Pyrus Species Research Articles

PHYLOGENETIC UTILITY AND MOLECULAR EVOLUTION OF THE NUCLEAR ALCOHOL DEHYDROGENASE GENE IN PYRUS

A total of 25 accessions from 18 Pyrus species and an outgroup accession from Malus (M. sieboldii) were used to investigate the phylogenetic utility of Adh and its molecular evolution in Pyrus. The Adh gene in Pyrus consists of six different-sized introns and in this study a 650 bp region, containing the first two introns, was used for sequence analysis. Four major Adh loci with particular length variation in the first intron were identified, and sequences of each locus were monophyletic in the dendrogram. Divergent copies, representing one locus, could be found in most individuals. Some of these polymorphic orthologs do not appear monophyletic possibly due to ancient hybridisation as well as lineage sorting. Sequence divergence among different Adh loci ranged from 8% to 10%, while polymorphism within the Adh loci was quite low (<2%) and failed to reconstruct a robust phylogeny. However, the clades received higher bootstrap value as compared to ITS, showing that introns of low copy number nuclear genes (LCNG) have great potential to improve phylogenetic reconstruction at the low taxonomic level.

Identification of European and Asian pears using EST-SSRs from Pyrus

Ten EST-SSRs previously isolated from Pyrus were used to identify 81 P. communis, 13 P. pyrifolia and 20 P. ussuriensis or P. × bretschneideri accessions. Cross-transference of these EST-SSRs was high in these species. PYC-008 and PYC-004 were the least informative SSRs in each of the pear species and were monomorphic in P. pyrifolia while PYC-013, PYC-002 and PYC-009b were the most informative in all species. EST-SSRs were very valuable for identification of incorrectly identified accessions, failed grafts and sets of synonyms in each of the species. Unsuspected relationships were uncovered, including a parental relationship between ‘Anjou’ and ‘Farmingdale’, a clonal relationship between ‘Berger’ and ‘Bartlett’, and a very close relationship between ‘Beurre Superfin’ and ‘Doyenne du Comice’. One SSR marker was different in one of three sports of ‘Doyenne du Comice’ (‘Doyenne du Comice Crimson Gem’) and in one of two sports of ‘Anjou’ (‘Gebhard Red’ red skin sport of ‘Anjou’). UPGMA cluster analysis separated the pear accessions into a large European cluster and an Asian group mostly according to common ancestry, geographical origin or time of ripening. High cross-transference of EST-SSRs in Pyrus species is very valuable for germplasm management in such a highly diverse collection as found at the NCGR Pyrus genebank in Corvallis, OR.

Karyotypic study of wild pear species of Fars Province, Iran

Introduction . Iran, with more than 10 species of Pyrus , is one of the important genetic resources for this genus in the world, and Fars province is one of the centers of origin. In our research, cytogenetical investigation of native wild pear species of Fars province, including P. glabra Boiss., P. syriaca Boiss. and, probably, a natural hybrid of them, (P. glabra × P syriaca ), was carried out using a video analysis system. Materials and methods . Seed germination of different species was done on damp filter paper in petri dishes, and root tips were collected for karyotypical investigation. After pretreatment, fixation, hydrolysis and staining, samples were prepared for observation under the microscope and chromosome morphology was studied. Analysis of the species genome (length of each chromosome, length of long and short arm, ratio of long arm to short arm and ratio of short arm to long arm) was done and the chromosomal type of each species was determined using Levan et al. 's method. Then the karyotypic symmetry of species was studied by Stebbins' method. Results and discussion . The results revealed that all of the genotypes, with 2n = 2x = 34, were diploid. Based on Stebbins' table, genotypes 1 and 2 of P. glabra were classified into group 1A, P. glabra × P. syriaca into group 2A and P. syriaca into group 2B. Genotype plots, according to the A1 and A2 parameters and Stebbins' cross classes, revealed the same results. Similarity and differences of species in chromosomal aspects were investigated and the results shown in a dendrogram.

