Subspecies Classification Research Articles

Classification and phylogeny of sika deer (Cervus nippon) subspecies based on the mitochondrial control region DNA sequence using an extended sample set

To further refine the classification and phylogeny of sika deer subspecies, the well-annotated sequences of the complete mitochondrial DNA (mtDNA) control region of 13 sika deer subspecies from GenBank were downloaded, aligned and analyzed in this study. By reconstructing the phylogenetic tree with an extended sample set, the results revealed a split between Northern and Southern Mainland Asia/Taiwan lineages, and moreover, two subspecies, C.n.mantchuricus and C.n.hortulorum, were existed in Northern Mainland Asia. Unexpectedly, Dybowskii’s sika deer that was thought to originate from Northern Mainland Asia joins the Southern Mainland Asia/Taiwan lineage. The genetic divergences were ranged from 2.1% to 4.7% between Dybowskii’s sika deer and all the other established subspecies at the mtDNA sequence level, which suggests that the maternal lineage of uncertain sika subspecies in Europe had been maintained until today. This study also provides a better understanding for the classification, phylogeny and phylogeographic history of sika deer subspecies.

A accurate identification method for Chinese materia medica——systematic identification of Chinese materia medica

This paper put forward a more accurate identification method for identification of Chinese materia medica (CMM), the systematic identification of Chinese materia medica (SICMM) , which might solve difficulties in CMM identification used the ordinary traditional ways. Concepts, mechanisms and methods of SICMM were systematically introduced and possibility was proved by experiments. The establishment of SICMM will solve problems in identification of Chinese materia medica not only in phenotypic characters like the mnorphous, microstructure, chemical constituents, but also further discovery evolution and classification of species, subspecies and population in medical plants. The establishment of SICMM will improve the development of identification of CMM and create a more extensive study space.

Phylogeography of amphi-boreal fish: tracing the history of the Pacific herring Clupea pallasii in North-East European seas

BackgroundThe relationships between North Atlantic and North Pacific faunas through times have been controlled by the variation of hydrographic circumstances in the intervening Arctic Ocean and Bering Strait. We address the history of trans-Arctic connections in a clade of amphi-boreal pelagic fishes using genealogical information from mitochondrial DNA sequence data. The Pacific and Atlantic herrings (Clupea pallasii and C. harengus) have basically vicarious distributions in the two oceans since pre-Pleistocene times. However, remote populations of C. pallasii are also present in the border waters of the North-East Atlantic in Europe. These populations show considerable regional and life history differentiation and have been recognized in subspecies classification. The chronology of the inter-oceanic invasions and genetic basis of the phenotypic structuring however remain unclear.ResultsThe Atlantic and Pacific herrings both feature high mtDNA diversities (large long-term population sizes) in their native basins, but an ocean-wide homogeneity of C. harengus is contrasted by deep east-west Pacific subdivision within Pacific C. pallasii. The outpost populations of C. pallasii in NE Europe are identified as members of the western Pacific C. pallasii clade, with some retained inter-oceanic haplotype sharing. They have lost diversity in colonization bottlenecks, but have also thereafter accumulated abundant new variation. The data delineate three phylogeographic groups within the European C. pallasii: herring from the inner White Sea; herring from the Mezen and Chesha Bays; and a strongly bottlenecked peripheral population in Balsfjord of the Norwegian Sea.ConclusionsThe NE European outposts of C. pallasii are judged to be early post-glacial colonists from the NW Pacific. A strong regional substructure has evolved since that time, in contrast to the apparent broad-scale uniformity maintained by herrings in their native basins. The structure only partly matches the previous biological concepts based on seasonal breeding stocks or geographical subspecies designations. The trans-Arctic herring phylogeography is notably similar to those of the amphi-boreal mollusk taxa Macoma and Mytilus, suggesting similar histories of inter-oceanic connections. We also considered the time dependency of molecular rates, critical for interpreting timing of relatively recent biogeographical events, by comparing the estimates from coding and non-coding mitochondrial regions of presumably different mutation dynamics.

