Headspace solid-phase microextraction combined with GC/MS (HS-SPME-GC/MS) is one of the strongest tools for comprehensive analysis of volatile compounds and has been used to analyze aromatic components of mango and investigate its varietal characteristics. In this study, profiling of aroma compounds in 17 mango cultivars, grown in the same green house to exclude the effect of environmental factors, was conducted and the patterns were subjected to principal component analysis (PCA) to identify the relationship between the aroma components and cultivars. Fifty-nine different volatile constituents were detected from the blends of these 17 mango cultivars. The cultivars were divided into four clusters using PCA based on the volatile components determined in the study. Aiko was found to mainly contain δ-3-carene and showed a composition more similar to its pollen parent, Irwin, than to its seed parent, Chiin Hwang No. 1.

The skin color of mature mango (Mangifera indica L.) fruit varies from green to red depending on cultivars. Red coloration of mango fruit skin is due to anthocyanin accumulation and is known to be induced by light exposure as in some other fruit crops. Recently, several investigations on the genes involved in anthocyanin biosynthesis in mango fruit skin have been conducted, to understand the molecular mechanism underlying red coloration of the mango fruit. In the present study, we investigated the characteristics of four R2R3-MYB transcription factors (TF) including MiMYB1, which is assumed to be the MYB TF regulating anthocyanin biosynthesis in mangoes. The deduced amino acid sequence of MiMYB1 showed a high degree of similarity with the anthocyanin-related R2R3-MYB TFs from other plant species and contained two conserved motifs defining the anthocyanin-related MYB TFs. Expression analysis in the mango fruit skin under different light conditions showed that the expression of MiMYB1 increased as the intensity of light exposure increased, parallel to anthocyanin accumulation and the expression of some structural genes of the anthocyanin biosynthetic pathway. In addition, a transient expression assay of tobacco leaves showed that co-infiltration of MiMYB1 and MibHLH2, which is a bHLH TF of mango, induced anthocyanin accumulation in tobacco leaves. These results suggest that MiMYB1 acts as the key regulator for anthocyanin biosynthesis in mango fruit skin, and that light-dependent red coloration of mango fruit skin is regulated by MiMYB1 in transcript levels.

Tomato yellow leaf curl virus (TYLCV), a monopartite begomovirus that originated in the eastern Mediterranean, has spread worldwide, becoming a serious threat to tomato (Solanum lycopersicum L.) production. Southeast Asia is considered one of the hotspots for begomovirus diversity, and a wide variety of local begomovirus species distinct from TYLCV have been identified. In this study, the protection effect of introgressions of single TYLCV Ty resistance genes, Ty-2 and Ty-3a, in tomato was examined against inoculations of the bipartite begomoviruses Tomato yellow leaf curl Kanchanaburi virus (TYLCKaV) and Pepper yellow leaf curl Indonesia virus (PepYLCIV) isolated from Indonesia. Our findings suggest that Ty-2 in the heterozygous state was found to be ineffective against PepYLCIV and TYLCKaV, whereas Ty-3a in the heterozygous state was effective against PepYLCIV and partially effective against TYLCKaV. Quantification of viral DNAs showed correlation between symptom expression and viral DNA accumulation. Moreover, mixed infections of TYLCKaV and PepYLCIV caused notably severe symptoms in tomato plants harboring Ty-3a. In cases of mixed infection, quantifying viral DNAs showed a relatively high accumulation of PepYLCIV, indicating that Ty-3a loses its effectiveness against PepYLCIV when TYLCKaV is also present. This study demonstrates the lack of effectiveness of Ty resistance genes against single and mixed infections of distinct local begomoviruses from Southeast Asia.

© 2019, Springer Nature B.V. We report on 14 microsatellites enriched in CT repeats obtained from a genomic library of lychee (Litchi chinensis Sonn.) cultivar “Hong Huay”. The polymorphisms revealed by these microsatellites were evaluated in a collection of 45 local Vietnamese lychee varieties and 4 Xerospermum noronhianum (Blume) Blume (Sapindaceae) collected from the wild. Samples were collected from local villages and forests in northern Vietnam. Genetic diversity parameters were estimated for the local Vietnamese varieties analyzed. The unweighted pair-group method of clustering using averages divided the lychee cultivars into three main groups: Cluster 1 (Group A) consisting of semi-natural lychees (“extremely early” lychee); Cluster 2 (Group B) consisting of cultivated cultivars (“intermediate” lychee); and Cluster 3 (Group C) representing X. noronhianum accessions. Using STRUCTURE, three subpopulations were also delimited among litchi accessions, including accessions with extremely early- and intermediate/late-maturing traits showing membership coefficients above 0.99 for Cluster 1 and Cluster 2, respectively. Accessions with early- and intermediate-maturing traits were identified as admixture forms with varying levels of membership shared between the two clusters, indicating their hybrid origin during litchi domestication. This is the first report on transferability of SSR markers developed from lychee (L. chinensis) to X. noronhianum. Results demonstrate the usefulness of microsatellites for identification, genetic diversity analysis and germplasm conservation in lychee and related Sapindaceae forest species.

During 2017, leaf samples of chili pepper (Capsicum annuum), tomato (Solanum lycopersicum), and tobacco (Nicotiana tabacum) plants exhibiting yellowing and curling symptoms were collected from Aceh province, Indonesia. These samples were used to isolate and sequence viral genomic DNA. Six isolates with complete DNA-A and DNA-B sequences of begomovirus were obtained, all of which showed >99% sequence identity to the others. DNA-A sequences shared the highest nucleotide sequence identity (89.3%-89.7%) with monopartite pepper yellow leaf curl Indonesia virus 2 (PepYLCIV2) and the second-highest sequence identity (87.3%-87.4%) with bipartite pepper yellow leaf curl Indonesia virus (PepYLCIV). The DNA-B sequences shared the highest nucleotide sequence identity (95%-97.5%) with PepYLCIV. Results of recombination analysis indicated that the novel begomovirus was a recombinant. In accordance with the guidelines for begomovirus species demarcation, these isolates should be assigned to a new species, and we have proposed the name ''pepper yellow leaf curl Aceh virus'' (PepYLCAV) for this virus.