Anatomy, ultrastructure and lignin distribution of stone cells in two Pyrus species

The statement “seeing is believing” expresses the importance of microscopy in basic and applied research. In this study, anatomy, ultrastructure and lignin distribution in stone cells from the fruit of two Pyrus species ( Pyrus bretschneideri cv. ‘Jingaisu’ and Pyrus pyrifolia cv. ‘Kousui’) were examined by using light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM) as well as autofluorescence analysis. To our knowledge, this is the first time this combined method has been used to analyze the stone cells in pear fruit. Sections stained with phloroglucinol–HCl revealed the presence of lignin in stone cells, and showed that stone cells were distributed in a mosaic pattern in the flesh, with larger stone cells concentrated around the core and smaller ones in the pericarp. There were no obvious differences in stone cell structure between the two varieties, but stone cell size and content was much greater in ‘Jingaisu’ than in ‘Kousui’. Further, lignin accounted for 29.8% of stone cell composition in ‘Jingaisu’, a significantly higher proportion than in ‘Kousui’ (24.6%); this result was confirmed by autofluorescence analysis. More detailed information on lignin distribution across the cell wall was obtained by TEM combined with the KMnO 4 staining technique. In TEM images, cell walls of both pear varieties were typically divided into four layers: compound middle lamella (CML), secondary wall 1 (S1), outer secondary wall 2 (S2L) and secondary wall 2 (S2), with different staining intensities for different lignin concentrations in those regions.

SELECTED PYRUS GENOTYPES AS POLLINIZERS FOR PYRUS COMMUNIS CULTIVARS

Probably the most important factors affecting the financial outcome of commercial fruit growing is the success of pollination and fertilization, which in turn are dependent on weather conditions, activity of pollinators and compatibility and flowering overlap of the pollinizers. Before introducing pollinizer accessions, it is obviously important to know the compatibility and flowering characteristics of the genotypes as well as tree characteristics like vigour, tree habit and pest and disease susceptibility. In order to find out these attributes, 25 accessions belonging to eight Pyrus species have been evaluated. Further, eight promising genotypes were tested in more detail for pollen production and germination, pollen compatibility with Pyrus communis cultivar 'Conference' and flowering phenology. As a result, four recommendable ornamental Pyrus accessions, both early and late flowering were found. To ensure maximal overlap of the flowering period of the main cultivar every year, a mix of 2 or 3 different pollinizers will be advised for commercial orchards

Non-concerted ITS evolution, early origin and phylogenetic utility of ITS pseudogenes in Pyrus

Molecular studies of 19 species of the genus Pyrus L. revealed different degrees of intra-individual polymorphism of the internal transcribed spacer (ITS 1, 5.8S rDNA and ITS 2) region due to the existence of putative non-functional copies (pseudogenes), putative recombinants and non-concerted evolution among functional copies. Different types of ITS pseudogenes displaying lower GC content and unstable secondary structure were preferentially amplified under normal PCR conditions. Functional ITS copies were successfully obtained in all investigated accessions under the modified PCR conditions. All pseudogenes were highly divergent from their corresponding functional copies and formed a monophyletic group in the phylogenetic tree based on all paralogs, indicating they were of relatively early origin. Functional ITS copies led to confused and poorly resolved phylogeny as a result of low sequence divergence, existence of unidentified ancient recombinants and a high degree of intra-individual functional ITS polymorphism, while certain types of pseudogenes and some relict pseudogenes offered more credible clues for the evolutionary history of Pyrus species.

