Development of DNA Barcodes for Citrus and Its Related Genera and Their Application in Species Identification
|Keywords||True citrus fruit trees DNA barcoding ITS sequences Chloroplast sequence Appraisal|
China is the origin of one of the world's most important citrus, with rich resources of citrus. The citrus plants belonging to Rutaceae Citrus subfamily (Rutaceae) (Aurantioideae). Since its citrus (Citrus L.) Linnaeus was founded in 1753, the domestic and foreign scholars from morphology, cytology to molecular level, a series of studies, has established a number of citrus classification system. Among them, the most famous U.S. Swingle system contains 16 kinds; Japan's Tanaka system contains 159 kinds and three variants; had Mian system of our country, contains 30 kinds. The citrus plant have easy hybridization between species, no integration of reproductive and vegetative mutation, the number of species of citrus plants, interspecific phylogenetic relationships and classification problems so far unsolved scientific problems. (DNA barcode) concept of DNA barcoding 2003 by Canadian zoologist Paul Hebert, a DNA-based molecular evolutionary principle, the use of short DNA fragments and the principles and methods of modern molecular phylogeny of the \The latest species identification, biology techniques. This technology as compared with the traditional species identification methods, has the advantages of high accuracy, high efficiency, from the environment of the object to be identified, the ontogeny and identification of individual experts factors. DNA barcode technology since its birth, scholars use of mitochondrial cytochrome C oxidase I (of cytochrome c oxidase subunit Ⅰ CO Ⅰ) gene as bar coding sequence for species identification and new species of insects, birds, fish and other animals, and hidden species found great success. But the slower rate of evolution of CO Ⅰ gene in plants, not suitable for the study of plant barcode. Currently, the plant DNA barcoding study at home and abroad in the stage of looking for the gene fragments, many scholars actively explored, reported a variety of plant barcode candidate fragment or fragments combination, but has not yet been found to meet all the standards of DNA barcoding characteristic fragment. In this study the true citrus fruit trees plant genera and 59 biological types for the experimental material, the use of the the chloroplast coding gene (matK, rpoB, rpoC1 rbcL) chloroplast interval sequences (trnH-psbA the trnG-trnS psbH-petB trnL- trnF) and nuclear genes transcribed spacer sequence (ITS) and intergenic regions (ITS1, ITS2) 9 DNA sequence as a barcode candidate sequences, sequence MEGA5.05 software base composition, sequence of nucleotide variation frequency transversion frequency and the conversion between the sequence and its ratio. Using SPSS17.0 software the wilcoxon inspection comparative analysis of the differences between the different coding sequences. Through comparative sequence intraspecific and interspecific differences in the distribution, the use of single fragments and combinations of fragments Swingle system of citrus plants the Tanaka plants and citrus and its related genera 6 species identification, comparing a single sequence and fragment combination the size of the identification rate. The results of the study are as follows: 1. Swingle single sequence of citrus system, Tanaka system 28 were identified to the Fuming Trifoliate dragon trifoliate, trifoliate build NJ distance tree outgroup compared the identification of a single sequence. The results show that: the nuclear genes transcribed spacer sequence (ITS) and intergenic regions (ITS1, ITS2) sequence of the highest average identification efficiency, chloroplast sequences (trnH-psbA the trnG-trnS psbH-petB trnL-trnF intervals) followed , identification of the chloroplast coding genes (matK, rpoB, rpoC1, rbcL) minimum. Swingle system, ITS1 identification ability strongest 100%, followed by psbH-petB, ITS sequence identification capacity, the identification rates were 93.8%, 12.5% ??rpoC1 sequence identification rate minimum. The the Tanaka system 28 materials psbH-petB, ITS identification rates were 100% and a minimum of 14.8% rpoC1 sequence. Paniculata, the wine cake tendons and oyster shell thorn outgroup citrus and its related species 6 genera were identified, building NJ distance tree, the comparison of a single fragment identification. The results show that: the ITS identification of the highest rates of 59.3% to 55.9% in, psbH-petB followed by the matK 50.8%, the remaining fragments are less than 50%. Integrated 1,2 As a result, the nuclear gene ITS the the chloroplast interval the sequence psbH-petB, chloroplast encoded the gene matK identified the highest rate in the DNA sequence of the same type, an important fragment of Citrus and its related genera of plant DNA barcoding study. 3. Combined and previous studies proposed a combination of programs and a single fragment of the third international bio-barcode identification results, use matK rbcL, the matK trnH-psbA, matK ITS, trnH-psbA ITS, ITS2 of trnG-trnS, matK ITS2, matK psbH-petB trnG-trnS, matK ITS trnG-trnS, matK rpoB rpoC1 fragment combination study to Fuming Trifoliate, the dragon trifoliate orange and trifoliate NJ distance tree built from 28 plants of citrus the Swingle, Tanaka system as outgroup, compare The combined fragment identification rate. The results show that:, trnH-psbA Identification of ITS the Swingle systems plant identification rate of up to 100%, the the matK trnH-psbA, matK ITS the trnG-trnS ITS2, followed by 93.8%, matK to the rpoB rpoC1 lowest identification rate of 81.3% . The Tanaka system 28 plant identification, matK ITS, trnG-trnS ITS2 identified the matK ITS trnG-trnS three rate up to 96.3%, trnH-psbA ITS, matK trnH-psbA identification ability decreased, respectively, 74.1 %, 51.9%. So the use of a combination of fragments of the citrus genus Swingle the Tanaka system 28 plant identification matK ITS ITS2 Identification of trnG-trnS the highest rate. To paniculata, wine cake ilicifolius and the oyster shell thorn outgroup combination of fragments of citrus and its related species identification, to build NJ distance tree, comparing the rate of identification of each fragment combinations. The results show that: the matK ITS trnG-trnS combination of identification rate of up to 83.1% from 81.4%, followed by matK of ITS. Little difference between the two identification rate, but the latter less trnG-trnS fragment, the sequence length is shorter, more efficient identification. In summary, in the single sequence identified citrus the Swingle systems, materials and citrus and its related species Tanaka system 28, psbH-petB, ITS has the highest rate of identification and the psbH-petB, ITS matK sequence in identified highest rate three citrus barcode study fragment; combined fragment compared with a single fragment, identification rate of a relatively substantial increase, matK ITS compared with other combinations of fragments in the DNA sequences of the same class, with higher identification rate, shorter sequence length, can be used to citrus and its related genera of plant DNA barcode. DNA barcode technology as a new means of species identification, can be used for identification of Citrus and its related genera of plants.