Genetic diversity and relationships among native Japanese horse breeds,the Japanese Thoroughbred and horses outside of Japan using genome‐wide SNP data

Eight horse breeds—Hokkaido, Kiso, Misaki, Noma, Taishu, Tokara, Miyako and Yonaguni—are native to Japan. Although Japanese native breeds are believed to have originated from ancient Mongolian horses imported from the Korean Peninsula, the phylogenetic relationships among these breeds are not well elucidated. In the present study, we compared genetic diversity among 32 international horse breeds previously evaluated by the Equine Genetic Diversity Consortium, the eight Japanese native breeds and Japanese Thoroughbreds using genome‐wide SNP genotype data. The proportion of polymorphic loci and expected heterozygosity showed that the native Japanese breeds, with the exception of the Hokkaido, have relatively low diversity compared to the other breeds sampled. Phylogenetic and cluster analyses demonstrated relationships among the breeds that largely reflect their geographic distribution in Japan. Based on these data, we suggest that Japanese horses originated from Mongolian horses migrating through the Korean Peninsula. The Japanese Thoroughbreds were distinct from the native breeds, and although they maintain similar overall diversity as Thoroughbreds from outside Japan, they also show evidence of uniqueness relative to the other Thoroughbred samples. This is the first study to place the eight native Japanese breeds and Japanese Thoroughbred in context with an international sample of diverse breeds.     

Discrimination of Portuguese grapevines based on microsatellite markers

A set of 46 grapevine denominations was genotyped at 11 microsatellite loci in order to discriminate them. Ninety four alleles with a mean number of 8.55 alleles per locus were observed in a total of 37 detected unique genotypes. Previously assumed synonyms were confirmed and several cases of homonymy resolved. Comparison of the data obtained in this study with data of 32 genotypes previously reported enabled the detection of three parent offspring relationships, and identified other putative parent/progeny relationships. These data allowed understanding the origin of some Portuguese cultivars. The integration of the obtained data with ampelographic data would be very important for the accurate identification of the Portuguese cultivars and can become a significant tool for the certification of quality wines produced in specific regions.     

Reworking the idea of chestnut (Castanea sativa Mill.) cultivation in Roman times: New data from ancient Campania

Abstract

Charcoal analysis was carried out in two archaeological sites on the north slope of the Somma-Vesuvius volcano, not far from Naples. Both sites were inhabited between the 2nd century AD and AD 472, when a great Vesuvius eruption (so called Pollena eruption) buried them. In both sites, Castanea sativa wood was largely used for architectural structures as well as firewood. Ten ¹⁴C dates, spanning between the 1st and the 5th century AD, testify to a continuative use of this wood throughout the life of the sites. The comparison between archaeobotanical and pollen data from other sites in southern Italy suggests that chestnut woods were close to the sites studied and had a restricted spreading, possibly in a refugium area on Vesuvius' foothills. The match between archaeological charcoals and pollen data suggests that the Romans did not spread chestnut cultivation in the entire Campania region and that chestnut probably became a common timber in Campania starting from the 5th–6th century AD.     

Rapid expansion of the distributional range and the population genetic structure of the freshwater amphipod <Emphasis Type="Italic">Crangonyx floridanus</Emphasis> in Japan

The freshwater amphipod Crangonyx floridanus (Amphipoda: Crangonyctidae) is considered to have been recently introduced from North America to Japan, and the recorded sites at which it has been collected now cover nearly all of Japan except for the northern part. In this study, we surveyed further areas outside of its known distribution range, and examined the population genetic structure and the phylogenetic relationships between Japanese and North American populations of this species based on nuclear (18S rRNA) and mitochondrial (COI) DNA sequences. We found that this amphipod has already reached Hokkaido, northernmost Japan, which suggests that it has undergone rapid expansion in a pattern of concentric circles from the central part of Japan. Genetic analysis showed that the Japanese population is genetically homogeneous, in contrast to the genetic diversification of this species seen in North American Crangonyx populations. The process of introducing, establishing, and expanding this amphipod in Japan may be explained as follows. A limited number of individuals from a North American native population were probably inadvertently introduced and established somewhere within the Kanto region. The local population size then increased and its distribution range expanded rapidly across Japan.     