Plant regeneration from in vitro leaves of four commercial Pyrus species

An efficient shoot regeneration from in vitro leaf sections of &lt;I&gt;Pyrus communis&lt;/I&gt; Bartlett, &lt;I&gt;P. pyrifolia&lt;/I&gt; Shenbuzhi, &lt;I&gt;P. bretschneideri&lt;/I&gt; Zaosu and &lt;I&gt;P. ussuriensis&lt;/I&gt; Manyuanxiang was successfully developed for use in future transgenic studies. On the basis of regeneration frequency and average shoot numbers, optimal shoot regeneration was obtained on leaf sections of &lt;I&gt;P. communis&lt;/I&gt; Bartlett when cultured on Murashige and Skoog complete medium containing 6.0 mg/l BA (6-benzyladenine) and 0.1 mg/l NAA (&amp;alpha;-naphthaleneacetic acid), while Quoirin and Lepoivre complete medium supplemented with 1.0 mg/l TDZ [thidiazuron (N-phenyl-N&lt;sup&gt;1&lt;/sup&gt;-1,2,3-thiadiazol-5-ylurea)] and 0.1 mg/l NAA was found best for &lt;I&gt;P. pyrifolia&lt;/I&gt; Shenbuzhi, and Nitsch and Nitsch complete medium containing 3.0 mg/l TDZ and 0.1 mg/l NAA or 0.2 mg/l IAA was suitable for&lt;I&gt;P. bretschneideri&lt;/I&gt; Zaosu or &lt;I&gt;P. ussuriensis&lt;/I&gt; Manyuanxiang, respectively. After cutting the leaves into three sections perpendicular to the midrib and culturing under the equivalent conditions, regeneration occurred more frequently on basal sections than middle sections, and no shoots formed on apical sections. A ratio of NH&lt;sup&gt;+&lt;/sup&gt;&lt;sub&gt;4&lt;/sub&gt;-N/NO&lt;sup&gt;-&lt;/sup&gt;&lt;sub&gt;3&lt;/sub&gt;- N of 1:2~1:7 was found beneficial for shoot regeneration. 75.0–87.5% of proliferating shoots formed roots after 4 weeks of transfer to 1/4 strength of Murashige and Skoog complete medium supplemented with 2.5 mg/l IBA (indole-3-butryric acid) and 30.0 g/l sucrose. Regenerated plants were successfully established under greenhouse conditions.

The Differences among Pear Genotypes to Fire Blight (Erwinia amylovora) Attack, Based on Observations of Natural Infection

Fire blight, caused by the bacterium Erwinia amylovora, is one of the most damaging diseases of pear in the world. In Cluj-Napoca area, situated in central Transylvania, Romania, fire blight was observed first in 1994, very late comparative with the other countries from occidental Europe. The response of the pear cultivars and species from National Pear Collection from Cluj-Napoca to fire blight attack, assessed in natural conditions of infection, range on a large scale of variability, which denotes a strong influence of the genotype in expression of resistance or sensitivity to disease. From all genotypes, about 20.5% have not presented symptoms of attack, among them being the following: 'Blanquet precoce', 'Klementinka', 'Severianka', 'Beurre Bachelier', 'Kieffer Seedling', 'Er Shi Shinge', 'Beurre Amanlis', 'Bristol Cross', 'Beurre Liegel', 'Beurre Lucon', 'Grand Champion', 'Magness', 'Mericourt' etc. and several ancient autochthonous cultivars ('Pere malaiete', 'De zahar de Bihor', 'Cu miez rosu', 'Clopotele', 'Garoafa mare', 'Craiese', 'Para de apa'). Also, there were identified several species of Pyrus with no attack, as P. pollveria, P. common pear, P. lindlezi, P. malifolia, P. persica, P. ussuriensis, P. variolosa. The remarked genotypes could be potential sources for further breeding programmes and increase the number of genotypes available for breeding new pear cultivars resistant to Erwinia attack.

Identification of S-genotypes and novel S-RNases in native Chinese pear

SummaryChinese pear (Pyrus spp.) exhibits gametophytic self-incompatibility (GSI), as do other fruit species in the family Rosaceae. This work determined S-locus diversity in 20 native Chinese pear cultivars or wild accessions using polymerase chain reaction (PCR) and pollination experiments. After cloning and sequencing the PCR products, the S-genotypes of all 20 pear cultivars or wild accessions were determined. Subsequent sequence analysis showed that P. sinkiangensis Yü ‘Aolian’ (SpS32), P. phaeocarpa Rehd. ‘Diaodan’ (SdSe), P. xerophila Yü ‘Shageda’ (S36Sd) and ‘Xingyeli’ (S22Sc), originating in China, shared some S-RNase genes with P. communis, providing evidence that oriental and occidental Pyrus species may share the same pool of alleles at the S-locus. Two novel S-RNase genes were also discovered in P. ussuriensis ‘Maili’ and ‘Neimenggushanli’, and deposited as S40 and S41, under the accession numbers DQ903313 and DQ988687, respectively. Their deduced amino acid sequences showed high similarity to S11-RNase (100% ) and S6-RNase (94.4%) between the C1 and C3 exons in Malus. The high similarity scores between S-RNases in Pyrus and Malus indicate that the existence of S-RNases predated speciation between Pyrus and Malus.

