CN112921114B - DNA sequence markers and their detection primers for identifying sex of cannabis plants - Google Patents
DNA sequence markers and their detection primers for identifying sex of cannabis plants Download PDFInfo
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Abstract
The invention relates to the technical field of plant variety identification and breeding, in particular to a DNA sequence marker for identifying the hemp plant sex and a detection primer thereof. The invention provides a group of insertion/deletion markers (Insert/Delete) which can identify the sex character of industrial hemp by screening and linking with the hemp sex, wherein the PCR amplification product of the marker is amplified to form a single strip in female strains of a plurality of hemp varieties, and is amplified to form a type containing two strips in male strains. The marker can be used for rapidly identifying the sex and the variety type of industrial hemp germplasm resources, and simultaneously provides a useful molecular marker for the auxiliary breeding of industrial hemp molecular markers.
Description
Technical Field
The invention relates to the technical field of plant variety identification and breeding, in particular to a DNA sequence marker for identifying the hemp plant sex and a detection primer thereof.
Background
Cannabis sativa L is Cannabis L belonging to the genus Cannabis of the family Cannabiaceae. It belongs to short day cross-pollination crop, most hemp germplasm resources are male and female cross plants, and few are male and female isoplants. Cannabis is classified into industrial cannabis and narcotic cannabis according to the level of its hallucinogenic addiction ingredient Tetrahydrocannabinol (THC). The industrial hemp fiber has good strength and good antibacterial property, and is widely used in the fields of textile, papermaking and the like. Cannabidiol (CBD) is an isomer of THC, has no addiction, develops rapidly in the fields of food, health care, medicine, cosmetics and the like in recent years, and shows good market prospect.
Generally, in industrial hemp production, the CBD content and the floral leaf yield of female plants are higher than those of male plants, which have a higher fiber quality than the female plants, within the same hemp material. According to different production purposes, in the production process, a proper male-female ratio is needed so as to improve the economic benefit per mu. Therefore, the method is very important for regulating and controlling the male-female ratio of the hemp by quickly and accurately identifying the sex of the hemp plant.
The hemp generally takes more than 3 months from the germination to the flowering of the seeds, the gender of the plant cannot be accurately judged morphologically before, the gender of the plant can be accurately judged morphologically only when the hemp blooms, the flowering time of the female plant and the male plant is inconsistent, and the male plant blooms about one month earlier than the female plant. However, in production, when the purpose of harvesting fiber is to harvest the female plants too much will affect the quality of the fiber and the fiber harvesting period; when CBD is harvested, the yield of the flower leaves and the CBD content are affected by too many male plants, and a great amount of manpower is needed for castration when the male plants bloom in production. The proper male-female ratio is very important in production.
Therefore, the sex of the plants is identified in the seedling stage, and the sex ratio is properly regulated according to the production purpose, so that the method has important significance for marijuana breeding and cultivation. The molecular marker linked with the sex can overcome the defect that the morphological and physiological and biochemical indexes are influenced by the environment and the development stage, and the sex of the plant is identified in the seedling stage on the molecular level. Insert/Delete markers refer to a certain number of nucleotide insertions or deletions in the whole genome of both parents, and PCR primers designed based on these different sites are Indel markers. InDel exists in plant genome widely, and InDel marker has the characteristics of wide distribution, high accuracy, stable variation and the like, and is widely used in the fields of genetic relationship analysis, genetic map construction, variety identification, gene positioning and the like. However, the cannabis sex-related InDel markers have not been reported yet.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a DNA sequence marker for identifying the hemp plant gender and a detection primer thereof.
The DNA sequence marker for identifying the hemp plant sex provided by the invention comprises a segment of a nucleotide sequence shown as SEQ ID NO. 1.
According to the invention, analysis shows that the genome of the male marijuana plant has a deletion with the length of 146bp relative to the genome of the female marijuana plant, and the type of the marijuana plant can be well identified by detecting the deletion fragment.
The invention also provides a primer pair for detecting the DNA sequence marker. The DNA sequence marker is identified in a nucleic acid amplification mode, and in order to realize accurate amplification of a fragment to be detected, primers are respectively designed on the upstream and downstream of the fragment to be detected by 100-200 bp. In some embodiments, the nucleic acid sequence of the primer pair for detecting the DNA sequence marker is shown as SEQ ID NO 2-3. Wherein SEQ ID NO.2 shows an upstream primer sequence, SEQ ID NO: 3 is the sequence of the downstream primer.
By adopting the primer pair provided by the invention, the sequencing result of the female plant genome amplification product is shown as SEQ ID NO.4 in the sequence table, and the sequencing result of the male plant genome amplification product is shown as SEQ ID NO.5 in the sequence table.
The invention also provides a kit for identifying the hemp plant sex, which comprises the primer pair.
The kit also comprises a DNA molecular weight marker; the DNA molecular weight marker comprises 2 DNA molecules with the length difference of 146 bp.
In the kit containing the primer pair with the nucleic acid sequence shown as SEQ ID NO. 2-3, the lengths of two markers are 397bp and 251bp respectively. In some embodiments, the nucleic acid sequences of the two DNA molecules of the marker are respectively shown in SEQ ID NO. 4-5.
The kit further comprises a PCR amplification reagent. The PCR amplification reagent comprises dNTPs, Taq enzyme and amplification buffer.
The invention provides a method for identifying the sex of hemp plants, which comprises the following steps:
the hemp genome DNA is used as a template, the primer group is used for PCR amplification, and the hemp sex is judged according to an amplification product.