DNA barcoding and species and subspecies classification within genus <I>Carassius</I>

The classification of Carassius has not been well established due to its great variability and wide distribution. Usually, Carassius is identified as three species: C. carassius, C. cuvieri and C. auratus, the latter including several subspecies, such as goldfish. Out of these subspecies, C. auratus gibelio have recently been thought of as a valid species of Carassius. In this study we collected the 5'end 651 bp segments of the mitochondrial cytochrome c oxidase I (COI) gene from 128 specimens, including C. carassius, C. cuvieri, C. auratus auratus, C. auratus gibelio and C. auratus langsdorfii. All three species of Carassius (C. carassius, C. cuvieri, C. auratus) were found to be valid, meanwhile genetic differentiation between the Eurasian C. auratus and Japanese C. auratus has reached a high level. However, several haplotypes were shared between C. auratus auratus and C. auratus gibelio. Consequently, C. auratus gibelio should be regarded as a subspecies of C. auratus rather than a valid species. Moreover, because both diploids and triploids exist in C. auratus auratus and C. auratus gibelio, ploidy level should not be used as criteria for the classification of species or subspecies in Carassius.

Multilocus Sequence Analysis (MLSA) of ‘Rickettsiella agriotidis’, an Intracellular Bacterial Pathogen of Agriotes Wireworms

Wireworms, the polyphagous larvae of click beetles belonging to the genus Agriotes (Coleoptera: Elateridae) are severe and widespread agricultural pests that affect numerous crops globally. A new bacterial specimen identified in diseased wireworms had previously been shown by microscopy and 16S ribosomal RNA (rRNA) gene-based phylogenetic reconstruction to belong to the taxonomic genus Rickettsiella (Gammaproteobacteria) that comprises intracellular bacteria associated with and typically pathogenic for a wide range of arthropods. Going beyond these earlier results obtained from rRNA phylogenies, multilocus sequence analysis (MLSA) using a four marker scheme has been employed in the molecular taxonomic characterization of the new Rickettsiella pathotype, referred to as 'Rickettsiella agriotidis'. In combination with likelihood-based significance testing, the MLSA approach demonstrated the close phylogenetic relationship of 'R. agriotidis' to the pathotypes 'Rickettsiella melolonthae' and 'Rickettsiella tipulae', i.e., subjective synonyms of the nomenclatural type species, Rickettsiella popilliae. 'R. agriotidis' forms, therefore, part of a Rickettsiella pathotype complex that most likely represents the species R. popilliae. As there are currently no genetic data available from the R. popilliae type strain, the respective assignment cannot be corroborated directly. However, an alternative taxonomic assignment to the species Rickettsiella grylli has been positively ruled out by significance testing. MLSA has been shown to provide a more powerful tool for taxonomic delineation within the genus Rickettsiella as compared to 16S rRNA phylogenetics. However, the limitations of the present MLSA scheme for the sub-species level classification of 'R. agriotidis' and further R. popilliae synonyms has been critically evaluated.

Characterization of culturable bacterial endophytes of switchgrass (Panicum virgatum L.) and their capacity to influence plant growth

AbstractSwitchgrass (Panicum virgatum L.) is a perennial warm season grass that is native to the plains of North America and is widely grown as a forage, bioenergy or groundcover crop. Despite its importance, a bottleneck in switchgrass production is poor seedling vigor, which as a perennial crop represents an important time for management. Herein, data identify a suite of culturable bacterial microflora extracted from switchgrass, and show their capability to influence host plant growth and development. A total of 307 bacterial isolates were cultured and isolated from surface sterilized switchgrass biomass and sequence identified into 76 strains (subspecies classification), 36 species and 5 phyla. Approximately 58% of bacterial strains, when reintroduced into surface‐sterilized switchgrass seeds, were documented to increase lamina length (cm from base to tip after 60 days growth) relative to uninoculated controls. Ecologically, Phylum Firmicutes was the most abundant bacterial classification and encompassed 75% of all isolates. Although the culturable bacterial community studies herein represent an unknown and assumedly minor proportion of the total microbiome, by focusing on culturable bacteria, we delineate functional feedback between the presence of isolated bacteria and switchgrass seedling growth.

