Kenji Nashima

S-genotype identification based on allele-specific PCR in Japanese pear.

Gametophytic self-incompatibility in Japanese pear (Pyrus pyrifolia Nakai) is controlled by the single, multi-allelic S-locus. Information about the S-genotypes is important for breeding and the selection of pollen donors for fruit production. Rapid and reliable S-genotype identification system is necessary for efficient breeding of new cultivars in Japanese pear. We designed S allele-specific PCR primer pairs for ten previously reported S-RNase alleles (S1–S9 and Sk) as simple and reliable method. Specific nucleotide sequences were chosen to design the primers to amplify fragments of only the corresponding S alleles. The developed primer pairs were evaluated by using homozygous S-genotypes (S1/S1–S9/S9 and S4sm/S4sm) and 14 major Japanese pear cultivars, and found that S allele-specific primer pairs can identify S-genotypes effectively. The S allele-specific primer pairs developed in this study will be useful for efficient S-genotyping and for marker-assisted selection in Japanese pear breeding programs.

Generation of expressed sequence tags for discovery of genes responsible for floral traits of Chrysanthemum morifolium by next-generation sequencing technology

BackgroundChrysanthemum morifolium is one of the most economically valuable ornamental plants worldwide. Chrysanthemum is an allohexaploid plant with a large genome that is commercially propagated by vegetative reproduction. New cultivars with different floral traits, such as color, morphology, and scent, have been generated mainly by classical cross-breeding and mutation breeding. However, only limited genetic resources and their genome information are available for the generation of new floral traits.ResultsTo obtain useful information about molecular bases for floral traits of chrysanthemums, we read expressed sequence tags (ESTs) of chrysanthemums by high-throughput sequencing using the 454 pyrosequencing technology. We constructed normalized cDNA libraries, consisting of full-length, 3′-UTR, and 5′-UTR cDNAs derived from various tissues of chrysanthemums. These libraries produced a total number of 3,772,677 high-quality reads, which were assembled into 213,204 contigs. By comparing the data obtained with those of full genome-sequenced species, we confirmed that our chrysanthemum contig set contained the majority of all expressed genes, which was sufficient for further molecular analysis in chrysanthemums.ConclusionWe confirmed that our chrysanthemum EST set (contigs) contained a number of contigs that encoded transcription factors and enzymes involved in pigment and aroma compound metabolism that was comparable to that of other species. This information can serve as an informative resource for identifying genes involved in various biological processes in chrysanthemums. Moreover, the findings of our study will contribute to a better understanding of the floral characteristics of chrysanthemums including the myriad cultivars at the molecular level.

Transcriptome analysis in petals and leaves of chrysanthemums with different chlorophyll levels

BackgroundChlorophylls (Chls) are magnesium-containing tetrapyrrole macromolecules responsible for the green color in plants. The Chl metabolic pathway has been intensively studied and nearly all the enzymes involved in the pathway have been identified and characterized. Synthesis and activity of these enzymes are tightly regulated in tissue- and developmental stage–specific manners. Leaves contain substantial amounts of Chls because Chls are indispensable for photosynthesis. In contrast, petals generally contain only trace amounts of Chls, which if present would mask the bright petal color. Limited information is available about the mechanisms that control such tissue-specific accumulation of Chls.ResultsTo identify the regulatory steps that control Chl accumulation, we compared gene expression in petals and leaves of chrysanthemum cultivars with different Chl levels. Microarray and quantitative real-time PCR analyses showed that the expression levels of Chl biosynthesis genes encoding glutamyl-tRNA reductase, Mg-protoporphyrin IX chelatase, Mg-protoporphyrin IX monomethylester cyclase, and protochlorophyllide oxidoreductase were well associated with Chl content: their expression levels were lower in white petals than in green petals, and were highest in leaves. Among Chl catabolic genes, expression of STAY-GREEN, encoding Mg-dechelatase, which is a key enzyme controlling Chl degradation, was considerably higher in white and green petals than in leaves. We searched for transcription factor genes whose expression was well related to Chl level in petals and leaves and found three such genes encoding MYB113, CONSTANS-like 16, and DREB and EAR motif protein.ConclusionsFrom our transcriptome analysis, we assume that a low rate of Chl biosynthesis and a high rate of Chl degradation lead to the absence of Chls in white chrysanthemum petals. We identified several candidate transcription factors that might affect Chl accumulation in chrysanthemum petals. Functional analysis of these transcription factors will provide a basis for future molecular studies of tissue-specific Chl accumulation.