<p>The red coloration of the mango 'Irwin' skin is an important factor determining its value in the Japanese domestic luxury fruit market. In the present study, to investigate the molecular mechanism underlying anthocyanin biosynthesis of mango fruit skin, UFGT-like genes were isolated and the expression profile of anthocyanin-related genes was determined. Several UFGT-like genes were identified in transcriptome data of red 'Irwin' mango skin and two genes, <i>MiUFGT1</i> and <i>MiUFGT3</i>, were considered to be involved in mango skin coloration. Deduced amino acid sequences of these genes exhibited high similarity to other plant UFGTs and contained the conserved PSPG box common to the glycosyltransferase family. The presence of a glutamine and a histidine residue at the C-terminus end of the PSPG box in <i>MiUFGT1</i> and <i>MiUFGT3</i>, respectively, implied that <i>MiUFGT1</i> and <i>MiUFGT3</i> use glucose and galactose, respectively, as a sugar donor; however, the actual function and sugar donor preference of these enzymes remain to be elucidated. Expression analysis of anthocyanin-related genes during skin coloration suggested that <i>MiCHS</i> and <i>MiANS</i>, as well as <i>MiUFGT1</i> and <i>MiUFGT3</i>, play important roles in the anthocyanin biosynthesis of mango fruit skin and that the expression of these genes is regulated by the MYB transcription factor, as reported in other plant species.</p>

© 2018 The Japanese Society for Horticultural Science (JSHS), All rights reserved. Indonesia is one of the world’s largest fresh pepper (Capsicum spp.) fruit-producing countries, and hot peppers are essential spices in Indonesian cuisine. During the last two decades, begomovirus, which is transmitted by the whitefly, Bemisia tabaci (Gennadius), and causes pepper yellow leaf curl disease, began to cause a huge economic loss by damaging pepper plants in Indonesia. In the present study, a highly efficient inoculation method was established for Pepper yellow leaf curl Indonesia virus (PepYLCIV), the most infectious bipartite begomovirus in pepper plants cultivated in North Sumatra, by combining agroinoculation and subsequent grafting. Partial tandem repeats of PepYLCIV DNA A and B were constructed and cloned into a binary pGreenII vector, and their infectivity was tested. Co-inoculation of Nicotiana benthamiana L. and Solanum lycopersicum L. ‘Momotaro’ with PepYLCIV DNA A and DNA B resulted in the production of typical begomoviral symptoms. Both the injection of the cotyledons with cultured agrobacteria and the inoculation of the hypocotyl with agrobacterial colonies induced viral symptoms in pepper No. 218 (C. annuum L.) seedlings in approximately 55–75%. When agroinoculated symptomatic No. 218 was grafted onto an uninfected ‘Takanotsume’ (C. annuum), all newly elongated shoots from the rootstock of ‘Takanotsume’ produced typical begomoviral symptoms. Agroinoculation combined with subsequent grafting provides a highly efficient method for introducing PepYLCIV into pepper plants.

Tomato yellow leaf curl disease caused by begomoviruses is a serious threat to tomato (Solanum lycopersicum L.) production. If begomoviruses, transmitted by whitefly (Bemisia tabaci), infect tomato plants during early growth, production can be almost entirely lost. Tomato yellow leaf curl Kanchanaburi virus (TYLCKaV), a bipartite Begomovirus, is emerging as an important threat to solanaceous crop production in Southeast Asia. The lack of mechanical transmission of some begomoviruses is a major experimental constraint. In this study, an agroinoculation method using TYLCKaV in tomato plants was established. Partial tandem repeats of TYLCKaV DNA A and DNA B were constructed and cloned to a binary pGreenII vector, and their infectivity was tested. Co-inoculation of TYLCKaV DNA A and DNA B to Nicotiana benthamiana L. produced typical begomoviral symptoms, and both of the viral DNA components accumulated in the upper uninoculated leaves, suggesting systemic infection of TYLCKaV. Two agroinoculation methods were conducted on tomatoes. First, excised sections of tomato shoots were agroinoculated with a soaking procedure. Although two Agrobacterium tumefaciens strains were tested, approximately 40% of inoculated plants only showed viral symptoms for EHA105. Second, agrobacterium from a cultured petri dish was directly inoculated with a colony inoculation procedure. When EHA105 was used, approximately 92% of inoculated plants showed viral symptoms. Sequencing the recovered viral DNA from the upper uninoculated leaf clarified that TYLCKaV had successfully infected the tomato plants. The colony inoculation procedure is labor-saving, and viral symptoms develop in susceptible tomato plants within approximately a month from sowing the seeds. This method could contribute to simple and speedy evaluation of TYLCKaV resistance of tomato plants.

A molecular marker analysis for Asian persimmon cultivar relationships and pollination status was conducted with 496 amplified fragment length polymorphism (AFLP) markers and 146 cultivars of Asian origin. Cultivars from China, Korea, and Japan were evaluated for marker composition and pollination status, which strongly influences fruit characteristics. Separation of Chinese, Korean, and Japanese cultivar groups and pollination type by neighbor-joining clustering, multidimensional scaling, and STRUCTURE was only weakly supported and not genetically significant. Significant differences for cultivar origin and pollination status were found for analysis of molecular variance (AMOVA), but most of the variation was among cultivars, not classification groups. All of the cultivar groups were genetically similar at the molecular level with most polymorphism due to individual cultivar differences.

Persimmon (Diospyros kaki Thunb) is hexaploid, and the pollination-constant, non-astringent (PCNA)/non-PCNA trait of Japanese origin is qualitatively controlled by the AST/ast alleles at a single locus and the PCNA trait is recessive to the non-PCNA trait. To avoid inbreeding depression led by repeated crosses among PCNA genotypes, non-PCNA genotypes should be used as cross parents. The marker-assisted selection system has been developed for the selection of PCNA offspring in the progeny derived from the cross of non-PCNA 'Taigetsu' (non-PCNA 'Kurokuma' X PCNA 'Taishu') to PCNA 'Kanshu'. The primer pairs E8.5/E9r and 7H9F/AST-R were used for detecting the molecular markers A(1) and A(3), respectively, which link AST alleles. Complete agreement was found between the sequence-characterized amplified region (SCAR) marker genotype and fruit astringency phenotype of the 48 offspring. The result confirmed that the marker-assisted selection using those markers was highly practical. In a larger offspring population (522 offspring) from the same cross, offspring segregated into 100 with both markers, 162 with only A(1), 179 with A(3), and 81 with neither, and this segregation ratio was significantly different from 2:3:3:2, which is the segregation ratio of random chromosome assortment in autohexaploid. The percentage of offspring expected to be PCNA was 15.5% (81 of 522), which was slightly lower than 20%.