Genetic Identification of Subspecies and Stocks of Minke Whale Meats Purchased from Korean Markets during 2005-2007

To identify subspecies and stocks of minke whale meats purchased from Korean markets during 2005-2007, we first obtained their complete sequences of mitochondrial DNA cytochrome b and control region sequences, and compared these sequences to the corresponding sequences of the common minke whale (Balaenoptera acutorostrata), obtained from GenBank. From analyses with partial cytochrome b sequences (383 bp) and non-coding, partial control region sequences (463 bp), Korean mink whale meats are identified as the products from the North Pacific minke whale (B. a. scammoni). In addition, the sequences of the partial control region from these meats showed G at site no. 298 and G or A at site no. 463, and the meats appear to be originated from the J stock within this subspecies. Thus, it seems to be urgent to fulfill strict regulation measures to reduce their accidental fisheries bycatch because the J stock has been protected since 1986. Additionally, B. a. scammoni is distinct from B. a. acutorostrata, with average Jukes-Cantor distance of 2.21% in the complete control region sequence analysis (935 bp) and 1.31% distance in the complete cytochrome b gene sequence analysis, and these present results support the present subspecies classification, although further sequencing analyses with nuclear genes are necessary.

Phylogeography and Demographic History of the Neotropical Otter (Lontra longicaudis)

The Neotropical otter (Lontra longicaudis) is a medium-sized semiaquatic carnivore with a broad distribution in the Neotropical region. Despite being apparently common in many areas, it is one of the least known otters, and genetic studies on this species are scarce. Here, we have investigated its genetic diversity, population structure, and demographic history across a large portion of its geographic range by analyzing 1471 base pairs (bp) of mitochondrial DNA from 52 individuals. Our results indicate that L. longicaudis presents high levels of genetic diversity and a consistent phylogeographic pattern, suggesting the existence of at least 4 distinct evolutionary lineages in South America. The observed phylogeographic partitions are partially congruent with the subspecies classification previously proposed for this species. Coalescence-based analyses indicate that Neotropical otter mitochondrial DNA lineages have shared a rather recent common ancestor, approximately 0.5 Ma, and have subsequently diversified into the observed phylogroups. A consistent scenario of recent population expansion was identified in Eastern South America based on several complementary analyses of historical demography. The results obtained here provide novel insights on the evolutionary history of this largely unknown Neotropical mustelid and should be useful to design conservation and management policies on behalf of this species and its habitats.

Design of a Bovine Low-Density SNP Array Optimized for Imputation

The Illumina BovineLD BeadChip was designed to support imputation to higher density genotypes in dairy and beef breeds by including single-nucleotide polymorphisms (SNPs) that had a high minor allele frequency as well as uniform spacing across the genome except at the ends of the chromosome where densities were increased. The chip also includes SNPs on the Y chromosome and mitochondrial DNA loci that are useful for determining subspecies classification and certain paternal and maternal breed lineages. The total number of SNPs was 6,909. Accuracy of imputation to Illumina BovineSNP50 genotypes using the BovineLD chip was over 97% for most dairy and beef populations. The BovineLD imputations were about 3 percentage points more accurate than those from the Illumina GoldenGate Bovine3K BeadChip across multiple populations. The improvement was greatest when neither parent was genotyped. The minor allele frequencies were similar across taurine beef and dairy breeds as was the proportion of SNPs that were polymorphic. The new BovineLD chip should facilitate low-cost genomic selection in taurine beef and dairy cattle.

Genetic and morphological divergence in island and mainland birds: Informing conservation priorities

Evolutionary processes can complicate conservation efforts for species with uncertain taxonomic classifications and discrete geographic populations. Discordant morphological and genetic patterns across the geographic range of species further calls for the identification of evolutionary significant units for conservation. Using island and mainland populations of a small Australian passerine (the superb fairy-wren, Malurus cyaneus), we examine the relationship between morphological and genetic divergence among two subspecies, M. c. ashbyi (Kangaroo Island, South Australia) and M. c. leggei (South Australia, mainland), using eight microsatellite markers. Island birds showed clear evidence for morphological divergence, with a larger body size and thinner bill compared to mainland birds. Two genetic clusters were found using Bayesian methods, comprising mainland and island regions. Estimates of recent migration rates between all sites were very low (<2%). Morphological and genetic differentiation between island and mainland sites correlated significantly, but not when controlling for isolation by distance. Genetic and morphological substructure was evident with three distinct genetic clusters in each region. Males, the highly sedentary sex, appeared to drive correlations between morphological and genetic differentiation. Our study provides evidence that the subspecies classification of M. cyaneus in island and mainland regions encapsulates two independently diverging populations that can be recognised in conservation planning.

