Selection Of DNA Barcoding For Root-Knot Nematodes And It’s Possible Application In Plant Quarantine Nematodes

Plant-parasitic nematodes are important pathogens of crops in the world and cause serious damage to yield. The invasion of plant quarantine nematodes not only can threat the security of ecosystem, but also decrease the agricultural production and international trades. The correct identification of nematode species is critical to plant qunrantine. The shortages of morphological identification made the urgent needs for fast and accurate techniques for molecular identification. Therefore, DNA barcoding is a techniques developed for species identification on basis of sequences variations in the specific fragments of genome DNA. The characters of standardization made the powerful ability in species classification and identification by using the global databases of DNA barcoding.The nucleotide sequences of28S (D2/D3),18S, ITS and IGS2fragments from ribosomal DNA (rDNA) were retrieved from GenBank and the feasibility as candidate DNA barcoding markers for Meloidogyne spp. was evaluated using pair-wise distance and phylogenetic methods. The results revealed that the average intraspecific divergences of28S (D2/D3),18S, ITS and IGS2is0.0043,0.0027,0.024and0.0014, respectively; for interspecific, is0.074,0.036,0.156and0.588, respectively. The genetic distances between intraspecific and interspecific of4loci showed some overlapping due to the existence of close sibling species of Meloidogyne. The neighbour-joining (NJ) trees constructed by K2P model demonstrated that28S (D2/D3) locus has higher species resolution. Due to the larger barcoding gap,28S (D2/D3) locus of rDNA can be suggested as a candidate DNA barcoding marker for Meloidogyne spp..The fragments of28S (D2/D3) of ribosomal DNA and COI gene of mitochondrial DNA were amplified from6species of Meloidogyne, with26and21populations, respectively. All fragments were sequenced and the feasibility as candidate DNA barcoding marker for Meloidogyne spp. was evaluated. The results revealed the genetic distances between intraspecific and interspecific of COI loci showed some overlapping and the application in barcoding needs further study. The genetic distances between intraspecific and interspecific of28S (D2/D3) loci showed some overlapping, but still have some barcoding gap. The NJ trees demonstrated that M. incognita, M. javanica and M. arenaria were mixed grouping and difficult to be separated each other into one branch. The rest species of Meloidogyne can be effectively distinguished by each other. Because of suitable fragment length, high success rate of amplification and sequencing, good ability of species discrimination, together with the genetic variation of intraspecific significantly smaller than interspecific,28S (D2/D3) locus can be used as an ideal DNA barcoding marker for Meloidogyne spp..The sequences of28S (D2/D3) of rDNA from plant quarantine nematodes were screened form GenBank and the feasibility as a candidate DNA barcoding marker was evaluated. The results revealed the genetic distances between intraspecific and interspecific of28S (D2/D3) loci from56species of15genera showed some overlapping, with94.8%of intraspecific genetic distance less than0.02and95.6%of interspecific genetic distance greater than0.02. Some barcoding gaps were still existed. The NJ tree of28S (D2/D3) sequences demonstrated that56species were separated into3branches which were classified and grouped in accordance with their taxon order Rhabditia, Dorylaimida and Triplonchida, respectively. Most populations from the same species of quarantine nematodes were formed a monophyletic with higher support rate, only a few of closely related species were clustered into same small branch and difficult to be distinguished. In summary, the28S (D2/D3) sequences can be used as an ideal DNA barcoding marker for plants quarantine nematodes.