Rearing,release and settlement prospect in Italy of <Emphasis Type="Italic">Torymus sinensis</Emphasis>, the biological control agent of the chestnut gall wasp <Emphasis Type="Italic">Dryocosmus kuriphilus</Emphasis>

Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae), is one of the most serious pests attacking chestnut trees. Recently it was incidentally introduced into Northwest Italy and it is now spreading throughout Europe. This pest was successfully controlled in Japan by introducing a parasitoid, Torymus sinensis Kamijo (Hymenoptera: Torymidae), from China’s mainland. Following this successful experience, the parasitoid was introduced into Italy from Japan. One year of preliminary studies led to a successful method of rearing imported galls with a synchronization between the parasitoid’s emergence and the presence of the target galls in the field. In two consecutive years, a total of 2,117 individuals were released in several sites covering most of the infested area. There are encouraging data about the settlement of the parasitoid and its synchrony with the host’s cycle: from about 64,000 host galls collected in the field over 200 T. sinensis were reared. The role of native parasitoids associated with the chestnut gall pest in its Italian distribution range is also discussed.     

Morphological traits and RAPD markers for characterization and identification of minor Spanish olive cultivars from the Extremadura region

A total of 91 wild olive accessions and 31 olive cultivars growing in the Extremadura region of central-western Spain were analyzed using morphological traits and RAPD markers. We focused on three main and 16 minor Spanish olive cultivars that are recognized as native or local to the Extremadura region. The five arbitrary 10-mer primers tested on the olive cultivars gave 67 polymorphic bands, representing 91% of the total amplification products. The number of bands per primer ranged from 9 to 18, whereas the number of polymorphic bands ranged from 8 to 17. All the cultivars could be identified by a combination of three primers (OPF-6, OPA-8, and OPK-16); four cultivar-specific markers were detected. The minor local "Jariego" and "Tempranillo" cultivars showed the most distal similarities. The resulting dendrogram, using the unweighted pair-group method with arithmetic mean clustering algorithm, depicted the pattern of relationships between the local Extremadura cultivars and the cultivars from geographically connected regions. This analysis showed a correlation between most of the minor local cultivars and the geographical origin; there was no apparent clustering according to morphological traits or fruit use of olive cultivars when these parameters were used as analysis criteria.     

Evaluation of introgressive hybridization among Cervidae in Japan's Kinki District via two novel genetic markers developed from public NGS data

Hybridization and backcrossing of native populations with introduced species can lead to introgression and genetic alteration. In this study, we evaluated introgression in 43 deer from a potential hybrid zone around Okinoshima Island, Kinki District, Japan. This region witnessed the migration of a hybrid population (cross between the Formosan sika deer [Cervus nippon taiouanus] and other deer species) that could potentially breed with the native Japanese sika deer (C. n. centralis). We used an existing genetic marker for the mitochondrial cytochrome b gene and two novel markers for nuclear DNA, developed using publicly available next‐generation sequencing data. We identified one mainland deer with a mitochondrial haplotype identical to that of the Formosan sika deer as well as nuclear heterozygous sequences identical to those of Formosan and Japanese sika deer. This suggests that the mainland deer is a hybrid offspring of the Okinoshima population and native deer. However, only Japanese sika deer sequences were found in the other 42 samples, indicating limited introgression. Nevertheless, hybridization pre‐ and postintroduction in the Okinoshima population could cause multispecies introgression among Japanese sika deer, negatively affecting genetic integrity. We developed a simple test based on polymerase chain reaction–restriction fragment length polymorphism to detect introgression in natural populations. Our method can accelerate genetic monitoring of Japanese sika deer in Kinki District. In conclusion, to prevent further introgression and maintain genetic integrity of Japanese sika deer, we recommend establishing fences around Okinoshima Island to limit migration, besides a continued genetic monitoring of the native deer.     