Phylogeography of Japanese Population ofPhelotrupes auratus(Coleoptera, Geotrupidae) Inferred from Mitochondrial DNA Sequences

A partial sequence of the mitochondrial cytochrome c oxidase subunit I (COI) gene (745 bp) was determined for 57 specimens of a geotrupid beetle (Phelotrupes auratus) from throughout the Japanese archipelago. Of the 57 beetles examined, 42 haplotypes were identified. Phylogenetic trees inferred using maximum parsimony, neighbor joining, and Bayesian inference methods were highly congruent. Reconstructed phylogenetic relationships indicated that P. auratus from the Japanese archipelago was separated into two distinct lineages: Group A, which consisted of 35 haplotypes from Honshu, Shikoku, and Hokkaido Islands, and Group B, which consisted of seven haplotypes from Kyushu and Yakushima Islands. In addition, two sublineages were also recognized within Group A: Subgroup A-1, which consisted of 11 haplotypes from eastern Honshu and Hokkaido, and Subgroup A-2, which consisted of 10 haplotypes from western Honshu and Shikoku. Average genetic distances within Group A were positively correlated to geographic distance between sampling localities. Phylogenetic relationships among haplotypes did not correspond to subspecies classification.

Genetic structuring and recent demographic history of red pandas (Ailurus fulgens) inferred from microsatellite and mitochondrial DNA

Clarification of the genetic structure and population history of a species can shed light on the impacts of landscapes, historical climate change and contemporary human activities and thus enables evidence-based conservation decisions for endangered organisms. The red panda (Ailurus fulgens) is an endangered species distributing at the edge of the Qinghai-Tibetan Plateau and is currently subject to habitat loss, fragmentation and population decline, thus representing a good model to test the influences of the above-mentioned factors on a plateau edge species. We combined nine microsatellite loci and 551 bp of mitochondrial control region (mtDNA CR) to explore the genetic structure and demographic history of this species. A total of 123 individuals were sampled from 23 locations across five populations. High levels of genetic variation were identified for both mtDNA and microsatellites. Phylogeographic analyses indicated little geographic structure, suggesting historically wide gene flow. However, microsatellite-based Bayesian clustering clearly identified three groups (Qionglai-Liangshan, Xiaoxiangling and Gaoligong-Tibet). A significant isolation-by-distance pattern was detected only after removing Xiaoxiangling. For mtDNA data, there was no statistical support for a historical population expansion or contraction for the whole sample or any population except Xiaoxiangling where a signal of contraction was detected. However, Bayesian simulations of population history using microsatellite data did pinpoint population declines for Qionglai, Xiaoxiangling and Gaoligong, demonstrating significant influences of human activity on demography. The unique history of the Xiaoxiangling population plays a critical role in shaping the genetic structure of this species, and large-scale habitat loss and fragmentation is hampering gene flow among populations. The implications of our findings for the biogeography of the Qinghai-Tibetan Plateau, subspecies classification and conservation of red pandas are discussed.

Phylogeographic analysis of nuclear and mtDNA supports subspecies designations in the ostrich (Struthio camelus)

We investigated the phylogeography and subspecies classification of the ostrich (Struthio camelus) by assessing patterns of variation in mitochondrial DNA control region (mtDNA-CR) sequence and across fourteen nuclear microsatellite loci. The current consensus taxonomy of S. camelus names five subspecies based on morphology, geographic range, mtDNA restriction fragment length polymorphism and mtDNA-CR sequence analysis: S. c. camelus, S. c. syriacus, S. c. molybdephanes, S. c. massaicus and S. c. australis. We expanded a previous mtDNA dataset from 18 individual mtDNA-CR sequences to 123 sequences, including sequences from all five subspecies. Importantly, these additional sequences included 43 novel sequences of the red-necked ostrich, S. c. camelus, obtained from birds from Niger. Phylogeographic reconstruction of these sequences matches previous results, with three well-supported clades containing S. c. camelus/syriacus, S. c. molybdophanes, and S. c. massaicus/australis, respectively. The 14 microsatellite loci assessed for 119 individuals of four subspecies (all but S. c. syriacus) showed considerable variation, with an average of 13.4 (±2.0) alleles per locus and a mean observed heterozygosity of 55.7 (±5.3)%. These data revealed high levels of variation within most subspecies, and a structure analysis revealed strong separation between each of the four subspecies. The level of divergence across both marker types suggests the consideration of separate species status for S. c. molybdophanes, and perhaps also for S. c. camelus/syriacus. Both the mtDNA-CR and microsatellite analyzes also suggest that there has been no recent hybridization between the subspecies. These findings are of importance for management of the highly endangered red-necked subspecies (S. c. camelus) and may warrant its placement onto the IUCN red list of threatened animals.

