Introgression between native and prehistorically naturalized (archaeophytic) wild pear (<Emphasis Type="Italic">Pyrus</Emphasis> spp.) populations in Northern Tohoku,Northeast Japan

Hybridization between native and cultivated species is a concern in conservation biology. However, detecting such hybridization and distinguishing true natives from prehistorically naturalized species based on phenotypic characteristics is difficult. Here, we report on introgression between native and prehistorically introduced pear (Pyrus) species in Northern Tohoku (northern end of Honshu Island), Japan. We analyzed 20 microsatellites in 226 wild, seemingly wild, or cultivated materials. Phenetic analysis showed that wild Japanese populations of P. ussuriensis var. ussuriensis in Northern Tohoku, previously considered true natives based on morphology and phytogeography, differed from those in continental Asia, confirming their nativeness. However, Bayesian inference of population structures showed that Japanese P. ussuriensis was genetically admixed with two genetic clusters: true native P. ussuriensis var. ussuriensis and prehistorically introduced P. pyrifolia. Even in the Kitakami Mountains, where true native populations of var. ussuriensis are believed to persist, most wild trees were at least somewhat admixed. Prehistorically introduced then naturalized plants are treated as natives in Japan’s conservation management, and some are considered endangered. However, introgression of prehistorically naturalized P. pyrifolia into threatened native P. ussuriensis var. ussuriensis has occurred. This paper examines the implications for conservation management.     

Genomic characterization of self-incompatibility ribonucleases in the Central Asian pear germplasm and introgression of new alleles from other species of the genus Pyrus

The Pyrus species exhibit the so-called S-RNase-based gametophytic self-incompatibility system, which is considered to be the most widespread self-incompatibility system among flowering plants. In this study, 57 Iranian pear (Pyrus communis L.) domestic cultivars and wild genotypes, plus 21 European pear cultivars used as references, were genotyped adopting a PCR-based genotyping assay using consensus and allele-specific primers. The results revealed traces of significant genetic contribution in the Iranian traditional varieties and genotypes from other Pyrus species; the genetic contribution of Japanese pear clearly emerged with the detection of some Pyrus pyrifolia S-RNase alleles. Moreover, our results highlighted the presence of three new S-RNase alleles (named S₁₂₆, S₁₂₇, and S₁₂₈) that were not previously identified in P. communis, possibly introduced in the germplasm of cultivated pear through gene transfer from other cultivated or wild species.     

A phylogenetic network of wild Ussurian pears (Pyrus ussuriensis Maxim.) in China revealed by hypervariable regions of chloroplast DNA

In order to understand the genetic diversity of wild Ussurian pears in China, chloroplast DNA (cpDNA) of 186 wild accessions from 12 populations in Inner Mongolia, Heilongjiang and Jilin Provinces and 51 Chinese and European pear cultivars including Pyrus ussuriensis, Pyrus pyrifolia, Pyrus bretschneideri, Pyrus sinkiangensis and Pyrus communis were investigated. Each accession was classified into one of three types (types A, B and C) based on two large deletions in the hypervariable regions between the accD–psaI and rps16–trnQ genes. Thirty haplotypes were identified by 32 mutations including 17 gaps (in/dels) and 15 base changes. Haplotype network analysis revealed that wild Chinese Ussurian pears could be grouped into subgroup I of type A. A haplotype, Hcp3, in subgroup I detected in Heilongjiang and Jilin Provinces was considered to be a divergent centre in Chinese Ussurian pears. However, the genetic diversity of wild accessions revealed by the two hypervariable regions was quite low. In particular, 98 % of wild Ussurian accessions in Inner Mongolia shared an identical haplotype Hcp1 and are, therefore, monomorphic. In comparison, Chinese pear cultivars were more divergent. These results suggest that the cpDNAs from wild Ussurian pears in Inner Mongolia have specifically differentiated compared to those from pears of other areas. The number of wild Ussurian pears has been decreasing because of desertification and land development, therefore conservation is needed.     