Pollination-constant, non-astringent (PCNA) type persimmons (Diospyros kaki Thunb.) steadily lose fruit astringency and are more desirable than other types of persimmons. The PCNA/non-PCNA trait is qualitatively controlled by AST/ast alleles at a single locus. Persimmons are hexaploid, and PCNA of Japanese origin and non-PCNA genotypes are denoted as aaaaaa and A----, respectively, if AST gene denotes A and ast gene denotes a. 'Taiten' is an F-1, non-PCNA persimmon offspring with genotype AAaaaa derived from PCNA x non-PCNA, and molecular markers linked AST alleles in 'Taiten' shows polymorphism as A(2) and A(3), so that the genotype of 'Taiten' can show A(2)A(3)aaaa using the alleles of molecular marker. Offspring from a backcross of 'Taiten' x 'Kanshu' (PCNA) were evaluated. The astringency type was analyzed phenotypically, by fruit sensory and brown specks tests, and genotypically by SCAR markers with PCR primer pairs (E4/A2r for detecting the SCAR marker A(2) and 7H9F/AST-R for both A(2) and A(3)). The ratio of PCNA offspring was 43/251 (17.1%), and there was discrepancy between the phenotypic evaluation and SCAR marker estimation in only 3 offspring. The ratio was not significantly different from the theoretical ratio of nulliplex in autohexaploid. The PCNA offspring segregation ratio was 36/151 (23.8%) for 'Fuyu' (PCNA) x 'Taiten' and 6/29 (20.7%) for 'Tenjingosho' (PCNA) x 'Taiten' (nearly the same ratio reported for 'Taiten' x 'Kanshu'). Marker-assisted selection using SCAR markers is thus effective and practical in persimmon breeding. (C) 2014 Elsevier B.V. All rights reserved.

In this study, a set of seven simple sequence repeat (SSR) markers were developed for Japanese maple, and the genetic diversity of 107 Japanese maple cultivars was estimated using these markers. A total of 87 alleles were detected, ranging from 4 to 25 per locus, with an average of 12.43 alleles per locus. Most cultivars showed unique SSR profiles, indicating that the markers were effective for cultivar identification, while some pairs of bud-sport cultivars and synonyms showed identical SSR profiles. Principal component analysis (PCA) indicated that 107 Japanese maple cultivars were divided into two groups: one consists of cultivars of Acer palmatum, and the other group includes cultivars of two varieties, A. amoenum var. amoenum and A. amoenum var. matsumurae. Analysis of molecular variance (AMOVA) revealed significant differentiation between the two species, while the genetic differentiation and distance between the two varieties of A. amoenum were very low, with FST = 0.026 and D = 0.057, respectively. Thus, the two varieties of A. amoenum are genetically indistinguishable from each other in domesticated cultivars, while most cultivars of A. palmatum have been developed separately from those of A. amoenum.

Mango (Mangifera indica L.) is one of the most important evergreen fruit trees, but it has a high tendency of biennial bearing because of irregular flowering. In this study, a FLOWERING LOCUS T-like (FT-like) gene was isolated from mango (MiFT) and characterized. The deduced amino acid sequence of MiFT showed high identity of the gene to other plant FT-like genes, and further, MiFT expression increased only in the leaves under floral-inductive conditions. Comparison of heavy crop load (HC) and no crop load (NC) mango trees showed that MiFT expression strongly increased only in NC trees under cool temperature. In NC trees, almost all the apical buds formed panicles, whereas in HC trees, only a few panicles were produced in the next season of fruit set. Further. HC trees had lower starch content in the shoots than NC trees. Furthermore, application of 250-ppm gibberellin 3 (GA(3)) completely inhibited flowering and MiFT expression in both HC and NC trees. GA metabolism genes were also isolated from mango and their expression patterns were investigated. Gibberellin-3-oxidase (GA3-ox) controls the final step of biosynthesis of active GA, and its gene expression surged only in HC trees under cool temperature. In conclusion, MiFT is considered a key factor in mango flowering, and regulation of MiFT expression through GA metabolism was discussed. (C) 2012 Elsevier B.V. All rights reserved.

Although Japanese mango is produced under a fairly unique cultivation system and fetches high prices at market, further growth of the mango industry in Japan requires the development of new cultivars. However, since individual mango flowers are quite small, no more than 1 cm in diameter, a large number of skilled workers is necessary for artificial pollination, thus making it difficult to obtain a sufficient number of progenies for breeding. We therefore evaluated a methodology whereby progenies were obtained by open pollination and their male parent was subsequently determined by DNA markers. Two mango cultivars ('Irwin' and 'Beni-Keitt') were arranged in a plastic house and honeybees were released as a pollen vector for open pollination. Harvested fruits were characterized and their seeds were sown in a bed. The male parent of the germinated seedlings was then identified by five simple sequence repeat (SSR) markers. As a result of SSR genotyping, the male parents of 185 of 239 'Irwin' seedlings were revealed; 79 were obtained by self-pollination and 106 from out-crossing. For 'Beni-Keitt', the male parent of 14 of the 20 seedlings was determined with two self-pollinated and 12 out-crossed. Preferential out-crossing in 'Irwin' was revealed using the chi-square test, although the considerable number of self-pollinated fruit obtained shows that a sufficient number of fruit can be set in a single-planted orchard. The effect of the male parent on fruit characteristics was subsequently examined, revealing that in 'Irwin' the Brix value of self-pollinated fruits was significantly higher than that of cross-pollinated fruits. Certain color values were, however, lower in self-pollinated 'Irwin' fruits.

Persimmon cultivars are classified into four types depending on the nature of astringency loss of the fruit, namely pollination constant non-astringent (PCNA), pollination variant non-astringent (PVNA), pollination variant astringent (PVA), and pollination constant astringent (PCA). Among these four types, PCNA is the most important cultivar for persimmon breeding due to the stable loss of natural astringency on the tree. The trait of natural astringency loss is recessive in Japanese PCNA cultivars, while that in the Chinese PCNA cultivar, 'Luo tian tian shi', is dominant and the latter locus, termed CPCNA, is different from that in Japanese cultivars. In order to develop a molecular marker for the selection of the CPCNA-type cultivar, we performed amplified fragment length polymorphism (AFLP) in combination with bulked segregant analysis for F-1 offspring derived from 'Luo tian tian shi', which was used as the maternal parent. A total of 384 primer combinations were tested, and three AFLP markers, namely EACT-MCCC-222 (RO1), EGGC-MCTC-309 (RO2), and EGCC-MCGA-105 (RO3), linked to the CPCNA dominant allele were obtained. Among these markers, EGGC/MCTC-309 (RO2) was converted into a sequence-characterized amplified region (SCAR) marker. PCR analysis using F-1 offspring (n = 264) revealed that the relevance ratio of the SCAR marker was 94%. The polymorphism of the RO2 marker, which was strongly linked to the CPCNA dominant allele, was detected in only two Chinese PCNA cultivars, namely 'Luo tian tian shi' and 'Tian bao gai', among the Chinese, Korean, and Japanese cultivars tested. These results indicate that the RO2 marker contributes to marker-assisted selection for breeding programs of new PCNA cultivars having the CPCNA trait.

