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      Species Identification of Oaks ( Quercus L., Fagaceae) from Gene to Genome

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          Abstract

          Species identification of oaks ( Quercus) is always a challenge because many species exhibit variable phenotypes that overlap with other species. Oaks are notorious for interspecific hybridization and introgression, and complex speciation patterns involving incomplete lineage sorting. Therefore, accurately identifying Quercus species barcodes has been unsuccessful. In this study, we used chloroplast genome sequence data to identify molecular markers for oak species identification. Using next generation sequencing methods, we sequenced 14 chloroplast genomes of Quercus species in this study and added 10 additional chloroplast genome sequences from GenBank to develop a DNA barcode for oaks. Chloroplast genome sequence divergence was low. We identified four mutation hotspots as candidate Quercus DNA barcodes; two intergenic regions ( matK-trnK-rps16 and trnR-atpA) were located in the large single copy region, and two coding regions ( ndhF and ycf1b) were located in the small single copy region. The standard plant DNA barcode ( rbcL and matK) had lower variability than that of the newly identified markers. Our data provide complete chloroplast genome sequences that improve the phylogenetic resolution and species level discrimination of Quercus. This study demonstrates that the complete chloroplast genome can substantially increase species discriminatory power and resolve phylogenetic relationships in plants.

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          A DNA barcode for land plants.

          DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF-atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK-psbI spacer, and trnH-psbA spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL+matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants.
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            Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants.

            A two-marker combination of plastid rbcL and matK has previously been recommended as the core plant barcode, to be supplemented with additional markers such as plastid trnH-psbA and nuclear ribosomal internal transcribed spacer (ITS). To assess the effectiveness and universality of these barcode markers in seed plants, we sampled 6,286 individuals representing 1,757 species in 141 genera of 75 families (42 orders) by using four different methods of data analysis. These analyses indicate that (i) the three plastid markers showed high levels of universality (87.1-92.7%), whereas ITS performed relatively well (79%) in angiosperms but not so well in gymnosperms; (ii) in taxonomic groups for which direct sequencing of the marker is possible, ITS showed the highest discriminatory power of the four markers, and a combination of ITS and any plastid DNA marker was able to discriminate 69.9-79.1% of species, compared with only 49.7% with rbcL + matK; and (iii) where multiple individuals of a single species were tested, ascriptions based on ITS and plastid DNA barcodes were incongruent in some samples for 45.2% of the sampled genera (for genera with more than one species sampled). This finding highlights the importance of both sampling multiple individuals and using markers with different modes of inheritance. In cases where it is difficult to amplify and directly sequence ITS in its entirety, just using ITS2 is a useful backup because it is easier to amplify and sequence this subset of the marker. We therefore propose that ITS/ITS2 should be incorporated into the core barcode for seed plants.
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              Highly Variable Chloroplast Markers for Evaluating Plant Phylogeny at Low Taxonomic Levels and for DNA Barcoding

              Background At present, plant molecular systematics and DNA barcoding techniques rely heavily on the use of chloroplast gene sequences. Because of the relatively low evolutionary rates of chloroplast genes, there are very few choices suitable for molecular studies on angiosperms at low taxonomic levels, and for DNA barcoding of species. Methodology/Principal Findings We scanned the entire chloroplast genomes of 12 genera to search for highly variable regions. The sequence data of 9 genera were from GenBank and 3 genera were of our own. We identified nearly 5% of the most variable loci from all variable loci in the chloroplast genomes of each genus, and then selected 23 loci that were present in at least three genera. The 23 loci included 4 coding regions, 2 introns, and 17 intergenic spacers. Of the 23 loci, the most variable (in order from highest variability to lowest) were intergenic regions ycf1-a, trnK, rpl32-trnL, and trnH-psbA, followed by trnSUGA-trnGUCC , petA-psbJ, rps16-trnQ, ndhC-trnV, ycf1-b, ndhF, rpoB-trnC, psbE-petL, and rbcL-accD. Three loci, trnSUGA-trnGUCC , trnT-psbD, and trnW-psaJ, showed very high nucleotide diversity per site (π values) across three genera. Other loci may have strong potential for resolving phylogenetic and species identification problems at the species level. The loci accD-psaI, rbcL-accD, rpl32-trnL, rps16-trnQ, and ycf1 are absent from some genera. To amplify and sequence the highly variable loci identified in this study, we designed primers from their conserved flanking regions. We tested the applicability of the primers to amplify target sequences in eight species representing basal angiosperms, monocots, eudicots, rosids, and asterids, and confirmed that the primers amplified the desired sequences of these species. Significance/Conclusions Chloroplast genome sequences contain regions that are highly variable. Such regions are the first consideration when screening the suitable loci to resolve closely related species or genera in phylogenetic analyses, and for DNA barcoding.
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                Author and article information

                Journal
                Int J Mol Sci
                Int J Mol Sci
                ijms
                International Journal of Molecular Sciences
                MDPI
                1422-0067
                26 November 2019
                December 2019
                : 20
                : 23
                : 5940
                Affiliations
                [1 ]Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China; pxb15633717296@ 123456163.com
                [2 ]Administration Bureau of Hongyashan State Owned Forest Farm in Yixian County, Yixian 074200, China; lhs13803120634@ 123456163.com (H.L.); wsr15830855155@ 123456163.com (S.W.); 18730272192@ 123456163.com (Y.Y.); lhj13831238335@ 123456163.com (H.L.); 13930286026@ 123456163.com (J.D.); yyc16603261128@ 123456163.com (Z.L.); Anchuanzhi2002@ 123456163.com (C.A.); szh13833020580@ 123456163.com (Z.S.)
                [3 ]State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing 100091, China
                [4 ]Key Laboratory of Tree Breeding and Cultivation of State Forestry Administration, Chinese Academy of Forestry, Beijing 100091, China
                Author notes
                [* ]Correspondence: libin1200@ 123456163.com
                Author information
                https://orcid.org/0000-0002-1582-2635
                Article
                ijms-20-05940
                10.3390/ijms20235940
                6928813
                31779118
                3424c817-fadb-4e45-9f65-a1ef61adaadb
                © 2019 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 24 September 2019
                : 08 November 2019
                Categories
                Article

                Molecular biology
                oak species identification,chloroplast genome,quercus,mutation hotspots
                Molecular biology
                oak species identification, chloroplast genome, quercus, mutation hotspots

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