Genetic Diversity of Some Pear Cultivars and Genotypes Using Simple Sequence Repeat (SSR) Markers

The genetic diversity and relationships among 47 pear cultivars and genotypes (Pyrus spp.), including 4 Japanese pears (Pyrus pyrifolia), 40 European pears (Pyrus communis), 1 Chinese pear (Pyrus bretschneideri) as well as 2 wild relatives (Pyrus salicifolia and Pyrus mazandaranica) were studied using 28 microsatellite primer pairs. A total of 174 alleles were produced at the 28 SSR loci with their sizes ranging from 81 to 290?bp. The number of observed alleles for each locus ranged from 3 (TsuENH014 and TsuENH046) to 12 (NB103a), with an average of 6.21 alleles per locus. In some SSR loci, more than two alleles were amplified in some cultivars and genotypes, suggesting that duplication has occurred in those accessions. This information suggests that at least two genomic regions exist for these loci in the pear genome. The observed heterozygosity (H _o) values of amplified loci ranged from 0.17 (TsuENH006) to 0.97 (NB103a). Shannon's information index (I) value was observed to be highest (2.14) in the NB103a locus, while the TsuENH006 locus had the lowest value with an average of 1.37 among SSR loci. The Dice genetic similarity coefficient ranged from 0.29 (??Nijisseiki?? and P. mazandaranica) to 0.91 (??Chojuro?? and ??Nijisseiki??) among samples. UPGMA cluster analysis showed two major groups corresponding to the Japanese and European pears.     

The southwestern origin and eastward dispersal of pear (<Emphasis Type="Italic">Pyrus pyrifolia</Emphasis>) in East Asia revealed by comprehensive genetic structure analysis with SSR markers

Pyrus pyrifolia is considered one of the most important cultivated Pyrus species. Hundreds of landraces and bred cultivars have been developed through the natural and artificial hybridizations necessary due to self-incompatibility. In this study, the genetic diversity of 478 Pyrus accessions, including Chinese landraces, bred cultivars, and wild samples, as well as introduced pear cultivars from Japan and Korea, was investigated with a set of 17 simple sequence repeat (SSR) markers distributed across all 17 linkage groups of the pear genome. A total of 121 alleles were detected, including 4 rare alleles with a frequency lower than 5%. Diversity statistics indicated a high level of genetic variation as quantified by the average values of the observed heterozygosity, the expected heterozygosity, and Wright’s fixation index, at 0.76, 0.78, and 0.02, respectively. Population structure and discriminant analysis of principal component analysis implied extensive genetic communication between sand pears in China and revealed four contiguous geographical clusters with overlapping geographical regions. The diversity of the four clusters and approximate Bayesian computation (ABC) indicated that sand pear spread from west to east along the Pearl River and Yangtze River valleys. High diversity and polyphyletic genetic components of cultivars in southwestern China further support southwestern China as the probable center of divergence for Pyrus species. A core collection of 80 out of 470 cultivars was selected, accounting for about 17% of accessions, and capturing 91% of all alleles, including all rare alleles. Our research provides a comprehensive understanding of sand pear germplasm in East Asia and constructs a preliminary core collection, which will be useful for association genetics studies, germplasm conservation, and breeding programs.     

Genetic variation and population structure of “Zangli” pear landraces in Tibet revealed by SSR markers

Pear (Pyrus L.) is one of the most important fruits in temperate regions. Even in the mountainous border region of Tibet, Sichuan, and Yunnan Provinces (China), a variety of highly adapted pear landraces were discovered in Tibetan Autonomous Prefecture and were named “Zangli pears.” Here, we analyzed genetic diversity of 29 genotypes of Zangli distributed in Tibetan Region, together with 30 genotypes of sand pear (P. pyrifolia) in border regions, and 8 genotypes of P. ussuriensis as out groups, using simple sequence repeats (SSR) markers. Twenty-eight SSR markers displayed high polymorphism with a total of 202 alleles. Cluster analysis revealed a close genetic relationship between Zangli and sand pears, while population structure analysis revealed that gene flow between the western and eastern side of the Jin-sha River was restricted by geographic isolation. Diversity statistics for five geographic groups implied a migration event from Sichuan and Yunnan Provinces to the border region of western Sichuan, eastern Tibet, and northwestern Yunnan Provinces. Six specific genetic alleles in Zangli pear landraces might be related to their adaption to severe environmental conditions.     

SSRs isolated from apple can identify polymorphism and genetic diversity in pear

Apple simple sequence repeats (SSRs) were intergenerically applied to the characterization of 36 pear accessions, including 19 Japanese pears (Pyrus pyrifolia), 7 Chinese pears (P. bretschneideri, P. ussuriensis), 5 European pears (P. communis), 3 wild relatives (P. calleryana), and 2 hybrids between P. pyrifolia and P. communis. All of the tested SSR primers derived from apple produced discrete amplified fragments in all pear accessions. Nucleotide repeats were detected in the amplified bands by both Southern blot and sequencing analysis, and nucleotide sequences of pear were compared with those of apple. The differences in fragment size among pear or between pear and apple were, in many cases, due to the differences in repeat number. Interestingly, the DNA sequence of flanking regions in apple was highly conserved in pear. Hybrids from P. pyrifolia×P. communis showed one fragment inherited from each parent in all scorable cases, which suggested that each primer pair amplified fragments originating from the same locus. A total of 79 alleles were detected from seven SSR loci in pear, and all pear varieties except for the mutants could be differentiated. In conclusion, SSRs isolated from apple are highly conserved in pear and could be utilized as DNA markers in the latter genus. Received: 17 July 2000 / Accepted: 22 September 2000     