Analysis of mtDNA sequences shows Japanese native chickens have multiple origins

In this study, we analysed the mitochondrial DNA D-loop region of Japanese native chickens to clarify their phylogenetic relationships, possible maternal origin and routes of introduction into Japan. Seven haplogroups (Types A-G) were identified. Types A-C were observed in Jidori, Shokoku and related breeds. However, Type C was absent in Shokoku, which was introduced from China, while most Indonesian native chickens were included in the Type C haplogroup. Types D-G were observed in Shamo and related breeds. Type E had a close genetic relationship with Chinese native chickens. Our results indicate that some breeds were not introduced into Japan as suggested in conventional literature, based on low nucleotide diversity of certain chicken breeds. Sequences originating from China and Korea could be clearly distinguished from those originating from Southeast Asia. In each group, domestic chickens were divided into the Jidori-Shokoku and Shamo groups. These results indicate that Chinese and Korean chickens were derived from Southeast Asia. Following the domestication of red junglefowl, a non-game type chicken was developed, and it spread to China. A game type chicken was developed in each area. Both non-game and game chickens formed the foundation of Japanese native chickens.     

Origin of cytoplasm substituted rice cultivars found in Japan

Genetic variation of Japanese rice cultivars were examined. Five of 450 lowland cultivars and another five of 200 upland cultivars were determined as the indica type by using isozyme genotypes and the remainder were of the japonica type. The major characteristics of these indica cultivars, revealed a slender shape of grains, a short apiculus hair length, a positive allele for Ph reaction, and allele-3 for the Pgd1 locus. Three of these indica cultivars showed a non-deletion ORF100, which is essential to the japonica-type plastid. The plastid subtype identity (PS-ID) sequences of these plastids is 6C7A, which is also a japonica-specific repeat unit. Thus, these cultivars were concluded to be naturally generated cytoplasm substituted lines. These plastids were introduced into a indica genetic background from japonica cultivars grown elsewhere. The rest of the indica cultivars revealed a deletion-type ORF100 and plastid subtype 8C8A, both of which are indica-specific. These cultivars carried indica-type allelic constitutions for diagnostic isozyme loci. However, other characters were identical to the cytoplasm-substituted cultivars in Japan. In East and Southeast Asia, cultivars carrying a indica-type nuclear genotype with a japonica-type plastid are restricted to Aus cultivars in the Bengal region. Genetic and historical records suggest that Japanese indica cultivars and the Aus cultivars are closely related. The Aus cultivars acquire necessary genetic constitutions from both indica and japonica cultivars through naturally occurring out-crossing to adapt to a particular cultivation condition in the region. The wide adaptability enabled them to be introduced into a northern region like Japan.     

Phylogeography of Japanese horse chestnut (<Emphasis Type="Italic">Aesculus turbinata</Emphasis>) in the Japanese Archipelago based on chloroplast DNA haplotypes

Japanese horse chestnut (Aesculus turbinata: Hippocastanaceae) is one of the typical woody plants that grow in temperate riparian forests in the Japanese Archipelago. To analyze the phylogeography of this plant in the Japanese Archipelago, we determined cpDNA haplotypes for 337 samples from 55 populations covering the entire distribution range. Based on 1,313 bp of two spacers, we determined ten haplotypes that are distinguished from adjacent haplotypes by one or two steps. Most of the populations had a single haplotype, suggesting low diversity. Spatial analysis of molecular variance suggested three obvious phylogeographic structures in western Japan, where Japanese horse chestnut is scattered and isolated in mountainous areas. Conversely, no clear phylogeographic structure was observed from the northern to the southern limit of this species, including eastern Japan, where this plant is more common. Rare and private haplotypes were also found in southwestern Japan, where Japanese horse chestnuts are distributed sparsely. These findings imply that western Japan might have maintained a relatively large habitat for A. turbinata during the Quaternary climatic oscillations, while northerly regions could not.     

