IDENTIFICATION METHOD OF TWO PARENTS OF NYMPHAEA HYBRID BASED ON SEQUENCES OF INTERNAL TRANSCRIBED SPACER (ITS) AND matK

An identification method of two parents of a Nymphaea hybrid based on sequences of internal transcribed spacer (ITS) and matK, comprising the following steps:

• (i) construction of a database for each of nucleotide sequences ITS and matK of Nymphaea:
• downloading existing nucleotide sequences of ITS and matK of Nymphaea;
• conducting stringent filtration to retain one sequence for each “species unit”, namely species/subspecies/variety/form/hybrid of the sequences of ITS and matK;
• conducting alignment, adjusting sequences with inconsistent sequence directions, and then conducting re-alignment to obtain the database for each of nucleotide sequences ITS and matK of Nymphaea;
• and arranging a sequence name for each of finally obtained sequences in a format of “species name/ACCESSION of sequence”;
• and
• (ii) construction of an ITS monoclonal sequence and an matK nucleotide sequence of a species for parents to be determined of the Nymphaea:
• extracting a genomic DNA of the Nymphaea of the parents to be determined;
• designing primers;
• conducting PCR sequence amplification;
• conducting Sanger sequencing;
• conducting sequence alignment and constructing a neighbor-joining tree based on a genetic distance, and then aligning sequences of ITS and matK obtained by monoclonal sequencing with the respective database;
• checking specific loci to obtain species information of male and female parents of the Nymphaea hybrid,
• wherein the construction of the database for each of the nucleotide sequences ITS and matK of the Nymphaea specifically comprises the steps of:
• (A) searching a nucleotide database of the National Center of Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov/nucleotide) with syntaxes “((Nymphaea[Organism]) AND 5.8S [Title]) NOT PREDICTED[Title]” and “((Nymphaea[Organism]) AND matK[Title]) NOT PREDICTED[Title]”, to obtain all data of the sequences of ITS and matK of the Nymphaea;
• acquiring 59 published chloroplast genomes of the Nymphaea with a syntax “chloroplast, complete genome[Title] OR plastid, complete genome[Title]) AND Nymphaea[Organism]”, and extracting a matK sequence in the chloroplast genomes;
• (B) conducting stringent filtration to retain one sequence for each “species unit”, namely species/subspecies/variety/form/hybrid of the sequences of ITS and matK;
• and
• (C) conducting alignment, adjusting the sequences with inconsistent sequence directions, and then conducting re-alignment to obtain the database of the nucleotide sequences ITS and matK of the Nymphaea;
• wherein the stringent filtration specifically comprises the steps of:
• (a) removing a species whose species name comprises unverified, sp., and cf., retaining a subspecies, a variety, a form, and a clear hybrid that are included in the species, which are referred to as “species units”, and regarding each of an isolate, a voucher, a genotype, and a strain as a cultivar and classifying the cultivar into each “species unit”;
• (b) if the “species unit” has only one sequence, omitting the filtration;
• after the filtration is conducted, retaining only one sequence for each “species unit”;
• (c) if a sequence of the cultivar in step a is quite different from that of the “species unit”, removing the sequence of the cultivar preferentially;
• (d) removing a sequence with a significantly high difference in a coding region 5.8S rRNA preferentially;
• removing a sequence having an unknown base and a degenerate base preferentially;
• and retaining a longer sequence preferentially;
• (e) using the matK sequence extracted from the chloroplast genomes only as a supplement;
• when the matK sequence obtained by sequencing already has the “species unit”, discarding the matK sequence extracted from the chloroplast genomes;
• and
• (f) retaining a sequence as a sequence that best represents the “species unit”, that is, a sequence being closest to a consensus sequence obtained from multiple sequences;
• wherein the construction of an ITS monoclonal sequence and the matK nucleotide sequence of the species for parents to be determined of the Nymphaea in step (ii) specifically comprises the steps of:
• (a′), designing an ITS primer suitable for the Nymphaea using conserved nucleotide sequences of 18S, 5.8S, and 25S rRNA of the Nymphaea and referring to a general plant barcode ITS primer;
• and designing a matK primer suitable for the Nymphaea using a conserved nucleotide sequence of matK of the Nymphaea and referring to a general plant barcode primer;
• (b′) extracting the genomic DNA of the Nymphaea of the parents to be determined;
• (c′) conducting PCR amplification using the genomic DNA of the Nymphaea of the parents to be determined obtained in step (b′) as a template and using the primers obtained in step a, to obtain amplification products of the sequences of ITS and matK, respectively;
• (d′) ligating the amplification product of the ITS sequence obtained in step (c′) using a pLB zero-background rapid cloning kit, and transforming into DH5α competent cells;
• conducting PCR amplification on an obtained selected bacterial plaque through a carrier primer, to obtain a PCR amplification product of the ITS sequence by cloning amplification;
• and
• (e′), subjecting the amplification product of the matK sequence obtained in step (c′) and the PCR amplification product of the ITS sequence by cloning amplification obtained in step (d′) to sequencing with a Sanger sequencer, and conducting sequence assembly on obtained sequencing results to obtain the matK sequence and the monoclonal ITS sequence for the parents to be determined of the Nymphaea;
• wherein
• the primers comprise:
• ITS-5, with a nucleotide sequence of AGTCGTAACAAGGTTTCCGT;
• ITS-3, with a nucleotide sequence of TAGTAACGGCGAGCGAACC;
• matK-5, with a nucleotide sequence of CGTACCGTACTTTTATGTTTACGAG;
• and
• matK-3, with a nucleotide sequence of ACCCAATCCATCTGGAAATCTTGCTTC.