Identification of Pyrus Single Nucleotide Polymorphisms (SNPs) and Evaluation for Genetic Mapping in European Pear and Interspecific Pyrus Hybrids

We have used new generation sequencing (NGS) technologies to identify single nucleotide polymorphism (SNP) markers from three European pear (Pyrus communis L.) cultivars and subsequently developed a subset of 1096 pear SNPs into high throughput markers by combining them with the set of 7692 apple SNPs on the IRSC apple Infinium® II 8K array. We then evaluated this apple and pear Infinium® II 9K SNP array for large-scale genotyping in pear across several species, using both pear and apple SNPs. The segregating populations employed for array validation included a segregating population of European pear (‘Old Home’בLouise Bon Jersey’) and four interspecific breeding families derived from Asian (P. pyrifolia Nakai and P. bretschneideri Rehd.) and European pear pedigrees. In total, we mapped 857 polymorphic pear markers to construct the first SNP-based genetic maps for pear, comprising 78% of the total pear SNPs included in the array. In addition, 1031 SNP markers derived from apple (13% of the total apple SNPs included in the array) were polymorphic and were mapped in one or more of the pear populations. These results are the first to demonstrate SNP transferability across the genera Malus and Pyrus. Our construction of high density SNP-based and gene-based genetic maps in pear represents an important step towards the identification of chromosomal regions associated with a range of horticultural characters, such as pest and disease resistance, orchard yield and fruit quality.     

Population genetic structure in a threatened tree, Pyrus calleryana var. dimorphophylla revealed by chloroplast DNA and nuclear SSR locus polymorphisms

Pyrus calleryana var. dimorphophylla, a variety of Callery pear (Pyrus calleryana), is endemic to the Tokai district of central Japan, and is currently listed as “Endangered”. The remnant habitats and trees are of limited number, and highly fragmented. As the first step in determining appropriate conservation units, genetic diversity and differentiation in this species were investigated using chloroplast DNA (cpDNA) and nuclear simple sequence repeat (SSR) polymorphisms. All possible remnant trees were genotyped, then six populations were defined based on the results of cpDNA haplotype determination and Bayesian clustering approaches performed using the SSR locus data. Some trees appeared to originate from artificial propagation. Some individuals were difficult to differentiate genetically from the related species, Pyrus × uyematsuana, which is considered to be a hybrid between P. calleryana var. dimorphophylla and a possibly naturalized species, Pyrus pyrifolia, implying that introgression between these species may have occurred. In P. calleryana var. dimorphophylla, anthropogenic factors such as propagation and related species planting are probably major causes of complexity in the genetic structure.     

Mesos components (CaCl2, MgSO4, KH2PO4) are critical for improving pear micropropagation

Pear accessions and species show a broad response to tissue culture media due to the wide genetic diversity that exists in the available pear germplasm. An initial study of mineral nutrition using a systematic response surface approach with five Murashige and Skoog medium mineral stock solutions indicated that the mesos factor (CaCl₂, MgSO₄, and KH₂PO₄) affected most plant responses and genotypes, suggesting that additional studies were needed to further optimize these three mesos components for a wide range of genotypes. Short stature, leaf spots, edge necrosis, and red or yellow coloration were the main symptoms of poor nutrition in shoot cultures of 10 diverse pear genotypes from six species. A surface response experimental design was used to model the optimal factor and factor levels for responses that included overall quality, leaf character, shoot multiplication, and shoot height. The growth morphology, shoot length, and multiplication of these pear shoots could be manipulated by adjusting the mesos components. The highest quality for the majority of genotypes, including five P. communis cultivars, P. koehnei, P. dimorphophylla, and P. pyrifolia ‘Sion Szu Mi’, required higher concentrations (>1.2× to 2.5×) of all the components than are present in Murashige and Skoog medium. ‘Capital’ (P. calleryana) required high CaCl₂ and MgSO₄ with low KH₂PO₄; for ‘Hang Pa Li’ (P. ussuriensis), low CaCl₂ and moderate to low MgSO₄ and KH₂PO₄ produced high-quality shoots. Suitable combinations of the meso nutrients produced both optimum shoot number and shoot length in addition to general good plant quality.     