In previous studies, we have developed molecular markers linked to the AST locus that controls fruit astringency type in persimmon; however, these markers are not feasible for practical application to breeding programs since they are not fully effective for discriminating the pollination constant and non-astringent (PCNA) genotype from the non-PCNA genotype in a progeny derived from 'Kurokuma'. Here we developed new sequence characterized amplified region (SCAR) markers that enable easy and reliable selection of the PCNA genotype from breeding populations. Genomic regions adjacent to probe 5R, which showed polymorphic fragments between PCNA and non-PCNA genotypes in restriction fragment length polymorphism (RFLP) analysis, were isolated from the genomic libraries of 'Nishimura-wase', 'Jiro', and 'Kurokuma'-derived offspring. The isolated genomic regions were characterized and 3 insertion/deletion mutations were observed between ast- and AST-linked regions. Several primers were designed in the flanking region of Indel-3 and, in multiplex PCR, it was shown that using 2 forward primers, AST-F and PCNA-F and a reverse primer, 5R3R, is the most useful and reliable primer set. The AST-linked 220-bp fragment proved to be a common marker of 'Kurokuma'-, 'Nisimura-wase'- and 'Aizumishirazu'- derived progenies. This multiplex PCR is considered the most practical tool for marker-assisted selection (MAS) and can enhance and accelerate progress in persimmon breeding.

Persimmon (Diospyros kaki Thunb.) is generally hexaploid, and a single AST locus controls the pollination-constant non-astringency trait on each of six corresponding chromosomes. The pollination-constant non-astringent (PCNA) genotype is nulliplex and requires homozygous recessive alleles (ast) at the AST locus. There are several non-PCNA cultivars/selections that could be cross parents; however, the probability of yielding nulliplex offspring depends on the number of recessive alleles (ast). In genotyping for the AST locus in hexaploid persimmon, in contrast to the situation in diploid plants, we need to detect the AST/ast allele dosage; this cannot be detected by common codominant markers. In this study, we detected the allele dosage of M(ast), which is a marker allele strongly linked to the ast allele among cultivars, by quantitative real-time polymerase chain reaction (qPCR) using three reference sites, actin (DkAct), anthocyanin reductase (DkANR), and L5R, whose sequences are conserved in the genome of persimmon cultivars. Based on the allele dosage of the M(ast), AST/ast genotypes were estimated for 63 non-astringent cultivars/selections, of which only five cultivars/selections were estimated to be simplex or duplex. The quantitative genotyping method using qPCR may be generally effective for polyploid plants.

Persimmon (Diospyros kaki Thunb.) is a polyploidy fruit tree species of economic importance to East Asia. Natural astringency loss is an important trait in persimmon breeding programs. Quantitative real-time PCR was used to determine the number of AST/ast alleles for fruit astringency in persimmon (D. kaki Thunb.). To this end, the cultivar Jiro was transformed with one or two copies of a gene encoding NADP-dependent sorbitol-6-phosphate dehydrogenase (S6PDH), which was used as a standard for measuring the allele number of a sequenced marker tightly linked to the recessive ast locus for nonastringency. Primers for markers linked to the AST or ast allele were then used to measure the AST to ast ratio directly in the progeny of a full-sib cross. From determination of the AST to ast ratio and the results of the S6PDH copy number, the number of AST and ast alleles at the AST/ast locus was estimated. This research supported the hypothesis that D. kaki is a hexaploid with six AST and/or ast alleles. In addition to the determination of the allelic status of the AST locus, the application of real-time PCR for confirmation of the ploidy level and allelic composition of target genes in autopolyploids or allopolyploids was demonstrated.

Persimmon (Diospyros kaki Thunb.) cultivars are classified into 4 types depending on the relationship between astringency of the mature fruit and the effect of seeds on the loss of astringency, and only pollination-constant and non-astringent (PCNA)-type persimmons stably lose fruit astringency as a part of fruit development. This is a recessive trait, regarded to be controlled by a single locus, namely, the AST locus, which has a polysomic nature. Thus far, we have identified 2 restriction fragment length polymorphism (RFLP) markers, namely, A1 and A2, each of which is separately linked to a different AST allele, and proved that the RFLP markers were useful for selecting PCNA-type persimmons. This study was conducted to convert the RFLP markers into polymerase chain reaction (PCR)-based markers. We isolated and characterized genomic regions corresponding to each RFLP marker by inverse PCR. Two primer pairs, E4/E9r and E4/A2r, were designed to generate 2 sequence characterized amplified region (SCAR) markers, i.e., PCR-A1 and PCR-A2, respectively. The PCR-A1 and PCR-A2 markers cosegregated with the A] and A2 markers, respectively, in 'Nishimura-wase'-derived progenies. Although the primer pair E4/A2r did not produce the PCR-A2 marker in the FU-275, which is a progeny derived from 'Aizumishirazu', all non-PCNA-type offspring but no PCNA-type offspring showed the PCR-A1 marker using the primer pair E4/E9r. Thus, it was revealed that the SCAR markers were useful for selecting PCNA-type offspring in these progenies. On the other hand, disruption of the relationship between the markers and the AST locus was observed in the KU-325, derived from 'Kurokuma', indicating that the selection of PCNA-type offspring using PCR-based markers is not effective for progeny derived from 'Kurokuma'. Herein, we discuss the possibility of applying PCR-based markers to genetic studies.

We present a new nursery grafting method for persimmon (Diospyros kaki Thunb.) propagation. This study examined the effects of grafting time and leaves of rootstock on the graft-take ratio and growth of grafted plants. Between April and September, we were able to graft a scion of 'Fuyu' persimmon onto a current seedling of 'Hourenbou' rootstock, which was between 110 and 195 days old. Although grafting onto a current seedling without leaves yielded a low graft-take ratio, 80 to 100% of scions grafted onto rootstock with leaves were established successfully. Between rootstock with and without leaves, a significant difference was noted in the callus-forming ratios 20 days after grafting. A callus was observed in all nursery plants grafted onto rootstock with leaves, irrespective of the bud-break feasibility.

To elucidate the relationships among Diospyros kaki and species closely related in previous studies, the nuclear ribosomal internal transcribed spacer (ITS) DNA sequence and the chloroplast matK gene were sequenced and compared with those of nine Diospyros species from Thailand, four species from temperate regions, and one species of southern Africa, D. lycioides. Maximum parsimony, maximum likelihood, and neighbor joining analyses of the matK and ITS data sets revealed that D. kaki is closely related to two diploid species, D. oleifera and D. glandulosa. D. kaki, D. glandulosa, and D. oleifera were placed differently in the trees obtained from ITS and matK data sets, suggesting that hybridization and/or introgression may have occurred during the development of these species. D. kaki was not found to be closely related to D. ehretioides, a diploid species from Thailand. These results differed from a prior analysis of this genus performed with chloroplast DNA ( cpDNA) restriction site mutations in 3.2- and 2.1-kb amplified sequences. The results supported Ng's hypothesis that D. glandulosa and D. kaki may share a common ancestor. D. oleifera was also closely associated with D. kaki.