Metabolomic and Functional Genomic Analyses Reveal Varietal Differences in Bioactive Compounds of Cooked Rice

Emerging evidence supports that cooked rice (Oryza sativa L.) contains metabolites with biomedical activities, yet little is known about the genetic diversity that is responsible for metabolite variation and differences in health traits. Metabolites from ten diverse varieties of cooked rice were detected using ultra performance liquid chromatography coupled to mass spectrometry. A total of 3,097 compounds were detected, of which 25% differed among the ten varieties. Multivariate analyses of the metabolite profiles showed that the chemical diversity among the varieties cluster according to their defined subspecies classifications: indica, japonica, and aus. Metabolite-specific genetic diversity in rice was investigated by analyzing a collection of single nucleotide polymorphisms (SNPs) in genes from biochemical pathways of nutritional importance. Two classes of bioactive compounds, phenolics and vitamin E, contained nonsynonymous SNPs and SNPs in the 5′ and 3′ untranslated regions for genes in their biosynthesis pathways. Total phenolics and tocopherol concentrations were determined to examine the effect of the genetic diversity among the ten varieties. Per gram of cooked rice, total phenolics ranged from 113.7 to 392.6 µg (gallic acid equivalents), and total tocopherols ranged between 7.2 and 20.9 µg. The variation in the cooked rice metabolome and quantities of bioactive components supports that the SNP-based genetic diversity influenced nutritional components in rice, and that this approach may guide rice improvement strategies for plant and human health.

Subspecies status of Common Eiders Somateria mollissima in Shetland based on morphology and DNA

Capsule Morphometrics, plumage and mitochondrial DNA of Common Eiders Somateria mollissima in Shetland indicate that these birds should be classified as subspecies faeroeensis. Aims To test whether molecular data support the suggestion based on measurements that Common Eiders in Shetland belong to the subspecies faeroeensis. Methods DNA extracted from Common Eiders found dead in Shetland was amplified and a section of the mitochondrial genome was sequenced to compare with data from birds previously sampled from Iceland, Faroes, east England and continental Europe. Results Shetland Common Eiders are closely similar to birds from the Faroe Islands classified as subspecies faeroeensis, and somewhat distinct from the nominate mollissima to which all Scottish eiders have been conventionally assigned. Eiders from southern Iceland have haplotypes more similar to faeroeensis despite differing in plumage and being assigned on plumage to a different subspecies borealis. Conclusion Molecular data indicate a need to revise the classification of common eider subspecies, with birds from Shetland considered as subspecies faeroeensis.

Listeria monocytogenes: Knowledge Gained Through DNA Sequence-Based Subtyping, Implications, and Future Considerations

The purpose of subtyping is to differentiate bacterial isolates beyond the classification of species or subspecies. Subtyping methods can be grouped into two broad categories based on the cellular components targeted: (1) phenotypic subtyping methods that differentiate isolates by the enzymes, proteins, or other metabolites expressed by the cell, and (2) molecular subtyping methods that discriminate isolates based on interrogation of nucleic acid sequences. The two major types of molecular subtyping methods include band-based methods based on fragment pattern data or DNA fingerprints, and methods that generate DNA sequence data. Molecular subtyping methods have shown that Listeria monocytogenes isolates can be classified into four genetic lineages or divisions. Although band-based molecular subtyping methods continue to serve as the gold standard for routine molecular subtyping of most clinically important foodborne pathogens, including L. monocytogenes, the explosion of recently completed and ongoing DNA sequencing projects, and thus available DNA sequence data, have stimulated efforts to develop highly discriminatory and high-throughput DNA sequence-based subtyping methods for L. monocytogenes. L. monocytogenes represents one of the most highly sequenced human pathogens; more than 20 genome sequences are currently available for this organism. This review provides an overview of the concepts behind subtyping and discusses the application of molecular subtyping methods, with an emphasis on DNA sequence-based subtyping methods to characterize L. monocytogenes.

