Shoot regeneration from cultured leaves of Japanese pear (Pyrus pyrifolia)

Several experiments were conducted to investigate in vitro regeneration of adventious shoots from cultured leaves of Japanese pear (Pyrus pyrifolia). A protocol was developed and regeneration achieved from six cultivars. Leaves harvested from shoot cultures which had been preconditioned on B5 medium with 5 μM thidiazuron plus 0.25 μM gibberellic acid were placed on regeneration medium of the same composition. Frequency of regeneration per leaf was as high as 23% but cultivar and environmental factors influenced the result. More mature (basal) leaves regenerated more frequently than younger ones from the shoot tip. Leaf orientation during regeneration and photoperiod was not a strong influence but regeneration from leaf pieces was less than from uncut leaves. An alternative regeneration procedure was developed in which first, shoot cultures were grown on the preconditioning medium. Leaves of the intact shoot cultures were then induced to regenerate directly when adventitious shoots formed on leaves of the intact shoot culture leaves without excision. Adventitious shoots from both procedures developed into typical shoot cultures when transferred to shoot culture maintenance medium. This revised version was published online in June 2006 with corrections to the Cover Date.     

Development of microsatellite markers in the Japanese pear (Pyrus pyrifolia Nakai)

Thirteen polymorphic microsatellite loci were developed in the Japanese pear (Pyrus pyrifolia Nakai) by using an enriched genomic library. The obtained microsatellite loci showed a high degree of polymorphism in the Japanese pear with 3–6 alleles per locus. The average values of observed and expected heterozygosities among these 13 loci were 0.69 and 0.71, respectively. Ten microsatellites could successfully amplify loci in the European pear (Pyrus communis L.), which were highly polymorphic as well.     

Highly conserved sequences of three major virion proteins of a Korean isolate of white spot syndrome virus (WSSV)

In Korea, mass mortality occurred among cultured shrimp with visible macroscopic white spots in 2000, and we confirmed the presence of white spot syndrome virus (WSSV) in the tissues of moribund shrimp by electron microscopy. In order to identify the characteristics of this Korean isolate of WSSV, we cloned and characterized its genomic DNA coding for VP24, VP26, and VP28. On the nucleotide level, VP24, VP26, and VP28 of the Korean isolate were found to be 100%, 100%, and 99% identical to those of Taiwan, Thailand and Chinese isolates, respectively. On the deduced amino-acid level, all 3 virion proteins showed 100% identity to those of the foreign isolates. The extent of sequence identity suggests that the Korean isolate originated from the same ancestor as the Taiwanese, Thai and Chinese isolates.     

Isolation and characterization of microsatellite markers in Japanese pear (Pyrus pyrifolia Nakai)

Fourteen microsatellite markers were developed from an enriched genomic library of Japanese pear (Pyrus pyrifolia) by selective hybridization. They were characterized using 17 Japanese pear cultivars. The expected heterozygosity and observed heterozygosity ranged from 0.21 to 0.74 and from 0 to 0.88, respectively. Two to 11 alleles were detected per locus, with IPPN09 and IPPN15 judged to amplify multiple loci. IPPN17 was the most informative locus with the lowest probability of identity (0.19). These primers exhibited a high cross‐species transferability between species and genera.     

Molecular cloning of beta-galactosidase from Japanese pear (Pyrus pyrifolia) and its gene expression with fruit ripening

We have cloned a cDNA fragment encoding a beta-galactosidase from Japanese pear (Pyrus pyrifolia) fruit (JP-GAL). It contained an untranslated sequence of 182 nucleotides at the 5' end, a presumptive coding sequence of 2,193 nucleotides and an untranslated sequence of 268 nucleotides including a polyadenylation signal and a poly (A) tail at the 3' end. It encoded a protein with a calculated molecular weight of 80.9 kDa which consists of 731 amino acids. Both the nucleotide and the deduced amino acid sequences showed a 98% sequence identity with that obtained from the apple beta-galactosidase cDNA. The peptide sequence obtained from the purified Japanese pear beta-galactosidase III matched the deduced amino acid sequence of SVSYDHKAIIINGQKRILISG (amino acid 25-45). Northern blot analysis showed that the probe derived from JP-GAL hybridized to a single 2.6 kb RNA. The mRNA was detected solely in the fruit; none was detected in the buds, leaves, roots or shoots of the Japanese pear. The steady-state level of the beta-galactosidase mRNA was measured during fruit ripening in three cultivars, Housui, Kousui (early ripening) and Niitaka (late ripening). The results showed that regardless of the cultivar, no JP-GAL mRNA was detected in the immature fruit. Increment of the mRNA level with fruit ripening coincided with the increase in the beta-galactosidase III activity. Our results showed that the expression of JP-GAL correlated with fruit softening and JP-GAL may be beta-galactosidase III.     