Polymorphism of microsatellite loci in cultivars and species of pear (<Emphasis Type="Italic">Pyrus</Emphasis> L.)

Using five SSR markers, polymorphism of microsatellite loci was examined in 46 cultivars and five species of pear (Pyrus ussuriensis, P. bretscgneideri, P. pyraster, and P. elaegnifolia). Most of the accessions examined showed the presence of unique allele sets. The degree of relationship between Russian and Western European pear cultivar was established. It was demonstrated that P. ussuriensis and its first generation progeny were genetically distant from typical cultivars of P. communis, as well as from the P. communis × P. ussuriensis hybrids of later generations. SSR estimates of the cultivar relatedness were shown to correlate with the corresponding pedigree-based estimates. A number of SSR alleles specific to P. ussuriensis were identified. Based on the analysis of microsatellite loci, the allelic composition was determined for each cultivar examined. These data can serve as a molecular certificate of the cultivar.     

Molecular markers derived from RAPD,SCAR, and the conserved 18S rDNA sequences for classification and identification in<Emphasis Type="Italic"> Pyrus pyrifolia</Emphasis> and<Emphasis Type="Italic"> P</Emphasis>.<Emphasis Type="Italic"> communis</Emphasis>

We generated RAPD, SCAR, and conserved 18S rDNA markers for classifying and identifying cultivars of Pyrus pyrifolia (Japanese pear) and P. communis (European pear). PCR amplification with selected specific primers—LCH327UP and LCH327DOWN—was performed using DNA extracted from 25 P. pyrifolia and P. communis cultivars. The 1,380-bp fragment was amplified from P. communis cvs. Beurre Giffard, Cascade, Conference, Clapps Favorite, Packhams Triumph, and Winter Nelis. RAPD has only a dominant single band of 1,380-bp, however, SCAR has one or more band of the same size. Amplification involving sequence-specific primer pairs LCH346UP and LCH346DOWN resulted in a loss of polymorphism. The 1,190-bp fragment was amplified from all P. pyrifolia cultivars. The conserved sequences of the 18S rDNA fragment of 25 pear cultivars were amplified and analyzed with 42 restriction enzymes. Compared with P. pyrifolia cultivars, they lacked the restriction enzyme site of KpnI and had one less RsaI site. Cultivar Gamcheonbae had a specific PstI restriction site, while cvs. Mansoo and Conference pear digested with AluI showed a different presentation than other cultivars. For the Okusankichi and Shinil pears TaqI was best marker for identification in P. pyrifolia. These results can be adopted for identifying pear cultivars; to date there is no standard marker for identifying the cultivars of fruit trees in Korean fruit tree breeding programs.Communicated by J.S. Heslop-Harrison     

Adventitious shoot regeneration in cotyledons from Japanese pear and related species

An efficient method for the generation of adventitious shoots from the cotyledons of Japanese pear and related species was developed. Cotyledons from seeds of the Japanese pear ??Okusankichi?? and the Asian pea pear ??Hokushimamenashi?? were used to determine the optimum concentrations of phytohormones in the medium. The rates of generation of adventitious shoots and the numbers of adventitious shoots per explant were highest when the media contained 5???M 1-naphthaleneacetic acid combined with 10 or 25???M 6-benzylaminopurine. These growth regulators were used to generate adventitious shoots from the cotyledons of 33 cultivars of Japanese, Chinese, Asian pea, and European pears. A high number of adventitious shoots per explant (1.3?C2.3) and high rates of regeneration of adventitious shoots (60?C76?%) were obtained from the cotyledons of Japanese pears ??Imamuraaki?? and ??Agenosho Shinanashi.?? Although pollination was not controlled, cotyledons from mother trees of old Japanese cultivars and Chinese pears tended to be more regenerable than those from other pear species. Since the rooting ability of the adventitious shoots was very low, micro-grafting was applied to obtain regenerated potted plants from adventitious shoots. Grafted regenerated plants were recovered at a rate of more than 60?%, regardless of cultivar. To our knowledge, this is the first report to evaluate shoot regeneration from cotyledons of major Pyrus species.     