Testing soybean antibiosis to three clones of soybean aphid, Aphis glycines (Hemiptera: Aphididae) using sprouts and leaflets

Antibiosis of eight soybean cultivars to three clones of soybean aphids (Aphis glycines Matsumura) was evaluated using both soybean sprouts and leaflets. Overall, the performance of soybean aphid was better on sprouts than on leaflets. We confirmed previous reports that Dowling and Jackson cultivars exhibited strong resistance to a clone of soybean aphids from the US, but not to either Japanese or Indonesian clones. The USA clone had delayed development, fewer offspring, and low emergence rates on these two cultivars. However, abnormal offspring were only investigated on the Tachinagaha cultivar. We confirmed that Bay and Himeshirazu cultivars were strongly resistant to the Japanese aphid clone; aphids produced fewer offspring and deformed offspring on Bay and had delayed development and a low rate of emergence on Himeshirazu. None of the eight soybean cultivars were resistant to the Indonesian clone, although abnormal offspring were produced on Jackson, Adams, and Tachinagaha cultivars. These data suggest that there are genetic differences among the three tested clones of soybean aphid and that the characteristics of local soybean aphid clones must be considered when developing resistant soybean cultivars for a given geographic area.     

Validation of microsatellite multiplexes for parentage analysis and species discrimination in two hybridizing species of coral reef fish (Plectropomus spp., Serranidae)

Microsatellites are often considered ideal markers to investigate ecological processes in animal populations. They are regularly used as genetic barcodes to identify species, individuals, and infer familial relationships. However, such applications are highly sensitive the number and diversity of microsatellite markers, which are also prone to error. Here, we propose a novel framework to assess the suitability of microsatellite datasets for parentage analysis and species discrimination in two closely related species of coral reef fish, Plectropomus leopardus and P. maculatus (Serranidae). Coral trout are important fisheries species throughout the Indo‐Pacific region and have been shown to hybridize in parts of the Great Barrier Reef, Australia. We first describe the development of 25 microsatellite loci and their integration to three multiplex PCRs that co‐amplify in both species. Using simulations, we demonstrate that the complete suite of markers provides appropriate power to discriminate between species, detect hybrid individuals, and resolve parent–offspring relationships in natural populations, with over 99.6% accuracy in parent–offspring assignments. The markers were also tested on seven additional species within the Plectropomus genus with polymorphism in 28–96% of loci. The multiplex PCRs developed here provide a reliable and cost‐effective strategy to investigate evolutionary and ecological dynamics and will be broadly applicable in studies of wild populations and aquaculture brood stocks for these closely related fish species.     

Ectomycorrhizal characterization of an American chestnut <Emphasis Type="SmallCaps">(</Emphasis><Emphasis Type="Italic">Castanea dentata</Emphasis><Emphasis Type="SmallCaps">)</Emphasis>-dominated community in Western Wisconsin

Circa 1900, a farmer from the eastern US planted 11 American chestnut (Castanea dentata) seeds on a newly established farm near West Salem in western Wisconsin. These trees were very successful, producing a large stand of over 6,000 trees. Since this area is well outside the natural range of chestnut, these trees remained free from chestnut blight until 1987. In the West Salem stand, chestnuts are the dominant species of a mixed forest community, reminiscent of the chestnut–oak ecosystems of pre-1900 Appalachia. To identify putative mycorrhizal associates of chestnut in this unique forest, our approach was twofold: (1) an extensive fruiting body survey was conducted for four seasons that yielded approximately 100 putative mycorrhizal species and (2) a belowground molecular approach was used to generate DNA sequences of the internal transcribed spacer region from ectomycorrhizae. Unexpectedly, chestnut did not appear to be the dominant underground ectomycorrhizal-forming plant species. This study highlights the need to identify the plant host species when conducting belowground molecular-based surveys and provides preliminary identification of ectomycorrhizal fungi associated with a disjunct stand of American chestnut. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Intra- and interbreed genetic variations of mitochondrial DNA major non-coding regions in Japanese native dog breeds [Canis familiaris)