Determination of S-genotypes of pear (Pyrus pyrifolia) cultivars by S-RNase sequencing and PCR-RFLP analyses

The pear (Pyrus pyrifolia) has gametophytic self-incompatibility (GSI). To elucidate the S-genotypes of Korean-bred pear cultivars, whose parents are heterozygotes, the PCR amplification using S-RNase primers that are specific for each S-genotype was carried out in 15 Korean-bred pear cultivars and 5 Japanese-bred pear cultivars. The difference of the fragment length was shown in the following order: S6 (355 bp) < S7 (360 bp) < S1 (375 bp) < S4 (376 bp) < S3 and S5 (384 bp) < S8 (442 bp) < S9 (1,323 bp) < S2 (1,355 bp). We analyzed the sequence of the S-RNase gene, which had introns of various sizes in the hypervariable (HV) region between the adjacent exons with a fairly high homology. The sizes of the introns were as follows: S1 = 167 bp, S2 = 1,153 bp, S3 = 179 bp, S4 = 168 bp, S5 = 179 bp, S6 = 147 bp, S7 = 152 bp, S8 = 234 bp, S9 = 1,115 bp. There were five conservative and five hypervariable regions in the introns of S1, S3, S4, S5, S6 and S-RNases. A pairwise comparison of these introns of S-RNases revealed homologies as follows: 93.7% between S1- and S4-RNases, 93.3% between S3- and S5-RNases and 78.9% between S6- and S7-RNases. PCR-RFLP and S-RNases sequencing determined the S-genotypes of the pear cultivars. The S-genotypes were S4S9 for Shinkou, S3S9 for Niitaka, S3S5 for Housui, S1S5 for Kimizukawase, S1S8 for Ichiharawase, S3S5 for Mansoo, S3S4 for Shinil, S3S4 for Whangkeumbae, S3S5 for Sunhwang, S3S5 for Whasan, S3S5 for Mihwang, S5S? for Chengsilri, S3S5 for Gamro, S3S4 for Yeongsanbae, S3S4 for Wonhwang, S3S5 for Gamcheonbae, S3S5 for Danbae, S3S4 for Manpoong, S3S4 for Soowhangbae and S4S6 for Chuwhangbae. The information on the S-genotypes of pear cultivars will be used for the pollinizer selection and breeding program.     

Studies on a virus causing stem grooving and graft-union abnormalities in Virginia Crab apple*

A virus was transmitted from apple trees to Nicotiana glutinosa and Chenopodium spp. and back to a range of woody indicators in which it affected only Virginia Crab; symptoms were grooves in the xylem, and swelling and necrosis of the scion immediately above the union with the stock. The virus was distinct from that causing stem pitting in Virginia Crab, because although easily detectable in several apple varieties, it was not found in many trees infected with stem pitting virus. The stem grooving virus has flexous particles 600–700 m/μ long, a heat inactivation point of 67 °C, a dilution end-point of 10^-3 in N. glutinosa sap and remains infective for at least 2 days at 20 °C.     

Cloning, characterization and promoter analysis of S-RNase gene promoter from Chinese pear (Pyrus pyrifolia)

The 5'-flanking region of the S(12)-, S(13)-, S(21)-RNase with a length of 854 bp, 1448 bp and 1137 bp were successfully isolated by TAIL-PCR from genomic DNA from 'Jinhua', 'Maogong' (Pyrus pyrifolia) and 'Yali' (Pyrus bretschneideri) genomic DNA. Sequence alignment and analysis of S(13)-, S(12)-, S(21)-RNase gene promoter sequences with S(2)-, S(3)-, S(4)-, S(5)-RNase 5'-flanking sequences indicated that a homology region of about 240 bp exists in the regions just upstream of the putative TATA boxes of the seven Chinese/Japanese pear S-RNase genes. Phylogenetic tree suggests that the homology region between the Chinese/Japanese pear and apple S-RNase gene promoter regions reflects the divergence of S-RNase gene was formed before the differentiation of subfamilies. Full length and a series of 5'-deletion fragments-GUS fusions were constructed and introduced into Arabidopsis thaliana plants. GUS activity were detected in S(12)-pro-(1 to 5)-GUS-pBll01.2 transgenic pistils and progressively decreased from S(12)-pro-1-GUS-pBI l01.2 to S(12)-pro-5-GUS-pBll01.2. No GUS activity was detected in S(12)-pro-6-GUS-pBll01.2 transgenic pistil and other tissues of non-transformants and all transgenic plants. The result suggested S(12)-RNase promoter is pistil specific expression promoter.     

