Genome-wide association study of individual sugar content in fruit of Japanese pear (Pyrus spp.).

BACKGROUND: Understanding mechanisms of sugar accumulation and composition is essential to determining fruit quality and maintaining a desirable balance of sugars in plant storage organs. The major sugars in mature Rosaceae fruits are sucrose, fructose, glucose, and sorbitol. Among these, sucrose and fructose have high sweetness, whereas glucose and sorbitol have low sweetness. Japanese pear has extensive variation in individual sugar contents in mature fruit. Increasing total sugar content and that of individual high-sweetness sugars is a major target of breeding programs. The objective of this study was to identify quantitative trait loci (QTLs) associated with fruit traits including individual sugar accumulation, to infer the candidate genes underlying the QTLs, and to assess the potential of genomic selection for breeding pear fruit traits. RESULTS: We evaluated 10 fruit traits and conducted genome-wide association studies (GWAS) for 106 cultivars and 17 breeding populations (1112 F1 individuals) using 3484 tag single-nucleotide polymorphisms (SNPs). By implementing a mixed linear model and a Bayesian multiple-QTL model in GWAS, 56 SNPs associated with fruit traits were identified. In particular, a SNP located close to acid invertase gene PPAIV3 on chromosome 7 and a newly identified SNP on chromosome 11 had quite large effects on accumulation of sucrose and glucose, respectively. We used 'Golden Delicious' doubled haploid 13 (GDDH13), an apple reference genome, to infer the candidate genes for the identified SNPs. In the region flanking the SNP on chromosome 11, there is a tandem repeat of early responsive to dehydration (ERD6)-like sugar transporter genes that might play a role in the phenotypes observed. CONCLUSIONS: SNPs associated with individual sugar accumulation were newly identified at several loci, and candidate genes underlying QTLs were inferred using advanced apple genome information. The candidate genes for the QTLs are conserved across Pyrinae genomes, which will be useful for further fruit quality studies in Rosaceae. The accuracies of genomic selection for sucrose, fructose, and glucose with genomic best linear unbiased prediction (GBLUP) were relatively high (0.67-0.75), suggesting that it would be possible to select individuals having high-sweetness fruit with high sucrose and fructose contents and low glucose content.

Development of an SSR marker set for efficient selection for resistance to black spot disease in pear breeding.

Black spot disease, which is caused by Alternaria alternata (Fries) Keissler Japanese pear pathotype, is one of the most harmful diseases in Japanese pear cultivation. Because of the potential harm of fungicides to consumers and the environment, resistant cultivars are desired. In this study, to enable efficient marker-assisted selection in pear breeding, we conducted comprehensive inoculation tests and genotyping with 207 pear cultivars. We identified a marker set (Mdo.chr11.27 and Mdo.chr11.34) suitable for selection for black spot resistance. In most susceptible cultivars, Mdo.chr11.27 amplified a 220-bp band and Mdo.chr11.34 amplified a 259-bp band. The genotype of Mdo.chr11.34 corresponds perfectly to the estimated genotype of Japanese pears susceptible to black spot disease. Using linkage analysis, we identified the positions of the gene for susceptibility to black spot disease in Chinese pear. Mdo.chr11.27 and Mdo.chr11.34 were tightly linked to susceptibility in Chinese pear, and the susceptibility gene was mapped at the top of linkage group 11, similar to that in Japanese pear. This marker set and the accumulation of phenotypic data will enable efficient marker-assisted breeding for black spot resistance in pear breeding.

A self-compatible pear mutant derived from γ-irradiated pollen carries an 11-Mb duplication in chromosome 17.

Self-compatibility is a highly desirable trait for pear breeding programs. Our breeding program previously developed a novel self-compatible pollen-part Japanese pear mutant (Pyrus pyrifolia Nakai), '415-1', by using γ-irradiated pollen. '415-1' carries the S-genotype S4dS5S5, with "d" indicating a duplication of S 5 responsible for breakdown of self-incompatibility. Until now, the size and inheritance of the duplicated segment was undetermined, and a reliable detection method was lacking. Here, we examined genome duplications and their inheritance in 140 F1 seedlings resulting from a cross between '515-20' (S1S3) and '415-1'. Amplicon sequencing of S-RNase and SFBB18 clearly detected S-haplotype duplications in the seedlings. Intriguingly, 30 partially triploid seedlings including genotypes S1S4dS5, S3S4dS5, S1S5dS5, S3S5dS5, and S3S4dS4 were detected among the 140 seedlings. Depth-of-coverage analysis using ddRAD-seq showed that the duplications in those individuals were limited to chromosome 17. Further analysis through resequencing confirmed an 11-Mb chromosome duplication spanning the middle to the end of chromosome 17. The duplicated segment remained consistent in size across generations. The presence of an S3S4dS4 seedling provided evidence for recombination between the duplicated S5 segment and the original S4haplotype, suggesting that the duplicated segment can pair with other parts of chromosome 17. This research provides valuable insights for improving pear breeding programs using partially triploid individuals.

Rapid and easy construction of a simplified amplicon sequencing (simplified AmpSeq) library for marker-assisted selection.