Transcriptome analysis of flower receptacles of the European pear (Pyrus communis L.) ‘La France’ and its giant fruit sport using next-generation sequencing technology

Summary We studied Giant La France (GLaF), a sport that sets large fruit and that appeared spontaneously on a European pear (Pyrus communis L.) ‘La France’ tree. It is known that GLaF exhibits DNA reduplication, specifically in the receptacle, before full bloom. To identify the genes related to DNA reduplication in GLaF, we performed 454 pyrosequencing to obtain mRNA sequences from GLaF and ‘La France’ and to examine their levels of expression. In total, 1,166,675 sequences were obtained from the poly(A)-enriched complimentary DNA libraries of GLaF and ‘La France’ and assembled into 49,181 contigs. A further 78,431 sequences were obtained as singletons. Comparisons of the total RNA sequence abundance between GLaF and ‘La France’ revealed that the levels of expression of 26 genes changed significantly in GLaF. However, none of these genes had an obvious functional role that could explain the difference in fruit size between the GLaF sport and ‘La France’. We also identified 61 core cell cycle genes by transcriptome analysis, but their involvement in the larger fruit size of GLaF remains to be elucidated. This study presents the first large-scale RNA sequence database for European pear using next-generation sequencing technology. The results obtained can assist in various studies concerning pear physiology, including clarification of the molecular basis for the GLaF sport mutation.

Molecular cloning and expression analysis of the WEE1 and CCS52A genes in European pear (Pyrus communis L.) and their possible roles in a giant fruit mutant

SUMMARY A giant-fruited mutant of European pear (Pyrus communis L.), Giant La France (GLaF), that arose from a bud sport of normal ‘La France’, exhibited receptacle-specific DNA reduplication resulting in an approx. 1.6-fold increase in fruit size compared with normal ‘La France’ fruit. To identify the genes responsible for this receptacle-specific DNA reduplication, we focussed on the regulatory genes WEE1 and CCS52A, which have been reported to increase the ploidy level of tomato fruit flesh. Full-length cDNAs of WEE1 1 and CCS52A, referred to as PcWEE1 and PcCCS52A, were cloned from P. communis L., ‘La France’. We determined the genomic sequences of these two genes, clarified their exon-intron structures, and compared cDNA sequences between ‘La France’ and GLaF. The predicted amino acid sequences were similar to other species and retained their conserved domains. No differences were found between the PcWEE1 and PcCCS52A cDNA sequences of ‘La France’ and GLaF. However, analysis revealed that the levels of expression of PcWEE1 and PcCCS52A were higher in the receptacles of GLaF than in those of ‘La France’ 1 week before the full-bloom stage. Our results suggest that PcWEE 1 and PcCCS52A may play important roles in the occurrence and persistence of DNA reduplication in the receptacles, and hence fruit, of GLaF.

Retrotransposon-based insertion polymorphism markers in mango

Retrotransposons are major components of eukaryotic genomes and are present in high copy numbers. We developed retrotransposon-based insertion polymorphism (RBIP) markers based on long terminal repeat (LTR) sequences and flanking genome regions by using shotgun genome sequence data of mango (Mangifera indica L.). Three novel LTR sequences were identified based on two LTR retrotransposon structural features; a 5′ LTR located upstream of the primer binding site and a 3′ LTR showing high sequence similarity to the 5′ LTR. Starting with 377 unique sequences containing both 3′ LTR and downstream genome region sequences, we developed 82 RBIP markers that were applied to DNA fingerprinting of 16 mango accession. Five RBIP markers were enough to distinguish all 16 accessions. Our result showed that LTR identification from shotgun genome sequences was effective for development of retrotransposon-based DNA markers without whole-genome sequence information. We discuss application of the developed RBIP markers for identification of genetic diversity and construction of a genetic linkage map.