We investigated marker-assisted selection of the pollination constant and non-astringent (PCNA) trait and inheritance modes of the marker locus in persimmon (Diospyros kaki Thunb.) using restriction fragment length polymorphism (RFLP) analysis. In two backcross progenies, FU-170 and FU-275, 5 of 71 and 23 of 101 offspring, respectively, were judged as PCNA genotype by the marker phenotype. The astringency type of individual offspring was determined by measuring the size of tannin cells and soluble tannin content in mature fruit, thus confirming complete co-segregation of RFLP markers and the non-PCNA phenotype in 32 and 68 fruit-obtainable offspring from FU-170 and FU-275, respectively; therefore, RFLP markers can be used for PCNA genotype detection. Their segregation ratio deviated significantly from that of disomic inheritance. We also discuss the possibility of polysomic inheritance of RFLP markers.

The phylogenetic relationships among 14 Mangifera L. species of Thailand were analyzed by comparing sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA). Parsimony and neighbor joining (NJ) analyses revealed that the common mango (M. indica L.) was closely related to M. laurina Bl., M. sylvatica Roxb., and Al. oblong folia Hook. f. Mangifera foetida Lour. and M. odorata Griff. were also related to M. indica in both parsimonious and NJ trees, although these two species are classified into a different subgenus (subgenus Limits) from the subgenus Mangifera to which M. indica belongs. ITS sequence analysis revealed that several species have nucleotide additivity (two different nucleotides at the same locus) at several sites in the ITS region. Also, M. indica had several polymorphisms among cultivars. This finding may suggest a possibility of hybrid origin of Mangifera species, although Mangifera species are all assumed to be diploid having chromosome number of 2n = 2x = 40.

The cultivation of the PCNA (pollination-constant and non-astringent)-type persimmon is advantageous because of its natural loss of astringency on the tree. We previously found RFLP markers linked to the dominant alleles controlling the PCNA/non-PCNA trait and showed that the PCNA genotype could be distinguished from non-PCNA genotype by these markers in the breeding populations. In this study, RFLP markers were investigated with 33 persimmon cultivars. All PCNA cultivars except for Chinese 'Luo Tian Tian Shi', had identical banding pattern, whereas the non-PCNA cultivars exhibited many polymorphic bands. Thus, we could distinguish PCNA cultivars of Japanese origin from non-PCNA cultivars by the RFLP analysis, although 'Luo Tian Tian Shi' was mistakenly classified as a non-PCNA type. These results suggest that this marker system could be applied to distinguish PCNA cultivars of Japanese origin from non-PCNA ones.

The genetic relationships among 19 pollination- constant and non- astringent (PCNA) cultivars and 14 non-PCNA cultivars of persimmon (Diospyros kaki Thunb.), including one Chinese PCNA cultivar 'Luo Tian Tian Shi', were analyzed by comparing 138 AFLP markers. A phylogenetic tree constructed based on the similarity indices of these AFLP markers indicated a close relationship among Japanese PCNA cultivars, but a more distant relationship with the Chinese PCNA cultivar 'Luo Tian Tian Shi'. The close relationship between PCNA cultivars native to Gifu prefecture was distinct, indicating that these cultivars developed from crosses among restricted sources in this region. The cultivars 'Gosho', 'Hana-gosho', 'Oo-gosho', and 'Yamato- gosho' showed a close relationship with some non-PCNA cultivars.

The phylogenetic relationships among 14 Mangifera L. species including three economically important species, i.e., common mango (M, indica L,), horse mango (M. foetida Lour.) and kwini (M. odorata Griff,), were analyzed by comparing 217 amplified fragment length polymorphism (AFLP) markers. The unweighted pair grouping method using arithmetic averages (UPGMA) and neighbor-joining (NJ) method were used and two outgroup tare, cashew nut (Anacardium occidentale L,) and gandaria (Bouea macrophylla Griff.), were added to both analyses. The common mango was closely related to banana mango (M. sylvatica Roxb.), M. laurina Bl., and M. oblongifolia Hook.f. Intraspecific variation among seven cultivars of common mango was much smaller than interspecific variation and these cultivars were classified into one M, indica group using both methods. Mangifera macrocarpa Bl., M. foetida, and M. odorata were also related to M. indica in both UPGMA and NJ trees, although these three species are classified into a different subgenus (subgenus Limus) from the subgenus Mangifera to which M. indica belongs. Also, in both UPGMA and NJ trees, M. gedebe Mig. and M. griffithii Hk.f. were placed in distant positions among the Mangifera species tested, indicating these two species are related distantly to M. indica. The AFLP technique was confirmed to be useful for phylogenetic analysis.

Phylogenetic relationships among seven species of the genus Durio, of which four produce edible fruits including the common durian (D. zibethinus), were investigated from RFLP analysis of PCR products from a variable region of cpDNA. The target region of cpDNA was reliably amplified from all Durio species tested, using total DNAs extracted by a modified CTAB method of Doyle and Doyle (1987) followed by an additional purification step. When restriction fragment polymorphisms of amplified products were examined with 19 restriction endonucleases (AseI, BamHI, BfaI, DraI, EcoRI, EcoRV, HindIII, KpnI, MspI, RsaI, ScaI, ScrFI, SalI, SmaI, SspI, StyI, TaqI, XbaI, XhoI), RFLPs were observed with 14 endonucleases. However, since all Durio species tested had length mutation on this amplified region of cpDNA, the most parts of diversities from 14 endonucleases were caused by the length mutations among species. Considering length mutation among species, a total of seven site mutations were detected as well as 12 length mutations, based on D. zibethinus. When unordered parsimony analysis was conducted using these data, the cultivated durian (D. zibethinus) did not show any close relationships to other three species producing edible fruits. D. zibethimus formed a cluster with wild species, D. griffthii, and other three species made two clusters with D. lowianus or D. mansoni. Our results indicated an independent evolutionary process among four edible species of the genus Durio.

The relationships among 17 Diospyros species from Thailand, with particular emphasis on the relationship of these species to temperate Diospyros species, including Diospyros kaki, were studied, using 81 cpDNA restriction site mutations detected in the 3.2- and 2.1-kb regions of amplified cpDNA and six different length mutations detected in the 2.1-kb region of amplified cpDNA. Parsimony and neighbor-joining analyses were conducted to identify relationships among species. Three temperate zone species, D. kaki, Diospyros lotus, and Diospyros virginiana, were monophyletic with one subtropical species, Diospyros ehretioides, suggesting a close evolutionary relationship among them. An immediate common progenitor for D. kaki and D. virginiana is suggested from cpDNA homology and the polyploidized karyotypes of both species. Our results did not support Ng's hypothesis that Diospyros glandulosa (synonym Diospyros roxburghii) is the progenitor of D. kaki. Two species, Diospyros rhodocalyx and Diospyros confertiflora, were so distant from the remaining species that additional study is needed to determine whether they should be placed in the same genus.Key words: cpDNA, Diospyros, PCR, phylogeny, persimmon, RFLP, taxonomy.