Mitochondrial DNA (mtDNA) major non-coding regions were amplified from 73 dogs of eight Japanese native dog breeds and from 21 dogs of 16 non-Japanese dog breeds by the polymerase chain reaction and their DNA sequences were determined. A total of 51 nucleotide positions within the non-coding region (969–972 base pairs) showed nucleotide variations of which 48 were caused by transition. These nucleotide substitutions were abundant in the region proximate to tRNA^Pro. In addition to the nucleotide substitutions, the dog mtDNA D-loop sequences had a heteroplasmic repetitive sequence (TACACGTÀCG) involving size variation. The DNA sequences of the non-coding region were classified into four different groups by phylogenetic analysis and the deepest branchpoints of this dog phylogeny was calculated to about 100 000 years before the present. Phylogenetic analysis showed that Japanese native dog breeds could not be clearly delimited as distinct breeds. Many haplotypes found in members of some clustering groups were seen in each dog breed, and interbreed nucleotide differences between Japanese dog breeds were almost the same as the intrabreed nucleotide diversities.     

Origins of Japanese flowering cherry (Prunus subgenus Cerasus) cultivars revealed using nuclear SSR markers

Japanese flowering cherry (Prunus subgenus Cerasus) cultivars, which are characterized by beautiful flowers, have been developed through hybridization among wild Prunus taxa. The long history of cultivation has caused significant confusion over the origins of these cultivars. We conducted molecular analysis using nuclear simple sequence repeat (SSR) polymorphisms to trace cultivar origins. Bayesian clustering based on the STRUCTURE analysis using SSR genotypes revealed that many cultivars originated from hybridization between two or more wild species. This suggests that morphological variations among flowering cherry cultivars probably arose through a complex sequence of hybridizations. Our findings generally supported estimates of the origins of cultivars based on morphological study, although there were some exceptions.     

Genetic diversity and structure of the endangered lady's slipper orchid Cypripedium japonicum Thunb. (Orchidaceae) in Japan

Cypripedium japonicum Thunb. (Orchidaceae), once a common perennial herb, is now designated as endangered throughout most of its distribution due to habitat destruction and fragmentation, and the impacts of horticultural collection. We investigated the genetic characteristics of this species for conservation purposes, using microsatellite markers to examine the genetic diversity and structure of 15 native and 5 ex situ populations in Japan. The results imply that although allelic variation is low in Japanese C. japonicum, sexual reproduction by seed, as well as clonal propagation, may occur in some populations. Both native and ex situ populations were found to be genetically differentiated, indicating that some populations may have experienced recent population declines, genetic fragmentation, or bottlenecks. The degree of genetic drift from the putative ancestral population, inferred through STRUCTURE analysis, was more pronounced in northern populations than in southern populations. Some of the ex situ conserved populations exhibited a low degree of differentiation from ancestral native populations. Our results imply that conservation of C. japonicum in Japan is best supported by maintaining individual populations and their unique genetic characteristics.     

Microsatellite marker-based identification and genetic relationships of olive cultivars from the Extremadura region of Spain

Seventy-seven olive accessions corresponding to 25 cultivars from the Extremadura region of Spain were studied using four microsatellite or SSR markers in order to fingerprint them, and evaluate genetic similarity and relationships between local and introduced olive cultivars. The number of alleles per locus ranged from 4 to 8, with a mean of 6.25 alleles per primer pair (a total of 25 alleles). The observed heterozygosity ranged from 0.58 to 0.95, while the expected heterozygosity varied between 0.68 and 0.83. The polymorphism information content values ranged from 0.63 to 0.79. The mean polymorphism information content value of 0.70 for the SSR loci provided sufficient discriminating ability to evaluate the genetic diversity among the cultivars. The SSR data allowed unequivocal identification of all the cultivars; a combination of three SSR markers was sufficient to discriminate all 25 olive cultivars. A dendrogram was prepared, using the unweighted pair-group method with arithmetic mean clustering algorithm; it depicted the pattern of relationships between the cultivars. Most of the local cultivars grouped according to their geographic origin. No clear clustering trends were observed when the morphological traits of fruit endocarps or fruit use of cultivars were employed as analysis criteria. We conclude that there is a high level of variability among local olive cultivars from the Extremadura region at both the morphological and molecular levels; these data should be useful for identifying and distinguishing local germplasm.     