Complete sequence of the chloroplast genome from pear (Pyrus pyrifolia): genome structure and comparative analysis

The chloroplast genome of Pyrus was found to be 159,922?bp in length which included a pair of inverted repeats (IRs) of 26,392?bp, separated by a small single-copy region of 19,237?bp and a large single-copy region (LSC) of 87,901?bp. A total of 130 predicted genes (113 unique genes and 17 genes, which were duplicated in the IR) including 79 protein-coding genes, four ribosomal RNA genes and 30 tRNA genes were identified based on similarity to homologs from the chloroplast genome of Nicotiana tabacum. Genome organization was very similar to the inferred ancestral angiosperm chloroplast genome. Comparisons between Pyrus, Malus, and Prunus in Rosaceae revealed 220 indels (??10?bp). Excluding ycf1 and ycf2, which contained deletions in the coding region, all of these were detected in the spacer or intron regions. Three insertions and 13 deletions were detected in Pyrus compared to the same loci in Malus and Prunus. After comparing 89 noncoding chloroplast DNA regions in Pyrus and Malus, highly variable regions such as ndhC-trnV and trnR-atpA were identified. In Pyrus and Malus, the IR/LSC borders were 62?bp shorter than those of Prunus. In addition, there were length mutations at the IRa/LSC junction and in trnH. A total of 67 simple sequence repeats (more than 10 repeated motifs) were identified in the Pyrus chloroplast genome. The indels and simple sequence repeats will be useful evolutionary tools at both intra- and interspecific levels. Phylogenetic analysis demonstrated a close relationship between Pyrus and Prunus in the Rosaceae.     

In vitro induction of tetraploid plants from a diploid Japanese pear cultivar (Pyrus pyrifolia N. cv. Hosui)

Tetraploid plants of a Japanese pear cultivar (Pyrus pyrifolia N.) were induced using an in vitro colchicine treatment. Proliferating shoots were transferred to a shoot proliferation medium (SPM) containing 0.1% or 0.01% colchicine, incubated for 1, 2, 4 or 8 days, then transferred to fresh SPM. The ploidy level of the colchicine-treated individuals was analysed by flow cytometry. Four months after colchicine treatment four mixoploids were selected and cultured on SPM for a further 5 months. The ploidy level of the proliferated shoots derived from selected mixoploids was analysed, and five tetraploid shoots were selected. The selected tetraploid shoots were rooted, and potted plants were grown in a greenhouse. The stomata of tetraploid plants were found to be longer than those in diploid plants.     

编码日本梨树花粉类植物防御素蛋白(PDN1)的cDNA核苷酸序列

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Gamma irradiation‐induced disease resistance of pear (Pyrus pyrifolia “Niitaka”) against Penicillium expansum

In this study, the effects of gamma irradiation on the resistance of pear fruit against Penicillium expansum, the causal agent of blue mould disease, were investigated. A low dose of gamma irradiation for 14 days increased the disease resistance and firmness of pear fruits. Remarkably, exposure to 200 Gy of gamma irradiation significantly maintained fruit firmness, markedly reduced disease incidence and enhanced the activity of defence‐related enzymes (e.g., β‐1,3‐glucanase, phenylalanine ammonia lyase, peroxidase and polyphenol oxidase) and expression of pathogenesis‐related (PR) genes (e.g., PR‐1, PR‐3 and PR‐4). Therefore, the gamma irradiation‐induced resistance against P. expansum involves both metabolic changes and the induction of expression of defence‐related genes. In addition, scanning electron microscopic analysis revealed that gamma irradiation significantly inhibits the growth of P. expansum. These results suggest that exposure of mature harvested pear fruits to artificial gamma irradiation confers fungal disease resistance; therefore, gamma irradiation represents an important strategy for controlling postharvest diseases in pear fruit.     