Diospyros species distributed widely in Thailand were classified into four ecotypes, according to their habitat constantly humid area, alternately dry and wet area, mountainous cool area and all area. Some of them inhabit near dwelling areas or in the paddy field in the village. The young fruit is covered with dense pubescence in most species. The size, shape, and color of mature fruit greatly vary greatly with the species. In most species, the mature fruit has a soft pulp and hard skin. The fruit of six species has been used for dying. Four species produce edible fruits, with color and flavor favorable for breeding of edible Diospyros species. The fruit of some species contains some chemicals useful as fish poisoning or of medicines, although the active components have not yet been identified. The edible fruit contained many tannin cells, but the fruit used as fish poisoning and medicines had only a few.

The phylogenetic relationships among eleven Artocarpus species, including two economically important species, the jackfruit (A. heterophyllus Lamk) and the breadfruit (A. altilis Fosberg), were estimated by comparing 30 restriction-site mutations in an amplified region of chloroplast DNA (cpDNA). Neighbor-joining and parsimony analyses were performed on the data to identify the relationships. The jackfruit and chempedak (A. integer Merr.) were monomorphic and therefore indistinguishable for all restriction sites. This confirmed the close relationship between these two species. The breadfruit and A. elasticus formed a monophyletic group with strong support from both neighbor-joining and parsimony analyses, indicating the possibility that the breadfruit was derived from A, elasticus or its close relatives. In general, our results were in agreement with the conventional classifications in Artocarpus species except for the position of A. chaplnsha. (C) 1997 Elsevier Science B.V.

RFLP analysis of PCR products from a variable region of the chloroplast genome was conducted to determine their potential for use in phylogenic studies of the genus <I>Diospyros</I>. The target region of cpDNA was reliably amplified by the procedure of Arnold et al. (1991) from all 14 Diospyros species tested. When restriction fragment polymorphisms of amplified products were examined at the intraspecific level with 10 endonucleases (<I>Ase</I> I, <I>Bfa</I> I, <I>Bst</I> NI, <I>Dde</I> I, <I>Msp</I> I, <I>Nco</I> I, <I>Rsa</I> I, <I>Scr</I> FI, <I>Sty</I> I, and <I>Taq</I> I), 15 cultivars of persimmon (<I>D. kaki</I>) and 3 horticultural varieties of <I>D. lotus</I> were monomorphic within species. However, restriction fragment polymorphisms were observed at the interspecific level. The 14 <I>Diospyros</I> species tested could be divided into 6 groups by the digestion patterns with ScrF I, 5 groups with <I>Bst</I> NI or <I>Taq</I> I, 4 groups with <I>Bfa</I> I or <I>Sty</I> I, 3 groups with <I>Ase</I> I, <I>Dde</I> I, or <I>Nco</I> I, 2 groups with <I>Msp</I> I or <I>Rsa</I> I. These results clearly indicated that the present analysis method is extremely valuable for phylogenetic and evolutionary studies at the interspecific level of the genus <I>Diospyros</I>.

ブラッドオレンジの果肉におけるアントシアニン形成に及ぼすショ糖濃度、温度、光の影響を調査し、温度がその形成に強い影響を及ぼしていることを明らかにした。

パッションフルーツ数種類を用いて、カルス誘導と個体再生およびプロトプラスト培養の可能性について検討した。

ウメ南高の鉢植え樹を用いて、 日長が葉芽および花芽の発達に及ぼす影響を検討し、 あまり影響を及ぼさないことを明らかにした。

ムラサキクダモノトケイソウを種子親としたパッションフルーツ類の交配の可能性と交配個体の生育特性について論じた。

The genetic relationships among 19 pollination-constant and non-astringent (PCNA) cultivars and 14 non-PCNA cultivars of persimmon (Diospyros kaki Thunb.), including one Chinese PCNA cultivar 'Luo Tian Tian Shi', were analyzed by comparing 138 AFLP markers. A phylogenetic tree constructed based on the similarity indices of these AFLP markers indicated a close relationship among Japanese PCNA cultivars, but a more distant relationship with the Chinese PCNA cultivar 'Luo Tian Tian Shi'. The close relationship between PCNA cultivars native to Gifu prefecture was distinct, indicating that these cultivars developed from crosses among restricted sources in this region. The cultivars 'Gosho', 'Hana-gosho', 'Oo-gosho', and 'Yamato-gosho' showed a close relationship with some non-PCNA cultivars.

This study was conducted to analyze the differences in the control mechanism of flower induction between mango cultivars and to elucidate the factors that induce flowering other than low temperature, and to obtain useful knowledge for new cultivation method and stable production of mangoes. The results showed that MiFT gene expression was involved in the induction of flowering induced by low temperature in other cultivars as well as in 'Irwin', but MiFT was not involved in summer flowering. In addition, 'Irwin' was found to flower in summer, which is thought to be required low temperature for floral induction. We plan to investigate the mechanism of summer flowering through comprehensive transcriptome analysis.

The AST locus controlling persimmon astringency was determined by comparative mapping with Diospyros lotus, a diploid relative of D. kaki, as a reference. The syntenic region of AST locus was 915-kb in physical map of D. lotus. In addition, transcriptome and coexpression network analyses between PCNA and non-PCNA progenies revealed that AST gene affects a few transcription factors centered in the PA regulatory network. On the other hand, transcriptome analysis of the gene conferring non-astringent trait in Chinese-type PCNA persimmon indicated that the genes conferring sugar transporter, flavonoid glucosyltransferase, and flavonoid/phenylplopaloid O-methyltransferase may associate with this trait. Finally, a new innovative strategy was considered for PCNA persimmon breeding including Chinese-type PCNA persimmons.

The accuracy of AFLP markers linked to the locus of natural astringency-loss in Chinese-type PCNA persimmon were confirmed and then, the markers were used as probes for fosmid/BAC library constructed from Diospyros lotus, which is a close diploid relative of D. kaki, for constructing contig to identify the locus of natural astringency-loss. However, the results showed that the fosmid/BAC library is hard to use directly for identifying the locus probably due to the difference of genome structure between D. kaki and D. lotus. On the other hand, RNA-seq analysis using segregated progeny between Chinese-type PCNA and non-PCNA indicated that the genes related to transportation in the cell were differently expressed among them, as well as the genes related to flavonoid biosynthesis pathway.

The survey was conducted in unfinished Northern mountainous area in Vietnam for obtaining new species related to D. kaki and we found several trees which bear pubescent fruits. However, these trees seem to be the same species which we collected previously in Vietnam, so that there are no new species from this survey. In addition, although we conducted resurvey of Diospyros spp. in germplasm repository of China and the campus of Kasetsart University in Thailand, we could not find new species closely related to D. kaki. The phylogenetic studies from DNA sequences of ITS and matK regions in “ye-mao-shi”, which was assumed to closely relate to D. kaki from morphological characteristics, is indicated that this species should be included with D. glandulosa which assumed to be ancestor of D. kaki, due to matK analysis, not due to ITS analysis. This contradiction of the results from each analysis should be corrected by the further analysis in the future.

