Target Amplicon Sequencing for Genotyping Genome-Wide Single Nucleotide Polymorphisms Identified by Whole-Genome Resequencing in Peanut.

Genome-wide genotyping data regarding breeding materials are essential resources for improving breeding efficiency, especially in plants with complex genomes with a high degree of polyploidy. Several current breeding efforts in cultivated peanut ( L.), which has a tetraploid genome, are devoted to developing high oleic acid cultivars. Genetic maps for such breeding programs have been developed using simple-sequence repeat (SSR) markers, the use of which requires time-consuming electrophoretic analyses. Next-generation sequencing (NGS) technology can overcome this technical hurdle. Initially, we attempted double-digest restriction site-associated DNA sequencing on peanut breeding materials used to develop high oleic acid cultivars. However, this method was not effective because few single nucleotide polymorphism (SNPs) were available because of low genetic diversity of the lines. The genome sequences of the probable diploid ancestors of cultivated peanut, Krapov. & W. C. Greg. and Krapov. & W. C. Greg., are available. Therefore, we next employed whole-genome resequencing analysis to obtain genome-wide SNP data. In this analysis, we observed large biases in the numbers and genomic positions of interspecific and intraspecific SNPs. For genome-wide genotyping, we selected a subset of SNPs covering the peanut genome as the targets of amplicon sequencing analysis. Using this technique, genome-wide genotypes of the breeding materials were easily and rapidly determined. The SNP information and analytic methods developed in this study should accelerate genetics, genomics, and breeding in peanut.

Transgenic tobacco lines expressing defective CMV replicase-derived dsRNA are resistant to CMV-O and CMV-Y.

Cucumber mosaic virus (CMV) is a tripartite, positive sense RNA virus causing infections and yield losses to many plant species. Here, we generated a construct containing inverted repeat of 1,793 bp fragment of defective CMV replicase gene derived from RNA2 of cucumber mosaic virus strain O (CMV-O). The replicase gene was modified by deleting a 9 bp region between nucleotides 1909-1918. This caused a deletion in the active centre motif of polymerases, producing defective translated product 9 nucleotides shorter than the full length protein. The RNAi construct containing inverted repeat of the defective gene was used to produce transgenic tobacco lines expressing CMV-derived double-stranded RNA via Agrobacterium-mediated transformation. Of the four transgenic lines inoculated with CMV-O or CMV-Y in vitro and ex vivo, three lines (T1, T4 and T5) showed immunity to both strains of CMV as no symptoms were detected, whereas one line (T7) exhibited high resistance with mild symptoms limited to inoculation portions. No virus could be detected in uninoculated new leaves of the transgenic lines after RT-PCR and Dot-immunobinding assay analyses. Small interfering RNAs present in transgenic lines before and after virus challenge indicates that the resistance was acquired through RNA silencing.

Construction of an integrated high density simple sequence repeat linkage map in cultivated strawberry (Fragaria × ananassa) and its applicability.

The cultivated strawberry (Fragaria × ananassa) is an octoploid (2n = 8x = 56) of the Rosaceae family whose genomic architecture is still controversial. Several recent studies support the AAA'A'BBB'B' model, but its complexity has hindered genetic and genomic analysis of this important crop. To overcome this difficulty and to assist genome-wide analysis of F. × ananassa, we constructed an integrated linkage map by organizing a total of 4474 of simple sequence repeat (SSR) markers collected from published Fragaria sequences, including 3746 SSR markers [Fragaria vesca expressed sequence tag (EST)-derived SSR markers] derived from F. vesca ESTs, 603 markers (F. × ananassa EST-derived SSR markers) from F. × ananassa ESTs, and 125 markers (F. × ananassa transcriptome-derived SSR markers) from F. × ananassa transcripts. Along with the previously published SSR markers, these markers were mapped onto five parent-specific linkage maps derived from three mapping populations, which were then assembled into an integrated linkage map. The constructed map consists of 1856 loci in 28 linkage groups (LGs) that total 2364.1 cM in length. Macrosynteny at the chromosome level was observed between the LGs of F. × ananassa and the genome of F. vesca. Variety distinction on 129 F. × ananassa lines was demonstrated using 45 selected SSR markers.