1. Application of psba-trnh igs for dna barcoding in genus Lathyrus.
- Author
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Nigda, A., Tynkevich, Y. O., and Volkov, R. A.
- Subjects
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GENETIC barcoding , *LATHYRUS , *NUCLEOTIDE sequence , *CHLOROPLAST DNA , *NUMBERS of species , *POLYMERASES - Abstract
Background. The psbA-trnH intergenic spacer (IGS) is one of the most commonly used in the molecular taxonomy and DNA-barcoding regions of the chloroplast genome. The advantages of this region are the possibility of direct sequencing of PCR products, a small size that facilitates PCR amplification and a moderately high nucleotide polymorphism, compared to other fragments of the cpDNA. However, the sequences of psbA-trnH for many plants are still absent in databases, which makes it difficult to identify them. This, in particular, concerns Lathyrus venetus (Mill.) Wohlf - the Red list species of the legume flora of Ukraine. The aim of current study was to amplify and sequence the IGS psbA-trnH of this species and to estimate the possibility of using it for DNA barcoding. Methods. PCR amplification was performed using the Maxima Hot Start PCR Master Mix (Thermo Scientific) polymerase. For the amplification, the primers complementary to the flanking IGS regions of psbA and trnH genes were used. The primers annealing temperature was 61 ° C. The obtained PCR products were sequenced and the nucleotide sequence was analyzed using bioinformatics methods. Finding the matches in the GenBank database was conducted using the Blast program. Results. The sequence of IGS psbA-trnH for L. venetus has the length of 241 bp. The search in the Genbank database showed that the sequence of IGS of L. niger L. is most closely related to L. venetus among the deposited sequences. The calculated percentage of similarity between IGS of these species was 98.7 %. The vast majority of other Lathyrus species have a similar percentage of similarity to IGS of L. venetus in the range of 97.1 to 97.9 %. In comparison to all the aforementioned species IGS psbA-trnH L. venetus has two oligonucleotide insertions and one polynucleotide deletion at the 3’-end. Unexpectedly, the same deletion was found to be characteristic for the IGS of the genus Pisum L. Also, several point nucleotide substitutions have been identified. In general, it is important for DNA-barcoding that the nucleotide sequence of IGS psbA-trnH allows a clear distinction between L. venetus and other species of Lathyrus genus. Conclusions. At the next stage of our study, there will be conducted a sequencing of IGS psbA-trnH for closely related to L. venetus, but more abundant species L. vernus (L.) Bernh., which often forms morphologically similar hybrids with L. venetus. This complicates the control of the latter. Also, in order to better identify the hybrid forms, we plan to pick up the DNA-barcode of nuclear localization and use them together. [ABSTRACT FROM AUTHOR]
- Published
- 2019