This study was conducted to analyze the expression pattern of MiFT, which is a main factor for floral induction in Mango, to investigate the effect of endogenous and exogenous factors on floral induction and expression of MiFT, and to analyze the floral related genes other than MiFT. It was revealed that experiencing cool temperature below 15℃ for 130 hours was enough to increase the expression of MiFT in mango. It was also revealed that expression of MiFT in young leaves was lower than old leaves. In addition, some genes that showed down- or up-regulation by the floral-inductive cool temperature in mango leaves were obtained. These genes are under investigation.

Fosmid library was constructed by a diploid relative, Diospyros lotus, of D. kaki (hexaploid) for isolation of AST gene which confers astringency trait in persimmon. After screening seed clone from fosmid library by using a probe of AST-linked marker, fosmid contig was developed by further screening of the library using end-sequences of the clone. In addition, BAC library of D. lotus was constructed and BAC contig was also developed. Each contig seemed to contain AST locus there, so that we were approaching very near to isolate the AST gene. Furthermore, this BAC library was shown to be effective to isolate the CPCNA gene which confers the trait of non-astringency in Chinese-type non-astringent persimmon. Using the BAC library, isolation of CPCNA gene may also be possible.

To develop an practical method for determining the male parent of mango seedlings, high-polymorphic SSR markers were selected and multiplex PCR method was developed. Progenies obtained from open pollinated 'Irwin' trees and 'Beni-Keitt' trees in a plastic house were used for determining male parents. The results showed that cross-pollinated seedlings were major in both cultivars. In addition, Myb transcription factor associated with anthocyanin biosynthesis were isolated from mango and its structure was analyzed.

There is a report to show that Diospyros kaki is closely related to D. lotus, D. oleifera, and D. glandulosa. In addition to these three species, we started to find some Diospyros spp. having close relationships to D. kaki in the area of South China and Northern mountain regions of Thailand and Vietnam where D. kaki (cultivated kaki) thought to be originated. Due to our exploration, we found several species of Diospyros, among which we clarified that some species have morphologically very close features to D. kaki. “Ye-mao-shi” from China and “Hong Coc” from Vietnam are two species in our interests. Especially, “Ye-mao-shi” is the most interesting species because of its very close morphology to D. kaki. When the distribution of “Ye-mao-shi” was investigated by specimen survey in botanical gardens in China, it clearly showed that this species aredistributed only in Yunnan province. We also showed a possibility that “Ye-mao-shi” is diploid, as the same as D. lotus, D. oleifera, and D. glandulosa. We need further investigations using molecular analyses to clarify the phylogenetic position of this species to D. kaki and to consider the involvement of this species for appearance of cultivated kaki.

Ploidy variants of wild A.arguta specifically occurred in Japan, were characterized. A wide range of ploidy variance from diploid to octaploid was observed. Geographic distribution of the variants was determined showing the localization of the higher ploidy population in Tohoku area. Morphology, fruit composition and reproductive character of the ploidy variants were determined. Cross compatibility of the ploidy variants with kiwifruit (A.deliciosa) was observed. The characteristics of the cross seedlings showed the potential of the variants as breeding materials.

The gene conferring the trait of astringency-loss in Japanese-type PCNA is shown to be located at 100kb away of fosmid contig constructed from Diospyros lotus, a close relative of D.kaki. This single gene controls the astringency trait in persimmon fruit and 6 alleles are responsible for the appearance of this trait. The DkMyb4, a regulatory gene, is also found to control tannin biosynthesis in the fruit and is indicated to determine whether an individual is PCNA or non-PCNA due to its strength of expression. Using the DNA sequences linked to this gene, we could construct a primer pair for PCR analysis for selecting PCNA offspring of Japanese-type. In addition, we also found an AFLP marker to distinguish Chinese-type PCNA among breeding population.

近年、日本でマンゴーのハウス栽培が拡大しつつあり、高級果物としての需要が高まっている。日本では主に‘Irwin'が栽培されており、成熟期に鮮やかな赤色を呈するのが特徴である。温帯果樹のリンゴやブドウでは、高温によりアントシアニン合成が阻害されることが示されており、30℃を超える条件で栽培すると、果実着色は不良となる。一方、マンゴー‘Irwin'は40℃を超える高温下で栽培されているにも関わらず、着色への影響はあまり見られない。本研究では、マンゴーの着色と気温との関係を明らかにするために、低温処理がマンゴーの着色に及ぼす影響について調査した。低温処理(果実周辺温度を20〜25℃に保つ)した果実では、アントシアニン含量は対照区(果実周辺気温25〜45℃)と差がなかったが、クロロフィル含量の減少が抑制され、果皮はくすんだような赤色になった。一方、アントシアニン合成系(PAL, CHS, ANS)の遺伝子発現は、低温処理によって増加していた。夜間のみ低温処理を行った果実でも、アントシアニン含量は対照区と同程度であり、低温によるアントシアニン合成の促進は見られなかった。それに対し、遮光処理を行うと、アントシアニン含量は著しく抑制され、また、夜間低温処理と遮光を組み合わせると、アントシアニン蓄積とクロロフィル分解が共に抑制されることが示された。マンゴー果皮におけるアントシアニン合成系遺伝子群の発現量はアントシアニン含量と相関が低く、低温や遮光が遺伝子発現に及ぼす影響は明確でない。以上の結果から、マンゴー‘Irwin'の果皮におけるアントシアニン蓄積は、30℃以上の高温でもほとんど阻害されることなく、気温の影響は光の影響と比較して小さいことが明らかとなった。20〜25℃程度の低温は、成熟を遅延させ、クロロフィルの分解を妨げることから、赤色を鮮やかに発色させるためには適さないと考えられた。

The analysis was performed to identify key genes for regulating tannin accumulation into fruits of pollination constant non-astringent (PCNA)-type of persimmon originated in Japan and China. For this purpose, we conducted the following three experiments: 1)We constructed genomic library from closely related diploid species, D. lotus, and a seed clone was screened from the library with a probe tightly linked to the trait of natural astringency-loss of Japanese-type PCNA fruits. Then, we constructed ca. 300 kbp contig of D. lotus. Due to the sequence analysis of this contig, we found that the sequences are very homologous to those of D. kaki and it can use for exploring the gene of persimmon (D. kaki). In addition, primer sets designed from the sequences of this contig were able to be applicable to distinguish Japanese-type PCNA from others. We could make a universal primer set to select PCNA persimmon. 2)We conducted the expression analysis of mRNA to identify the genes involved in tannin accumulation of persimmon. For this purpose, suppression subtractive hybridization (SSH) analysis was performed between astringency-removed fruits with ethanol treatment on the tree and non-treated fruits. According to this analysis, we were able to obtain several candidates for the genes involved in tannin accumulation into the fruits. 3)As for investigation of a dominant gene conferring the trait of Chinese-type PCNA, we looked for molecular markers by AFLP method combined with bulked segregant analysis (BSA) using progenies derived from a Chinese PCNA 'Luo Tian Tian Shi'. By this analysis, we found three markers closely linked to the Chinese-PCNA trait. Using a marker among them, we screened genomic library of `Luo Tian Tian Shi' and obtained a genomic clone as the seed for constructing contig for the survey of the gene.