East–west genetic differentiation in Musk Ducks (Biziura lobata) of Australia suggests late Pleistocene divergence at the Nullarbor Plain

Musk Ducks (Biziura lobata) are endemic to Australia and occur as two geographically isolated populations separated by the Nullarbor Plain, a vast arid region in southern Australia. We studied genetic variation in Musk Duck populations at coarse (eastern versus western Australia) and fine scales (four sites within eastern Australia). We found significant genetic structure between eastern and western Australia in the mtDNA control region (ΦST = 0.747), one nuclear intron (ΦST = 0.193) and eight microsatellite loci (FST = 0.035). In contrast, there was little genetic structure between Kangaroo Island and adjacent mainland regions within eastern Australia. One small population of Musk Ducks in Victoria (Lake Wendouree) differed from both Kangaroo Island and the remainder of mainland eastern Australia, possibly due to genetic drift exacerbated by inbreeding and small population size. The observed low pairwise distance between the eastern and western mtDNA lineages (0.36%) suggests that they diverged near the end of the Pleistocene, a period characterised by frequent shifts between wet and arid conditions in central Australia. Our genetic results corroborate the display call divergence and Mathews’ (Austral Avian Record 2:83–107, 1914) subspecies classification, and confirm that eastern and western populations of Musk Duck are currently isolated from each other.

Relationships of Panicle Type Index with Subspecies Characteristics and Yield Traits in Filial Generation of Crosses between Indica and Japonica Rice

Two recombinant inbred line (RIL) populations, one derived from a cross between Zhongyouzao 8 (indica) and Toyonishiki (japonica) and the other from a cross between Qishanzhan (indica) and Akihikari (japonica), were grown in Liaoning and Sichuan Provinces, China, to study the panicle type index (PTI, the ratio of a number of node position on panicle axis where the secondary branch with the most numerous secondary branch grains is located to the number of primary branches) and its relationships with subspecies characteristics and yield traits. With continuous distribution of PTI and numerous of lines with different PTIs in the progenies, indica-japonica hybridization could be a method for breeding cultivars with ideal panicle type. PTI was significantly influenced by environments, with a significant decrease from Liaoning to Sichuan. A significantly negative correlation was found between PTI and most of subspecies characteristics. PTI varied remarkably with different subspecies types. As a whole, it showed a trend of indica (H)>indicalinous (H')>japonicalinous (K')>japonica (K). However, it is not appropriate to regard PTI as a parameter for subspecies classification in rice because it was recombined in the filial generations of cross between indica and japonica rice. The negative correlations were found between PTI and most of panicle characteristics, as well as yield-related traits. Based on PTI, most of lines from the two populations were middle dominant panicle type (the number of grains on the secondary branch is numerous in middle position of the panicle) and lower dominant panicle type (the number of grains on the secondary branch is numerous in lower position of the panicle and becomes less towards the top of panicle) with significant yield advantages.

Chapter 5: Subspecies are for Convenience

Abstract The century-long debate over the meaning and utility of the subspecies concept has produced spirited print but only superficial consensus. I suggest that genuine consensus about subspecies is an impossible goal, because trinomial epithets will inevitably be applied to a heterogeneous mix of evolutionary phenomena, thereby precluding genuine standardization of the concept. Populations that have intermediate levels of phenotypic differentiation and geographic isolation from one another often fall into a region I refer to as the zone of art, where even skilled experts can disagree about the validity of any one subspecific treatment. The trinomial system cannot accurately represent the kind of information now available about genetic and character variation across space. Instead, ever more accurate tools are being perfected for quantitative, standardized descriptions of variation. These analyses—not subspecies classifications—will keep providing new scientific insights into geographic variation. Eve...

Comparison of Morphological and Genetic Differentiations in Filial Generation of Cross Between Indica and Japonica Rice

Abstract A recombinant inbred line (RIL) population of F 8 and F 9 generations derived from a cross between a typical indica rice (Qishanzhan) and a typical japonica rice (Akihikari) was used to study the difference between morphological differentiation based on phenotype characters and genetic differentiation using indica and japonica specific SSR markers, and to evaluate the relationship between vascular bundle characters and morphological and genetic differentiations. The results showed that the frequency distributions of morphological and genetic differentiations were all inclined to japonica type in the filial generation. The population was more inclined to japonica type based on genetic differentiation than on morphological differentiation. The consistent degrees of classification based on the Cheng's index, the ratio of large vascular bundle number to small vascular bundle number in panicle neck (RLSVB) and the ratio of large vascular bundle number in the second internode from the top to that in the panicle neck (RLVB) were all about 50% compared with the genetic differentiation, and the consistent degree of the total scores of the Cheng's index combined with the vascular bundle number ratios was significantly increased to about 80% compared with the genetic differentiation. Therefore, the vascular bundle characters could be used as a helpful supplement for subspecies classification.