomic traits of lisianthus. To develop simple sequence repeat (SSR) markers, we used 454-pyrosequencing technology to obtain genomic shotgun sequences and subsequently identified 8263 putative SSRs. A total of 3990 primer pairs were designed in silico and 1189 unique primer pairs were extracted through a BLAST search. Amplification was successful for more than 1000 primer pairs, and ultimately 278 SSR markers exhibited polymorphism between the two lisianthus accessions evaluated. Based on these markers, a genetic linkage map was constructed using a breeding population derived from crosses between the two accessions, for which flowering time differed (>140 days when grown under 20°C). We detected one QTL associated with flowering time (phenotypic variance, 27%; LOD value, 3.7). The SSR marker located at this QTL may account for variation in flowering time among accessions (i.e., three accessions whose nodes of the first flower were over 30 had late-flowering alleles of this QTL).

TASUKE+: a web-based platform for exploring genome-wide association studies results and large-scale resequencing data.

Recent revolutionary advancements in sequencing technologies have made it possible to obtain mass quantities of genome-scale sequence data in a cost-effective manner and have drastically altered molecular biological studies. To utilize these sequence data, genome-wide association studies (GWASs) have become increasingly important. Hence, there is an urgent need to develop a visualization tool that enables efficient data retrieval, integration of GWAS results with diverse information and rapid public release of such large-scale genotypic and phenotypic data. We developed a web-based genome browser TASUKE+ (https://tasuke.dna.affrc.go.jp/), which is equipped with the following functions: (i) interactive GWAS results visualization with genome resequencing data and annotation information, (ii) PCR primer design, (iii) phylogenetic tree reconstruction and (iv) data sharing via the web. GWAS results can be displayed in parallel with polymorphism data, read depths and annotation information in an interactive and scalable manner. Users can design PCR primers for polymorphic sites of interest. In addition, a molecular phylogenetic tree of any region can be reconstructed so that the overall relationship among the examined genomes can be understood intuitively at a glance. All functions are implemented through user-friendly web-based interfaces so that researchers can easily share data with collaborators in remote places without extensive bioinformatics knowledge.

Genomic dissection of a 'Fuji' apple cultivar: re-sequencing, SNP marker development, definition of haplotypes, and QTL detection.

'Fuji' is one of the most popular and highly-produced apple cultivars worldwide, and has been frequently used in breeding programs. The development of genotypic markers for the preferable phenotypes of 'Fuji' is required. Here, we aimed to define the haplotypes of 'Fuji' and find associations between haplotypes and phenotypes of five traits (harvest day, fruit weight, acidity, degree of watercore, and flesh mealiness) by using 115 accessions related to 'Fuji'. Through the re-sequencing of 'Fuji' genome, total of 2,820,759 variants, including single nucleotide polymorphisms (SNPs) and insertions or deletions (indels) were detected between 'Fuji' and 'Golden Delicious' reference genome. We selected mapping-validated 1,014 SNPs, most of which were heterozygous in 'Fuji' and capable of distinguishing alleles inherited from the parents of 'Fuji' (i.e., 'Ralls Janet' and 'Delicious'). We used these SNPs to define the haplotypes of 'Fuji' and trace their inheritance in relatives, which were shown to have an average of 27% of 'Fuji' genome. Analysis of variance (ANOVA) based on 'Fuji' haplotypes identified one quantitative trait loci (QTL) each for harvest time, acidity, degree of watercore, and mealiness. A haplotype from 'Delicious' chr14 was considered to dominantly cause watercore, and one from 'Ralls Janet' chr1 was related to low-mealiness.

TASUKE: a web-based visualization program for large-scale resequencing data.

SUMMARY: Because an enormous amount of sequence data is being collected, a method to effectively display sequence variation information is urgently needed. tasuke is a web application that visualizes large-scale resequencing data generated by next-generation sequencing technologies and is suitable for rapid data release to the public on the web. The variation and read depths of multiple genomes, as well as annotations, can be shown simultaneously at various scales. We demonstrate the use of TASUKE by applying it to 50 rice and 100 human genome resequencing datasets. AVAILABILITY AND IMPLEMENTATION: The tasuke program package and user manual are available from http://tasuke.dna.affrc.go.jp/. CONTACT: taitoh@affrc.go.jp.