PCR-based method for identifying the S-genotypes of Japanese pear cultivars

 Japanese pear (Pyrus pyrifolia Nakai), a member of the Rosaceae, shows gametophytic self-incompatibility that is controlled by the S-locus. The S-genotype of Japanese pear cultivars is an important factor for crossing and breeding. We report a rapid reliable method to identify these S-genotypes. It consists of PCR amplification of the S-RNase gene from genomic DNA and subsequent digestion of the PCR fragments with S-allele-specific restriction endonucleases. Using this method, we determined the unknown S-genotypes of nine Japanese pear cultivars and selected self-compatible varieties from the offspring of the self-compatible cultivar, ‘Osa-Nijisseiki’. Received: 8 June 1998 / Accepted: 19 October 1998     

Genetic relationships between wild progenitor pear (Pyrus L.) species and local cultivars native to Georgia,South Caucasus

The genetic diversity of 108 individuals of wild pear species (Pyrus communis subsp. caucasica, P. balansae, P. salicifolia, P. syriaca, P. demetrii, P. bulgarica, P. ketzkhovelii, P. sachokiana) and 35 samples of local and introduced cultivated pears from the country of Georgia were compared to 73 individuals of wild P. communis subsp. caucasica and P. communis subsp. pyraster in the collection of USDA-ARS National Plant Germplasm System (NPGS). Pyrus communis subsp. caucasica from both Georgia and the NPGS, P. communis subsp. pyraster from the NPGS, and P. salicifolia from Georgia were differentiated, based on analysis of eleven microsatellite markers. In addition, accessions of P. communis subsp. caucasica from Georgia were genetically distinct from accessions of the same subspecies in the NPGS collection that originated from other European and Middle Eastern Asian countries. Local pear cultivars in Georgia were genetically similar to P. communis subsp. caucasica and P. balansae growing wild in Georgia suggesting that they may have originated from native pear trees that could serve as unique genetic resources for pear breeding programmes.     

Chloroplast DNA-based genetic diversity and phylogeography of Pyrus betulaefolia (Rosaceae) in Northern China

Pyrus betulaefolia Bunge, considered as an intermediate between oriental and occidental pear groups, is one of the most important wild pear species. The number of its populations is decreasing because of habitat destruction, fragmentation, and continuous exploitation, so protection and conservation measures are urgently needed. Assessment of its genetic diversity and phylogeography are imperative for its efficient conservation. Two chloroplast DNA intergenic fragments were used to detect genetic diversity and phylogeography of 320 individuals from 18 wild P. betulaefolia populations. Haplotype variation, genetic differentiation, and historical events of the populations were estimated. The results showed that P. betulaefolia populations sampled in northern China contained a high level of genetic diversity (H _T?=?0.826). A significant isolation-by-distance value (r?=?0.587, P?<?0.001, 1,000 permutations) among all 18 populations indicated a correlation between genetic divergence and geographic distance. Four population groups were identified in a neighbor-joining tree based on the genetic distance. Analyses of molecular variation showed that the genetic variation mainly existed among population groups, representing 64.61 % of the total variation. Phylogeographic analyses indicated that the populations of P. betulaefolia experienced a scenario of rapid range expansion, which probably occurred between 608,000 and 204,580 years ago. Meanwhile, both the restricted gene flow with isolation by distance and allopatric fragmentation were crucial processes responsible for shaping the genetic patterns of P. betulaefolia. The occurrence of specific haplotypes might be ascribed to an ancestral introgression or joint retention of an ancestral polymorphism with other Pyrus species at the northern edge of the distribution of P. betulaefolia. Three populations displaying a high level of haplotype diversity and unique haplotypes were assumed to be relict populations of Quaternary glaciation and should have conservation priority. Three additional large populations should also be preferentially protected by building natural preservation zones.     

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.     

Analysis of genetic diversity and population structure of rice cultivars from Korea, China and Japan using SSR markers

A total of 29 simple sequence repeat (SSR) markers were used to analyze the genetic diversity of 150 accessions of cultivated rice (Oryza sativa L.) from Korea, China, and Japan. A total of 375 alleles were detected with an average of 12.9 per locus. The averaged values of gene diversity and polymorphism information content (PIC) for each SSR locus were 0.7001 and 0.6683, respectively. Alleles per locus in Korean rice were 8.8, whereas 8.1 and 7.2 alleles per locus were found in Chinese and Japanese rice, respectively. The mean gene diversity in Korean, Chinese, and Japanese rice was 0.6058, 0.6457, and 0.5174, respectively, whereas the mean PIC values for each SSR locus were 0.5759, 0.6138, and 0.4881, respectively. The genetic diversity of the Korean and Chinese cultivars was higher than that of the Japanese cultivars, and the genetic diversity ofjaponica was higher than that ofindica. The model-based structure analysis revealed the presence of three subpopulations, which was basically consistent with clustering based on genetic distance. An AMOVA analysis showed that the between-population component of genetic variance was less than 22% in contrast to 78% for the within-population component. The overallFST value was 0.2180, indicating a moderate differentiation among groups. The results could be used for designing effective breeding programs aimed at broadening the genetic bases of commercially grown varieties.