Rapid identification of 1-aminocyclopropane-1-carboxylate (ACC) synthase genotypes in cultivars of Japanese pear (Pyrus pyrifolia Nakai) using CAPS markers

In Japanese pear (Pyrus pyrifolia Nakai), fruit storage potential is closely related to the amount of ethylene produced. We have developed a rapid and accurate method for analyzing genes involved in high ethylene production during fruit ripening in Japanese pear. This involves cleaved-amplified polymorphic sequences (CAPS) of two 1-aminocyclopropane-1-carboxylate (ACC) synthase genes (PPACS1 and PPACS2). Two CAPS markers (A for PPACS1 and B for PPACS2), associated with the amount of ethylene produced, were identified. Marker A was associated with high ethylene producers and marker B with moderate ethylene producers. The absence of these two markers enabled the identification of low ethylene producers. Using these markers, we have identified ethylene genotypes for 40 Japanese pear cultivars and two Chinese pear (P. bretschneideri) cultivars that are commercially important and used in breeding programs. Furthermore, we performed linkage analysis of these two genes in the F(2) population, which revealed that the recombination frequency between the two markers was 20.8 +/- 3.6%. This information is critical to the selection of parents and in breeding strategies to improve storage ability of Japanese pears.     

Partitioning of (13)C-photosynthate from spur leaves during fruit growth of three Japanese pear (Pyrus pyrifolia) cultivars differing in maturation date

BACKGROUND AND AIMS: In fruit crops, fruit size at harvest is an important aspect of quality. With Japanese pears (Pyrus pyrifolia), later maturing cultivars usually have larger fruits than earlier maturing cultivars. It is considered that the supply of photosynthate during fruit development is a critical determinant of size. To assess the interaction of assimilate supply and early/late maturity of cultivars and its effect on final fruit size, the pattern of carbon assimilate partitioning from spur leaves (source) to fruit and other organs (sinks) during fruit growth was investigated using three genotypes differing in maturation date. METHODS: Partitioning of photosynthate from spur leaves during fruit growth was investigated by exposure of spurs to (13)CO(2) and measurement of the change in (13)C abundance in dry matter with time. Leaf number and leaf area per spur, fresh fruit weight, cell number and cell size of the mesocarp were measured and used to model the development of the spur leaf and fruit. KEY RESULTS: Compared with the earlier-maturing cultivars 'Shinsui' and 'Kousui', the larger-fruited, later-maturing cultivar 'Shinsetsu' had a greater total leaf area per spur, greater source strength (source weight x source specific activity), with more (13)C assimilated per spur and allocated to fruit, smaller loss of (13)C in respiration and export over the season, and longer duration of cell division and enlargement. Histology shows that cultivar differences in final fruit size were mainly attributable to the number of cells in the mesocarp. CONCLUSIONS: Assimilate availability during the period of cell division was crucial for early fruit growth and closely correlated with final fruit size. Early fruit growth of the earlier-maturing cultivars, but not the later-maturing ones, was severely restrained by assimilate supply rather than by sink limitation.     

De novo assembly of a wild pear (Pyrus betuleafolia) genome

China is the origin and evolutionary centre of Oriental pears. Pyrus betuleafolia is a wild species native to China and distributed in the northern region, and it is widely used as rootstock. Here, we report the de novo assembly of the genome of P. betuleafolia‐Shanxi Duli using an integrated strategy that combines PacBio sequencing, BioNano mapping and chromosome conformation capture (Hi‐C) sequencing. The genome assembly size was 532.7 Mb, with a contig N50 of 1.57 Mb. A total of 59 552 protein‐coding genes and 247.4 Mb of repetitive sequences were annotated for this genome. The expansion genes in P. betuleafolia were significantly enriched in secondary metabolism, which may account for the organism's considerable environmental adaptability. An alignment analysis of orthologous genes showed that fruit size, sugar metabolism and transport, and photosynthetic efficiency were positively selected in Oriental pear during domestication. A total of 573 nucleotide‐binding site (NBS)‐type resistance gene analogues (RGAs) were identified in the P. betuleafolia genome, 150 of which are TIR‐NBS‐LRR (TNL)‐type genes, which represented the greatest number of TNL‐type genes among the published Rosaceae genomes and explained the strong disease resistance of this wild species. The study of flavour metabolism‐related genes showed that the anthocyanidin reductase (ANR) metabolic pathway affected the astringency of pear fruit and that sorbitol transporter (SOT) transmembrane transport may be the main factor affecting the accumulation of soluble organic matter. This high‐quality P. betuleafolia genome provides a valuable resource for the utilization of wild pear in fundamental pear studies and breeding.