A wide range of genetic diversity in pear (Pyrus ussuriensis var. aromatica) genetic resources from Iwate, Japan revealed by SSR and chloroplast DNA markers

Iwateyamanashi (Pyrus ussuriensis var. aromatica) is one of the Pyrus species which grows wild in Japan. The number of Iwateyamanashi trees has been decreasing, so conservation and evaluation is urgently needed. Over 500 accessions of Pyrus species collected from Iwate in northern Tohoku region are maintained at Kobe University as an Iwateyamanashi germplasm collection. In order to investigate the genetic diversity, five SSR (simple sequence repeat) markers, developed from Japanese and European pear were examined for 86 Pyrus individuals including 58 accessions from Iwate. These SSR loci could discriminate between all the Iwate accessions except for 10 that bear seedless fruit, as well as determine the genetic diversity in Iwateyamanashi germplasms. High levels of variation were detected in 41 alleles and the mean observed heterozygosity across 5 loci was 0.50 for the Iwate accessions. Seedless accessions sharing identical SSR genotype with the local pear variety “Iwatetanenashi” were supposed to have been propagated vegetatively via grafting. In an UPGMA phenogram, Japanese pear varieties (P. pyrifolia) were clustered into two groups with some Iwate accessions including seedless ones. Another 38 Iwate accessions were not clustered clearly, and there was no clear relationship between these accessions and geographical distribution or morphological characters. Allele frequency revealed that the Iwate accessions were genetically more divergent than the Japanese pear varieties. Most Japanese pears possessed a 219 bp deletion at a spacer region between the accD and psaI genes in the chloroplast DNA (cpDNA), but other Pyrus species and two Iwateyamanashi trees did not. In the Iwate accessions, 79.3% had a deletion type cpDNA and others had a standard type cpDNA without deletion. These results are indicative of the wide range of genetic diversity in the Iwate accessions which include Japanese pear varieties. A combination of SSR and cpDNA analyses revealed high heterogeneity in Iwateyamanashi and coexistence of Iwateyamanashi and hybrid progeny with P. pyrifolia. These could be reasons for the wide range of continuous morphological variation described previously.

Isolation and characterization of polymorphic microsatellite markers from European pear (Pyrus communis L.)

AbstractThis study reports the development and characterization of 19 microsatellite primer pairs developed from genomic DNA of European pear (Pyrus communis) and their transferability to other Pyrus and Malus material. The primers were designed from two different genomic libraries enriched for di‐ and trinucleotide repeats. When tested in six P. communis cultivars and 15 other Pyrus species, 13 primers revealed single‐locus polymorphism and six showed more complex patterns that suggest multiple loci. Two to 18 alleles were detected per locus and two primer pairs were sufficient to discriminate all accessions. Transferability of nine primer pairs to Malus was demonstrated through amplification of discrete products in two accessions.