Marker-assisted selection (MAS) is fundamental for plant breeding programs, as it can identify desirable seedlings at a young stage and reduce the cost, time and space needed for plant maintenance, especially for perennial crops. To facilitate the process of genotyping, which is time consuming and laborious, we developed a simplified amplicon sequencing (simplified AmpSeq) library construction method for next-generation sequencing that can be applied to MAS in breeding programs. The method is based on one-step PCR with a mixture of two primer sets: the first consisting of tailed target primers, the second of primers that contain flow-cell binding sites, indexes and tail sequences complementary to those in the first set. To demonstrate the process of MAS using s implified AmpSeq, we created databases of genotypes for important traits by using cultivar collections including triploid cultivars and segregating seedlings of Japanese pear (Pyrus pyrifolia Nakai), Japanese chestnut (Castanea crenata Sieb. et Zucc.) and apple (Malus domestica Borkh.). Simplified AmpSeq has the advantages of high repeatability, ability to estimate allele number in polyploid species and semi-automatic evaluation using target allele frequencies. Because this method provides high flexibility for designing primer sets and targeting any variant, it will be useful for plant breeding programs.

Development of SSR Databases Available for Both NGS and Capillary Electrophoresis in Apple, Pear and Tea.

Developing new varieties in fruit and tea breeding programs is very costly and labor-intensive. Thus, establishing a variety discrimination system is important for protecting breeders' rights and producers' profits. Simple sequence repeat (SSR) databases that can be utilized for both next-generation sequencing (SSR-GBS) and polymerase chain reaction-capillary electrophoresis (PCR-CE) would be very useful in variety discrimination. In the present study, SSRs with tri-, tetra- and pentanucleotide repeats were examined in apple, pear and tea. Out of 37 SSRs that showed clear results in PCR-CE, 27 were suitable for SSR-GBS. Among the remaining markers, there was allele dropout for some markers that caused differences between the results of PCR-CE and SSR-GBS. For the selected 27 markers, the alleles detected by SSR-GBS were comparable to those detected by PCR-CE. Furthermore, we developed a computational pipeline for automated genotyping using SSR-GBS by setting a value "α" for each marker, a criterion whether a genotype is homozygous or heterozygous based on allele frequency. The set of 27 markers contains 10, 8 and 9 SSRs for apple, pear and tea, respectively, that are useful for both PCR-CE and SSR-GBS and suitable for automation. The databases help researchers discriminate varieties in various ways depending on sample size, markers and methods.

Genetic structure analysis of cultivated and wild chestnut populations reveals gene flow from cultivars to natural stands.

Japanese chestnut (Castanea crenata Sieb. et Zucc.), the only fruit tree species domesticated in Japan, has been cultivated alongside natural stands since prehistorical times. Understanding the genetic diversity of this species and the relationships between cultivated and wild chestnut is important for clarifying its breeding history and determining conservation strategies. We assessed 3 chestnut cultivar populations and 29 wild chestnut populations (618 accessions). Genetic distance analysis revealed that wild populations in the Kyushu region are genetically distant from other populations, whereas other wild and cultivar populations are comparatively similar. Assignment tests suggested that cultivars were relatively similar to populations from central to western Honshu. Bayesian structure analyses showed that wild individuals were roughly classified according to geographical distribution along the Japanese archipelago, except that some wild individuals carried the genetic cluster prevalent in cultivars. Parentage analyses between cultivars and wild individuals identified 26 wild individuals presumed to have a parent-offspring relationship with a cultivar. These results suggested that the genetic structure of some wild individuals in natural stands was influenced by gene flow from cultivars. To conserve wild individuals carrying true "wild" genetic clusters, these individuals should be collected and preserved by ex situ conservation programs.

Genetic evidence that Chinese chestnut cultivars in Japan are derived from two divergent genetic structures that originated in China.

The Chinese chestnut (Castanea mollissima Bl.) was introduced into Japan about 100 years ago. Since then, a number of Chinese chestnut cultivars and Japanese-Chinese hybrid cultivars have been selected by farmers and plant breeders, but little information has been available about their origins and genetic relationships. A classification based on simple sequence repeat markers was conducted using 230 cultivars including Japanese chestnut (Castanea crenata Sieb. et Zucc.) cultivars originated in Japan, Japanese-Chinese hybrid cultivars, and Chinese chestnut cultivars originated in both Japan and China. First, a search for synonyms (cultivars with identical genotypes) revealed 23 synonym groups among the Chinese chestnut cultivars, and all but one cultivar from each synonym group was omitted from further analyses. Second, genetic structure analysis showed a clear division between Japanese and Chinese chestnut, and most of the Japanese and Chinese cultivars had a simple genetic structure corresponding to the expected species. On the other hand, most Japanese-Chinese hybrid cultivars had admixed genetic structure. Through a combination of parentage and chloroplast haplotype analyses, 16 of the 18 hybrid cultivars in this study were inferred to have parent-offspring relationships with other cultivars originated in Japan. Finally, Bayesian clustering and chloroplast haplotype analysis showed that the 116 Chinese chestnut cultivars could be divided into two groups: one originated in the Hebei region of China and the other originated in the Jiangsu and Anhui regions of China. The Chinese chestnut cultivars selected in Japan showed various patterns of genetic structure including Hebei origin, Jiangsu or Anhui origin, and admixed. The chestnut cultivar genetic classifications obtained in this study will be useful for both Japanese and Chinese chestnut breeding programs.