The purpose of this study is 1) to clarify whether pollination constant non-astringent (PCNA) persimmons exist in China except for 'Luo Tian Tian Shi', in addition to clarify an uniqueness of `Luo Tian Tian Shi', and 2) to survey ancestral species of persimmon (Diospyros kaki Thunb.). For this purpose, we conducted the following investigations: 1)We constructed survey trips to Luotian county in Hubei province, where 'Luo Tian Tian Shi' was found. As the results, we were able to find new PCNA cultivars named `Qiuyan Tian Shi', 'Baogai Tian Shi', and 'Xiaoguo Tian Shi'. In addition, we were able to investigate the original tree of `Luo Tian Tian Shi', which showed us `Luo Tian Tian Shi' originated from a seedling. 2)The uniqueness of PCNA trait in `Luo Tian Tian Shi' was clarified to investigate the F_1 progenies derived from crossings between 'Luo Tian Tian Shi' and Japanese PCNA or non-PCNA cultivars. In F_1 progenies from all crossings, PCNA and non-PCNA individuals were segregated at the ratio of 1:1. It means that the trait of Chinese PCNA is dominant. It is quite different from recessive trait of Japanese PCNA. 3)As for consideration of ancestral species of persimmon, the survey of specimens was conducted in Botanical Garden in Kunming, Wuhan, and Beijing. We found an interesting species named "ye mao shi" classified as Diospyros kaki var. sylvestris. This species has a pubescent fruit surface, while wild persimmon (D. kaki) named "ye shi" has glabrous fruits. The morphological characteristic of "ya mao shi" was very similar to D. galndulosa, which is supposed to be a progenitor of D. kaki. We also could find the trees of this species in the forest of Xishuangbanna Tropical Botanical Garden, the Chinese Academy of Science.

本研究では完全甘ガキの自然脱渋性を支配する遺伝子の探索を進めるために1)ゲノムライブラリーの作成と甘渋性識別マーカー周辺領域の単離、2)甘渋性に連鎖した新規マーカーの探索、を試みた。以下、その概要を記す。1)ゲノムライブラリーの作成と甘渋性識別マーカー周辺領域の単離カキ品種'次郎'(完全甘ガキ)由来のゲノムライブラリーを作成し、甘渋性識別マーカーを用いてスクリーニングを行った結果、約27kbのインサートを持つクローンが単離された。このインサートについて甘渋性識別マーカー周辺領域の簡単な制限酵素マップを作成した。現在、単離されたゲノム断片について、より詳細な調査を行っている。2)甘渋性に連鎖した新規マーカーの探索これまでに同定されている甘渋性に連鎖したマーカーのみでは、遺伝子の探索を進めるにあたり十分な情報が得られないため、ゲノミックサブトラクション法を用いて新規マーカーの探索を試みた。サブトラクション後のPCR増幅断片をいくつか単離し、プローブに用いてRFLP分析を行っているが、現在までに甘渋性に連鎖した新規のマーカーは得られていない。今後は異なる制限酵素を用いたゲノミックサブトラクションを試みていく予定である。

So far, it was thought that no non-astringent-type persimmons exist in China. However, a pollination constant non-astringent (PCNA) persimmon was recently found in Lou Tian Tian province of China, and this PCNA-type cultivar, 'Luo Tian Tian Shi', have showed very unique trait for astringency. When 'Luo Tian Tian Shi' was crossed wish a Japanese PCNA cultivar, several offspring bore astringent fruits. In order to confirm this phenomenon, tannin cell size that is a good index to distinguish PCNA from non-PCNA was determined. As the result, we were able to confirm that non-PCNA offsping by a cross between 'Luo Tian Tian Shi' and a Japanese PCNA cultivar were yielded wish a relatively high rate in the offspring. We also clarified that the chemical properties of tannins in the fruit is quite different between 'Luo Tian Tian Shi' and Japanese PCNA cultivars. Tannins from 'Luo Tian Tian Shi' showed higher molecular weight and faster coagulation reaction with acetaldehyde than in Japanese PCNA cultivars. The chemical properties of tannins of 'Luo Tian Tian Shi' were resemble to those of non-PCNA cultivars. Furthermore, gene expressions involved in flavonoid biosynthesis, which is the first step for tannin biosynthesis, were also different between 'Luo Tian Tian Shi' and Japanese PCNA cultivars. As a conclusion, we could demonstrated a unique trait of astringency in 'Luo Tian Tian Shi', which was thought to be due to a difference of gene expressions involved in flavonoid biosynthesis.

The pollination-constant and non-astringent (PCNA)-type fruit is the most desirable persimmon for fresh consumption, and new PCNA-type cultivars with good fruit quality have been awaited worldwide. However, the inheritance of PCNA-type is qualitative and the PCNA-type is recessive to non-PCNA (PVNA, PVA, and PCA) types. Molecular marker closely linked to the trait of natural astringency-loss is a powerful tool for the breeding project. So, we tried to establish it by the following experiments to make the breeding process more efficient. 1. Bulked segregant analysis (BSA) of 6 PCNA-type and 10 non-PCNA-type offspring in breeding population was performed by AFLP method using 128 primer combinations. As a result, 4 potential AFLP markers were detected for discriminating PCNA- and non-PCNA types. 2. The most reliable marker among 4 potential AFLP markers was selected and sequenced. When this marker was used as a probe for Southern analysis after genomic DNA was digested HindlII, all progenies examined could be distinguished between PCNA-type and non-PCNA type by the existence or absence of 6.5kb or 8.0 kb band. Furthermore, segregation rate of these bands indicated that persimmon shows polysomic inheritance. Persimmon seemed to be an auto- or autoallo-hexaploid. 3. When Southern analysis using AFLP maker as a probe was examined to existing cultivars, PCNA cultivars could be distinguished from non-PCNA culitvars with 100% accuracy. This result indicates a high reliability of this analysis for selecting PCNA-type among wider breeding populations. 4. The sequencing analysis of two RFLP markers (6.5 kb and 8.0 kb) for characterizing them and making primers for PCR analysis for easier detection of these makers was not completely successful. We could sequence 8.0 kb band but not 6.5 kb band. So, further study was needed to design PCR primers for more easier method than Southern analysis to distinguish PCNA-type from non-PCNA-type.