Pear (Pyrus Species) Genetic Resources in Iwate, Japan

Iwateyamanashi (Pyrus ussuriensis Maxim. var. aromatica (Nakai et Kikuchi) Rehd.) is one of the Pyrus species grown wild in Japan. The origin of the Japanese pear (P. pyrifolia) is uncertain but it has been suggested that Iwateyamanashi is the possible progenitor of the modern Japanese cultivar. During the last few decades, the number of Iwateyamanashi trees has been decreasing and therefore, conservation is urgently needed. After 13 explorations in the northern Tohoku region of Japan, 615 pear trees and 30 local names were recognized mainly in Iwate Prefecture. The center of the distribution seemed to be somewhere around Mt. Hayachine to the northern area of Kitakami highlands (from lat. 39 °20′ to 40 °10′N, and from long. 141 °20′ to 141 °50′E). Four morphological characters concerning fruit shape, measured for 85 trees, showed a wide range of continuous variation. For the skin colour of fruit, 51% of trees bore russet type fruits, 22% smooth and 27% intermediate ones. Most of the fruits had five loculi but Sanenashi fruits (seedless pear), one of the old cultivars, had three, and fruits of another two trees had four. More than 80% of trees tended to produce fruits with a calyx but some trees bore fruits without a calyx. These observations indicate a wide range of genetic diversity in Pyrus species which is caused, not only by high heterogeneity in Iwateyamanashi itself, but also by the coexistence of Iwateyamanashi, P. pyrifolia (Burm.) Nakai and hybrid progeny in this area. Already 250 trees have been conserved as genetic resources by grafting at Kobe University.

Salt Tolerance in Pyrus Species is Linked to Levels of Na and Cl Translocation from Roots to Leaves

Salt tolerance was tested in five Asian Pyrus rootstock species: Pyrus betulaefolia Bunge (strains C and N; P. betulaefolia C and P. betulaefolia N, respectively), P. calleryana Dcne. (strain No. 8; P. calleryana 8), P. pyrifolia Nakai, P. fauriei Schneid., and P. dimorphophylla Makino (strain No. 6; P. dimorphophylla 6). Four-month-old seedlings were subjected to each of 50 mM, 100 mM, 150 mM, and 200 mM NaCl solutions, and leaf injury, shoot growth, leaf water potential, and mineral uptake were evaluated. P. betulaefolia C showed the highest salt tolerance with no visible symptoms of injury even in the 200 mM NaCl treatment. P. betulaefolia N and P. dimorphophylla 6 also exhibited higher salt tolerance. In contrast, P. calleryana 8, P. fauriei, and P. pyrifolia exhibited lower salt tolerance with severe leaf injuries leading to defoliation and death. Leaf water potential decreased via NaCl treatment in all species and there was no difference among species. Sodium and Cl contents in the roots of all species increased within 2 weeks after NaCl treatment, although the differences among species and NaCl treatment was small. On the other hand, Na and Cl contents in leaves were different among species. The salt-sensitive species P. calleryana 8 and P. pyrifolia accumulated higher concentrations of Na and Cl in the leaves over 150 mM NaCl at 6 weeks after treatment compared to the salt tolerant species P. betulaefolia C. These results imply that P. betulaefolia C might have a key mechanism such as storage exclusion and/or transport-restriction between the shoot and root to depress the transport of Na and Cl to the upper plant parts, enabling a higher tolerance to NaCl.

Selection of Dwarfing Pear Rootstock Clones from Pyrus betulaefolia and P. calleryana Seedlings

A rootstock trial has been established to develop dwarfing pear rootstock clones from Pyrus species. Seedlings that were raised from the open-pollinated seeds of P. betulaefolia Bunge (PB) and P. calleryana Decne (PC); and showed significant genetic dwarfing growth and good traits based on ease of clonal propagation were screened. Among those, four seedlings of PB named SPRB1, SPRB13, SPRB15, and SPRB22 (SPRB = Shinshu University Pear Rootstock P. betulaefolia clone) and six seedlings of PC named SPRC3, SPRC5, SPRC8, SPRC13, SPRC15, and SPRC20 (SPRC = Shinshu University Pear Rootstock P. calleryana clone) were selected. Clones that were generated from the selected seedlings had significantly higher rooting ability by softwood cuttings compared with the control during an 8-year test. Japanese pear ‘Kosui’ and European pears ‘La France’ and ‘Conference’ revealed a dwarfing growth habit when grafted onto the selected clones. All scion/stock combinations interacted significantly to affect shoot growth, trunk cross-sectional area (TCSA), and canopy spread. There was no noticeable difference in the fruit yield per tree of three cultivars grafted on PB and SPRB1, but the estimated yields per ha increased more than 20% in all cultivars on SPRB1. ‘La France’ trees on SPRB15 and SPRC20 began to bear early with significantly higher yields. Fruit quality characteristics of ‘La France’ and ‘Conference’ did not differ because of the influence of stock clones, however, ‘Kosui’/SPRB1 had lower average fruit weight that corresponded to the lighter fruit skin color.