Use of blood typing to confirm principles of coat-color genetics in horses.

The chestnut rule in equine coat-color genetics asserts that the inter se mating of chestnut horses never produces bay, black, brown or gray offspring. The gray rule asserts that a gray offspring must have at least one gray parent. Nine alleged exceptions to the chestnut rule, all involving bay offspring, and eight alleged exceptions to the gray rule, including four offspring that were also exceptions to the chestnut rule, were examined for parent-offspring genetic incompatibilities in as many as 17 genetic systems of blood-group markers. In all except one of the 17 cases it was possible to show that parentage had been incorrectly assigned. In 9 of the 16 exclusions it was possible to exclude the stallion irrespective of the mare and in one of the 16 exclusions it was possible to exclude the mare irrespective of the stallion. The percentage of exclusions, i.e., 94, was closely in line with expectation based on the established efficacy of these tests, about 90 percent, in excluding the incorrect stallion or stallions in paternity cases. Although the results strongly uphold the validity of the chestnut and gray rules in equine coat-color inheritance, they do not completely exclude the possibility that there could be rare exceptions to one or the other or both color rules. Insofar as equine registries may be concerned, the results clearly indicate that no alleged exception to the color rules should be considered eligible for registry in the absence of these tests.     

Mapping and pedigree analysis of the gene that controls the easy peel pellicle trait in Japanese chestnut (Castanea crenata Sieb. et Zucc.)

The Japanese chestnut (Castanea crenata Sieb. et Zucc.) has a pellicle that is difficult to peel, which increases the labor and cost for removing the pellicle from the nut during processing. Thus, a pellicle that is easier to peel has been an important objective of Japanese chestnut breeding programs. A newly released cultivar (“Porotan”) exhibits a unique, easily peeled pellicle. A previous study indicated that this trait is controlled by recessive gene p, and that several of the ancestors of Porotan (e.g., “Tanzawa” and 550-40) were P/p heterozygotes. Two F₁ populations from intra-specific crosses of Japanese chestnut, Tanzawa (P/p) × Porotan (p/p) and 550-40 (P/p) × Tanzawa (P/p), were used for genetic mapping of the gene that controls this characteristic. A total of 11 simple sequence repeat (SSR) markers were obtained that showed significant linkages to the p gene, and genetic linkage maps for the region around the p gene were established. Pedigree analysis was conducted for eight ancestors of Porotan around the pellicle-peeling locus using graphical genotypes based on the 11 SSR loci. The two recessive p alleles and surrounding haplotypes of Porotan were inherited through different intermediate cultivars: one allele was derived from “Otomune” (P/p) via Tanzawa and the other was derived from Otomune via Tanzawa, “Kunimi” (P/p), and breeding line 550-40. A recombination event was found in the flanking region close to the p gene in Kunimi. Molecular identification of the easy peel pellicle trait will lead to marker-assisted selection and will greatly improve Japanese chestnut breeding.     

One-step reconstruction of multi-generation pedigree networks in apple (Malus × domestica Borkh.) and the parentage of Golden Delicious

The increasing availability of genomic tools improves our ability to investigate the patterns of genetic diversity and relatedness among individuals. The pedigrees of many apple cultivars are completely unknown, often reducing the efficiency of breeding programs. Using a multilocus simple sequence repeat dataset, we applied a novel multi-generation pedigree-network reconstruction procedure based on the software FRANz in a Malus × domestica collection (101 cultivated and 22 wild apples) with partially known pedigree relationships. The procedure produced 78 parent–offspring relationships organized into three networks and showed high power for detecting real pedigree links (98.5 %) and a low false-positive rate (9.0 %). The largest reconstructed pedigree network spanned four generations and involved 65 cultivars. The availability of detailed pedigree connections confirmed that recent genealogical relationships affect population genetic structure in apple. Finally, our analysis enabled us to confirm or discard several pedigrees known only anecdotically, among which the cultivar Grimes Golden was validated as a parent of the widely grown cultivar Golden Delicious. The pedigree reconstruction protocol here described will be of broad applicability to other collections and crop species.