Development of polymorphic microsatellite markers in Camellia chekiangoleosa (Theaceae) using 454-ESTs.

PREMISE OF THE STUDY: A set of microsatellite markers for Camellia chekiangoleosa was developed and characterized using 454 sequencing technology to study the population genetic structure and the diversity of germplasm collections. METHODS AND RESULTS: Eighteen polymorphic microsatellite markers were identified and tested in 150 individuals from three natural populations of C. chekiangoleosa. Alleles numbered from two to seven, and the observed and expected heterozygosities ranged from 0.100 to 0.760 and 0.133 to 0.809, respectively. CONCLUSIONS: These markers will potentially be conducive to further genetic studies on C. chekiangoleosa.

Resequencing and genome-wide association studies of autotetraploid potato.

Potato is the fourth most important food crop in the world. Although with a long history for breeding approaches, genomic information and association between genes and agronomic traits remain largely unknown particularly in autotetraploid potato cultivars, which limit the molecular breeding progression. By resequencing the genome of 108 main cultivar potato accessions with rich genetic diversity and population structure from International Potato Center, with approximate 20-fold coverage, we revealed more than 27 million Single Nucleotide Polymorphisms and ~ 3 million Insertion and Deletions with high quality and accuracy. Domestication analysis and genome-wide association studies (GWAS) identified candidate loci related to photoperiodic flowering time and temperature sensitivity as well as disease resistance, providing informative insights into the selection and domestication of cultivar potato. In addition, GWAS with GWASploy for 25 agronomic traits identified candidate loci by association signals, especially those related to tuber size, small-sized tuber weight and tuber thickness that was also validated by transcriptome analysis. Our study provides a valuable resource that facilitates the elucidation of domestication process as well as the genetic studies and agronomic improvement of autotetraploid potato.

Whole-genome sequencing reveals rare off-target mutations in CRISPR/Cas9-edited grapevine.

The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) system is a powerful tool for targeted genome editing, with applications that include plant biotechnology and functional genomics research. However, the specificity of Cas9 targeting is poorly investigated in many plant species, including fruit trees. To assess the off-target mutation rate in grapevine (Vitis vinifera), we performed whole-genome sequencing (WGS) of seven Cas9-edited grapevine plants in which one of two genes was targeted by CRISPR/Cas9 and three wild-type (WT) plants. In total, we identified between 202,008 and 272,397 single nucleotide polymorphisms (SNPs) and between 26,391 and 55,414 insertions/deletions (indels) in the seven Cas9-edited grapevine plants compared with the three WT plants. Subsequently, 3272 potential off-target sites were selected for further analysis. Only one off-target indel mutation was identified from the WGS data and validated by Sanger sequencing. In addition, we found 243 newly generated off-target sites caused by genetic variants between the Thompson Seedless cultivar and the grape reference genome (PN40024) but no true off-target mutations. In conclusion, we observed high specificity of CRISPR/Cas9 for genome editing of grapevine.

Transcriptome analysis of Ginkgo biloba kernels.

Ginkgo biloba is a dioecious species native to China with medicinally and phylogenetically important characteristics; however, genomic resources for this species are limited. In this study, we performed the first transcriptome sequencing for Ginkgo kernels at five time points using Illumina paired-end sequencing. Approximately 25.08-Gb clean reads were obtained, and 68,547 unigenes with an average length of 870 bp were generated by de novo assembly. Of these unigenes, 29,987 (43.74%) were annotated in publicly available plant protein database. A total of 3,869 genes were identified as significantly differentially expressed, and enrichment analysis was conducted at different time points. Furthermore, metabolic pathway analysis revealed that 66 unigenes were responsible for terpenoid backbone biosynthesis, with up to 12 up-regulated unigenes involved in the biosynthesis of ginkgolide and bilobalide. Differential gene expression analysis together with real-time PCR experiments indicated that the synthesis of bilobalide may have interfered with the ginkgolide synthesis process in the kernel. These data can remarkably expand the existing transcriptome resources of Ginkgo, and provide a valuable platform to reveal more on developmental and metabolic mechanisms of this species.