SELECTION FOR NEW PEAR ROOTSTOCKS: IN VITRO SCREENING AND FIELD EVALUATION FOR TOLERANCE TO IRON CHLOROSIS

Crosses have been made between the new INRA pear rootstock selection 'Pyriam' and four Mediterranean Pyrus species: Pyrus communis cordata, P. amygdaliformis, P. amygdaliformis persica, and P. elaeagrifolia. An in vitro test to evaluate tolerance to lime-induced chlorosis has been applied to these progenies. Embryos were dissected out of fruits and cultured in a basal medium to ensure proper plantlet development. One week later, plantlets with roots and leaves emerging out of the cotyledons were transferred to a paper bridge in a Magenta flask containing a liquid medium with 2μM Fe3+DTPA and 10mM NaHCO3. After two weeks of culture, different levels of iron deficiency chlorosis on the newly formed leaves were observed. All evaluated plants were acclimated, grown in the greenhouse, and transferred to a field plot with a clay loam soil with 26% of calcium carbonate equivalent. Total chlorophyll content of each seedling was measured twice, during the springs of 2000 and 2001, and once after grafting with 'Conference' during the fall of 2001. No significant correlations between in vitro and field conditions were observed at the individual seedling level of tolerance. However, a significant correlation was found comparing hybrid crosses. The in vitro tolerance to iron deficiency was similar to the known differential tolerance of each Pyrus species, being P. amygdaliformis, Pyrus communis cordata, and P. amygdaliformis persica more tolerant than P. elaeagrifolia and Pyrus communis. The results also confirmed a significant correlation between in vitro seedling evaluation for tolerance to iron deficiency conditions and field tolerance of grafted plants to lime-induced chlorosis. To solve the lack of correlation at the individual seedling level, the next assays will investigate in vitro cloning of each individual prior to the in vitro test. In vitro propagation of some preliminarily selected clones was satisfactory.

Comparative analysis of chloroplast DNA in Pyrus species: physical map and gene localization

A physical map of chloroplast DNA (cpDNA) of pear [Pyrus ussuriensis var. hondoensis (Nakai et Kikuchi) Rehder] was constructed using five restriction enzymes, SalI, XhoI, BamHI, SacI and PstI. This information will make it possible to investigate the phylogenetic relationships between Pyrus species. Pear cpDNA was found to be a circular molecule with a total size of about 156 kb in which two inverted repeats of 24.8 kb divide the molecule into small (17 kb) and large (90 kb) single-copy regions. The endonuclease recognition sites in the physical map were determined by single and double digestion of 13 lambda phage clones which covered the entire sequence of the pear cpDNA. Twenty nine genes were localized on the physical map of the pear cpDNA. The structure of pear cpDNA was almost the same in terms of genome size and gene order as that of tobacco cpDNA. RFLP analysis was carried out on cpDNAs from five Pyrus species (Pyrus pyrifolia, Pyrus ussuriensis, Pyrus calleryana, Pyrus elaeagrifolia and Pyrus communis). Two mutations, a recognition-site mutation and a length mutation (deletion), were found only in the cpDNA of P. pyrifolia cultivars. These mutations were localized on the physical map of pear cpDNA. The number of mutations of cpDNA in Pyrus species are small in comparison with those of other angiosperms, suggesting a high degree of genome conservatism in Pyrus species.

Genetic Relationships of Pyrus Species and Cultivars Native to East Asia Revealed by Randomly Amplified Polymorphic DNA Markers

