Toshiya Yamamoto

Potential assessment of genome-wide association study and genomic selection in Japanese pear Pyrus pyrifolia

Although the potential of marker-assisted selection (MAS) in fruit tree breeding has been reported, bi-parental QTL mapping before MAS has hindered the introduction of MAS to fruit tree breeding programs. Genome-wide association studies (GWAS) are an alternative to bi-parental QTL mapping in long-lived perennials. Selection based on genomic predictions of breeding values (genomic selection: GS) is another alternative for MAS. This study examined the potential of GWAS and GS in pear breeding with 76 Japanese pear cultivars to detect significant associations of 162 markers with nine agronomic traits. We applied multilocus Bayesian models accounting for ordinal categorical phenotypes for GWAS and GS model training. Significant associations were detected at harvest time, black spot resistance and the number of spurs and two of the associations were closely linked to known loci. Genome-wide predictions for GS were accurate at the highest level (0.75) in harvest time, at medium levels (0.38–0.61) in resistance to black spot, firmness of flesh, fruit shape in longitudinal section, fruit size, acid content and number of spurs and at low levels (<0.2) in all soluble solid content and vigor of tree. Results suggest the potential of GWAS and GS for use in future breeding programs in Japanese pear.

A practical method for apple cultivar identification and parent-offspring analysis using simple sequence repeat markers

Differentiation of cultivars with simple sequence repeat (SSR) markers is a very useful technique for the true-to-type characterization of cultivars and clarification of parent-offspring relationships. We developed an SSR marker set for cultivar identification comprising 15 markers that were screened from 46 previously published SSRs. This marker set could be used for apple varieties including Malusxa0×xa0domestica and/or other Malus species. These SSRs successfully characterized 95 apples, including the leading and major founding cultivars used worldwide for modern apple breeding. Therefore, this marker set could be applied to almost all apple cultivars. We also analyzed the parent-offspring relationships of 69 cultivars by considering allele transmissions. This analysis revealed the true parentage of the following seven cultivars: ‘Kizashi’, ‘Chinatsu’, ‘Honey Queen’, ‘Haruka’, ‘Seirin’, ‘Ozenokurenai’, and Morioka #48. This analysis also revealed a parentage discrepancy for ‘Hacnine’. From the parent-offspring analysis, two microsatellite mutation events at alleles inherited from pollen parents were observed.

Genetic mapping of the crown gall resistance gene of the wild apple Malus sieboldii

Crown gall, caused by Agrobacterium tumefaciens, causes severe damage to apple saplings resulting in weak growth and loss of commercial value. Developing molecular markers linked to crown gall resistance genes, and establishing a marker-assisted selection (MAS) for such a trait would be an effective way to improve rootstock breeding for crown gall resistance. The wild apple Malus sieboldii Sanashi 63 carries the crown gall resistance gene Cg effective against the A. tumefaciens strain Peach CG8331 (biovar 2). Applying the genome scanning approach on the mapping population JM7 (cgcg) × Malus sieboldii Sanashi 63 (Cgcg), Cg was mapped on the linkage group (LG) 2. The constructed linkage map of LG 2 of Sanashi 63 spans 59.8xa0cM and has an average marker density of 3.5xa0cM per marker. The 191xa0bp allele of the simple sequence repeat (SSR) NZmsEB119405 co-segregated perfectly with Cg in a segregating population of 119 individuals. Quantitative trait loci, accounting for 75.3% to 84.3% of phenotypic variation were detected in the same position. Testing eight additional rootstocks with the NZmsEB119405 SSR marker revealed that the 191xa0bp allele is also present in crown gall-susceptible rootstock accessions. Only the markers CH03b01 and NZmsPal92 mapping at 0.9 and 4.3xa0cM from Cg, respectively, showed “private” alleles associated to Cg.

The genome sequence of sweet cherry (Prunus avium) for use in genomics-assisted breeding

We determined the genome sequence of sweet cherry (Prunus avium) using next-generation sequencing technology. The total length of the assembled sequences was 272.4u2009Mb, consisting of 10,148 scaffold sequences with an N50 length of 219.6u2009kb. The sequences covered 77.8% of the 352.9u2009Mb sweet cherry genome, as estimated by k-mer analysis, and includedu2009>96.0% of the core eukaryotic genes. We predicted 43,349 complete and partial protein-encoding genes. A high-density consensus map with 2,382 loci was constructed using double-digest restriction site-associated DNA sequencing. Comparing the genetic maps of sweet cherry and peach revealed high synteny between the two genomes; thus the scaffolds were integrated into pseudomolecules using map- and synteny-based strategies. Whole-genome resequencing of six modern cultivars found 1,016,866 SNPs and 162,402 insertions/deletions, out of which 0.7% were deleterious. The sequence variants, as well as simple sequence repeats, can be used as DNA markers. The genomic information helps us to identify agronomically important genes and will accelerate genetic studies and breeding programs for sweet cherries. Further information on the genomic sequences and DNA markers is available in DBcherry (http://cherry.kazusa.or.jp (8 May 2017, date last accessed)).

CRISPR/Cas9-mediated targeted mutagenesis in grape

RNA-guided genome editing using the CRISPR/Cas9 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has been applied successfully in several plant species. However, to date, there are few reports on the use of any of the current genome editing approaches in grape—an important fruit crop with a large market not only for table grapes but also for wine. Here, we report successful targeted mutagenesis in grape (Vitis vinifera L., cv. Neo Muscat) using the CRISPR/Cas9 system. When a Cas9 expression construct was transformed to embryonic calli along with a synthetic sgRNA expression construct targeting the Vitis vinifera phytoene desaturase (VvPDS) gene, regenerated plants with albino leaves were obtained. DNA sequencing confirmed that the VvPDS gene was mutated at the target site in regenerated grape plants. Interestingly, the ratio of mutated cells was higher in lower, older, leaves compared to that in newly appearing upper leaves. This result might suggest either that the proportion of targeted mutagenized cells is higher in older leaves due to the repeated induction of DNA double strand breaks (DSBs), or that the efficiency of precise DSBs repair in cells of old grape leaves is decreased.

Genome-wide association study and genomic prediction using parental and breeding populations of Japanese pear ( Pyrus pyrifolia Nakai)

Breeding of fruit trees is hindered by their large size and long juvenile period. Genome-wide association study (GWAS) and genomic selection (GS) are promising methods for circumventing this hindrance, but preparing new large datasets for these methods may not always be practical. Here, we evaluated the potential of breeding populations evaluated routinely in breeding programs for GWAS and GS. We used a pear parental population of 86 varieties and breeding populations of 765 trees from 16 full-sib families, which were phenotyped for 18 traits and genotyped for 1,506 single nucleotide polymorphisms (SNPs). The power of GWAS and accuracy of genomic prediction were improved when we combined data from the breeding populations and the parental population. The accuracy of genomic prediction was improved further when full-sib data of the target family were available. The results suggest that phenotype data collected in breeding programs can be beneficial for GWAS and GS when they are combined with genome-wide marker data. The potential of GWAS and GS will be further extended if we can build a system for routine collection of the phenotype and marker genotype data for breeding populations.