Molecular Distinguishment of Trapa natans L. Varieties in Taihu Lake Region of China and Development of a RAPD-SCAR Marker for Authentication of ‘Heshangling’

in HortScience

Water chestnut (Trapa natans L.) is a group of annual, floating-leaved aquatic plants that serves as food and medical resources in many countries. However, the molecular method for distinguishing different T. natans L. resources is lacking. In this study, we detected genetic diversity of several chloroplast and nuclear genic or intergenic sequences in four varieties of T. natans and one wild type of Trapa incisa Siebold & Zuccarini to evaluate their potential as molecular markers. Our data revealed that the three chloroplast fragments (rbcL, matK, and pbsA-trnH) show no sequence difference among all tested samples. Only one nucleotide substitution is detected for the nuclear ribosomal internal transcribed spacer (ITS) in the T. natans variety Shuihongling. Four nucleotide substitutions are detected for the nuclear carotenoid isomerase (CRTISO) gene in the variety Hongxiuxie. In contrast, a total of 29 polymorphic sites are detected for a Toll and interleukin-1 receptor-nucleotide binding site–leucine rich repeat (TNL) gene in the five samples, among which six are nucleotide substitutions and the rest are insertions/deletions. The five samples could be fully distinguished from each other based on the TNL gene. To specifically authenticate ‘Heshangling’, 33 randomly amplified polymorphic DNA (RAPD) markers were adopted to amplify genomic sequences from the five samples. A pair of sequence characterized amplified region (SCAR) primers were designed based on the results of RAPD markers, which could specifically amplify one target band from all eight individuals of ‘Heshangling’, but none from any individuals of other T. natans varieties or one T. incisa. Taken together, a TNL sequence was provided in this study to distinguish four T. natans varieties and one T. incisa. Furthermore, a RAPD-SCAR marker was developed for efficient authentication of ‘Heshangling’.

Contributor Notes

This work was supported financially by the earmarked fund for Jiangsu Agricultural Industry Technology System to X. Shen, grant no. JATS (2018) 064.

Corresponding author. E-mail: hangyueyu@cnbg.net.

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    Amplification profiles of four varieties of Trapa natans and one Trapa incisa using RAPD26 marker. Lanes A to E in order are ‘Laowuling’, ‘Hongxiuxie’, ‘Shuihongling’, ‘Heshangling’, and ‘Yeling’. M, DL2000 DNA marker; the arrow represents a specific band of ‘Heshangling’. bp = base pairs.

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    The nucleotide sequence of ‘Heshangling’-specific fragment amplified using primer RAPD26. The underlined sequences are designed sequence characterized amplified region primers.

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    Verification of the ‘Heshangling’-specific randomly amplified polymorphic DNA–sequence characterized amplified region by polymerase chain reaction (PCR) in eight individuals from each of four varieties of Trapa natans and one Trapa incisa. A to E are PCR results from eight individuals of ‘Laowuling’, ‘Hongxiuxie’, ‘Shuihongling’, ‘Heshangling’, and ‘Yeling’, respectively. M = DL2000 DNA marker. Lanes 1 to 8 are PCR products from eight individuals. bp = base pairs.

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    The morphological features of four varieties of Trapa natans and one wild type of Trapa incisa in this study. A to E in order are ‘Laowuling’, ‘Hongxiuxie’, ‘Shuihongling’, ‘Heshangling’, and ‘Yeling’.

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    Phylogeny of Nucleotide binding site–leucine rich repeat (NBS-LRR) genes from Trapa bispinosa and Arabidopsis thaliana.

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