A total of 118 Pyrus sp. (pear) and cultivars native mainly to east Asia were subjected to randomly amplified polymorphic DNA (RAPD) analysis to evaluate genetic variation and relationships among the accessions. Two hundred fifty RAPD markers were scored from 20 decamer primers. RAPD markers specific to species were identified. Clustering analysis revealed two divisions: one comprising cultivars of P. communis L., and the other including all accessions of Pyrus native to east Asia. The grouping of the species and cultivars by RAPD data largely agrees with morphological pear taxonomy. However, some noted incongruence existed between two classification methods. Pyrus calleryana Dcne. clustered together with P. koehnei Schneid., P. fauriei Schneid. and P. dimorphophylla Makino. Pyrus betulaefolia Bge. clustered with P. ×hopeiensis Yu and P. ×phaeocarpa Rehd. A noncultivated clone of P. aromatica Kikuchi et Nakai grouped with P. aromatica cultivars. Pyrus hondoensis Nakai et Kikuchi and cultivars of P. ussuriensis Max. formed a single group. Some accessions from Korea (named Korean pear) had species-specific RAPD markers and comprised an independent group. Most of the Chinese white pears clustered together with most of the Chinese sand pears. Based on the present results, the new nomenclature P. pyrifolia var. sinensis (Lindley) Teng et Tanabe for Chinese white pear was suggested. Most accessions of Japanese pears fell into one main group, whereas pear cultivars from Kochi Prefecture of Japan subclustered with some Chinese sand pears and one accession from Korea. Our results infer that some local Japanese pear cultivar populations may have been derived from cultivars native to Kochi Prefecture in Shikoku region, and that the latter may have been introduced from ancient China and/or Korea.

Characterization of copia-like Retrotransposons in Pear.

Fifty-one non-redundant fragments of copia-like retrotransposons were cloned and identified in pear. Based on phylogenetic analysis, the retroelements were classified into 15 distinct groups and were highly heterogeneous in the pear genome. However, most of the groups could be clustered with some registered copia-like retrotransposons. Several clones showed relatively low identities to the known sequences. A Southern blot analysis, using 8 representative clones as probes, demonstrated that these retroelements were distributed in Pyrus species. Polymorphisms were detected among as well as within the Pyrus species, suggesting that the transposition of retrotransposons took place during evolution leading to diversification. Based on the Southern blot analysis, the copy number of copia-like retrotransposons was estimated to be at least 1, 000 in pear genomes.

Identification of Asian Pear Varieties by SSR Analysis

Sixty Asian pear accessions from 6 Pyrus species were genetically identified by 9 SSR markers with a total of 133 putative alleles. Among them, 58 varieties could be successfully differentiated except for 2 pairs of synonymous or clonal varieties. All the SSR markers produced 1 or 2 discrete amplified fragments for all the diploid accessions, whereas a triploid variety showed 3 fragments with some SSRs. The number of putative alleles ranged from 7 to 20, with an average value of 14.8. The observed heterozygosity and the power of discrimination were 0.63 and 0.91, respectively. A phenogram based on the SSR genotypes was obtained, showing 3 major groups corresponding to the Japanese, Chinese and European groups. The SSR markers were highly polymorphic and could be utilized as a reliable tool for cultivar identification in Asian pears.

Pear Seeds Retain Viability after Liquid Nitrogen Immersion

Cryopreservation in liquid nitrogen (LN) is relatively routine for many small, desiccation-tolerant (orthodox) seeds. Seeds of Pyrus species are considered orthodox but have not been evaluated for LN storage. Seeds of freshly collected P. communis L. (`Bosc') were evaluated for germinability and by TZ staining after exposure to four LN treatments: 1) direct immersion and direct removal; 2) direct immersion and 1 minute in LN vapor phase before removal; 3) 2 minutes in vapor phase before immersion and direct removal; and 4) 2 minutes in vapor phase before immersion and 1 minute in vapor phase before removal. Fresh `Bosc' seed viability evaluated by TZ and greenhouse germination tests remained high (83% to 100%) following four types of LN treatments, compared to the controls (77% to 87%). Differences in `Bosc' seed viability were small and TZ results showed no significant differences among the LN treatments. Direct LN immersion and removal resulted in significantly more greenhouse-germinated `Bosc' seeds than the other treatments and fewer control seeds germinated than any LN treated seeds. Fresh `Bosc' seed cryopreserved at 7.9% moisture exhibited high germinability by both TZ and germination tests. LN exposure caused no physical damage to the seeds. Chemical name used: 2,3,5-triphenyltetrazolium chloride (TZ).