Objective  To screen the optimal DNA barcode for identifying Celosiae semen and Celosia cristatae semen using the molecular biology identification techniques, and to establish a rapid, accurate, and convenient approach for identifying species in the Celosia L..

Methods  A total of 21 samples of Celosiae semen and Celosia cristatae semen were collected, and total DNA was extracted from the samples. The sequences of ITS, psbA-trnH, matK, rbcLand trnL-trnF were amplified and sequenced, MEGA-X software was used to analyze and process the data, the Kimura-2-parameter genetic distance was calculated, an neighbor-joining method cluster evolutionary tree was established, and the comparative analysis was conducted. The BestMatch and BestCloseMatch was calculate based on TaxonDNA to evaluate the discriminative ability of DNA barcodes. The ITS2 database and RNAfold database were used to predict the secondary structure of the barcode sequence.

Results   The barcode ITS, matK, psbA-trnH, rbcL, and trnL-trnF sequences were successfully amplified and had high sequencing success rates. Among them, the psbA-trnH had the most mutation sites, followed by ITS. The psbA-trnH, ITS2 and trnL-trnF had significant barcoding gap, and psbA-trnH was the most significant (100%). According to the phylogenetic tree, the IITS, ITS2, psbA-trnH, matK and trnL-trnF sequences could cluster Celosiae semen and Celosia cristatae semen into a separate branch, the support rates of each branch point were above 60%, with psbA-trnH and trnL-trnF having the highest support rate (99%). Analysis of molecular variance results showed that the psbA-trnH had the highest population genetic differentiation index and was the most suitable for distinguishing differences between Celosiae semen and Celosia cristatae semen. Except for matK, there were differences in the secondary structure of psbA-trnH, ITS2 and trnL-trnF sequences between Celosiae semen and Celosia cristatae semen.

Conclusion  With psbA-trnH as the main component ITS and trnL-trnF as the auxiliary ones, accurate identification of Celosiae semen and Celosia cristatae semen can be achieved.

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