In the method for identifying the hemp plant gender of the invention, the judgment comprises the following steps:
amplifying to obtain two strips, wherein the difference between the lengths of the two strips is 146bp, the two strips are male plant samples, the amplification only obtains one strip, and the size of the one strip is consistent with that of a longer strip in the male plant amplification strips, and the one strip is female plant samples;
or sequencing the amplified product, wherein the sequencing result shows that the sample containing the nucleotide sequence shown in SEQ ID NO.5 is from a female strain; the samples containing the nucleotide sequences shown by SEQ ID NO:4(397bp) and SEQ ID NO:5(251bp), respectively, were derived from male strains as a result of sequencing.
The invention also provides a method for identifying the variety of cannabis, which comprises the following steps:
the marijuana genome DNA is used as a template, the primer group is used for PCR amplification, and marijuana varieties are judged according to amplification products.
In the method for identifying a variety of cannabis of the invention, the judging comprises:
amplifying to obtain two strips, wherein the difference between the lengths of the two strips is 146bp, the two strips are female and male variant strains, the amplification only obtains one strip, and the size of the strip is consistent with that of the longer strip of the male strain amplification, and the strip is an all-female strain;
or sequencing the amplified product, wherein the sequencing result shows that the sample containing the nucleotide sequence shown in SEQ ID NO.5 is from a female strain; the samples containing the nucleotide sequences shown by SEQ ID NO:4(397bp) and SEQ ID NO:5(251bp), respectively, were derived from male strains as a result of sequencing.
The invention provides a group of insertion/deletion markers (Insert/Delete) which can identify the sex character of industrial hemp by screening and linking with the hemp sex, wherein the PCR amplification product of the marker is amplified to form a single strip in female strains of a plurality of hemp varieties, and is amplified to form a type containing two strips in male strains. The marker can be used for rapidly identifying the sex and the variety type of industrial hemp germplasm resources, and simultaneously provides a useful molecular marker for the auxiliary breeding of industrial hemp molecular markers.
Drawings
To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described as follows:
FIG. 1 shows the electrophoresis of the amplification results of F2 population (A) constructed from Yun Ma No. 7 XH 4 and H4 variety (B) female strain (F) and male strain (M) with Indel marker I1-10, wherein M is DL 2000;
FIG. 2I 1-10 shows the electrophoresis chart of the amplification results of Hanma No. 24 and ZY1 all-female strain in the heterogynic strain variety, wherein M is DL 2000.
Detailed Description
The invention provides a DNA sequence marker for identifying the hemp plant sex and a detection primer thereof, and a person skilled in the art can realize the identification by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market. The hemp in the invention is industrial hemp. The DNA sequence marker provided by the invention has good universality, and can realize good sex identification effect on various industrial cannabis sativa. The yunnan 7 is one of representative varieties of industrial hemp in China at present, and in the embodiment, the yunnan 7 is used as a subject for verification.
The DNA sequence marker provided by the invention is tatgcataaggtaaatatgtccatatctagagtaatcgtcaatgaaagtgacaaagtactcataaccacctcgagcttttacattcaaaggtccgcagacatcagaatgcactaaccctaggggttgtttggcacgctctcccttt;
the primer sequence is acctaacgatttccaaaagc, accaaacgttctttctttgc;
the fragment with the length of 397bp obtained by female strain amplification is acctaacgatttccaaaagcgtgcgattccgtctttttgcaactccattttgctgtggagtgctaggggcggttaattgggattcaattccaagttcaattaaataatctttgaactgcatatccatatattctccacccttatcagttcgcaagatctttaatgatttacctaattggttttgagccaatgcgtgaaattcctgaaactttgcaaatgtttcagatttcttatgcataaggtaaatatgtccatatctagagtaatcgtcaatgaaagtgacaaagtactcataaccacctcgagcttttacattcaaaggtccgcagacatcagaatgcactaaccctaggggttgtttggcacgctctccctttgcaaagaaagaacgtttggt
The 251bp fragment obtained by male strain amplification is acctaacgatttccaaaagcgtgcgatttcttctttctgcaattacattctactatggagtgctaggggcagttaattgagattaaatgccaggttcgatcaaaagatctttgaactgcatatccatatattctccacccctatcaattcgcaagatctttaaagttttacccaattagttttgagctaatgcaggctattcctgaaacttttgaaacgtttcagtggtccgcaaagaaagaacgtttggt
The invention is further illustrated by the following examples:
example 1 identification of sex of offspring and sex of different hemp varieties (lines) and hybrids
1.1 InDel marker development: NCBI first downloads cannabis whole genome sequence information (https:// www.ncbi.nlm.nih.gov/bioproject/PRJNA73819) and further uses the MUMer software to detect sites of difference in the female and male genomic sequences. Based on the Indel detection result of MUMer, Primer I1-10 was designed using Primer3 based on the reference sequence.
1.2 Yun Ma No. 7 XH 4F2DNA of individual male and female plants of colony and H4 variety
Respectively selecting Yuma No. 7 multiplied by H4F according to appearance forms26 individuals of the male and female individuals of the H4 variety are extracted by a CTAB methodGenomic DNA.
1.3 verification of Indel marker I1-10 in F2 population and H4 variety male and female single strains constructed by Yunmen No. 7 XH 4
The primer sequences are SEQ ID NO.2 and SEQ ID NO.3, and the F2 population and the male and female plants of the H4 variety are amplified.
The PCR reaction system comprises: 50 μ M dNTPs,0.2 μ M primer, 0.5U Taq polymerase (TaKaRa, Large ligation) and 30ng of DNA template;
PCR amplification procedure: 5min at 95 ℃; 30s at 94 ℃ (Tm value) for 1min, 40s at 72 ℃, and 35 cycles; extending at 72 deg.C for 5min, and storing at 10 deg.C for 10 min.
The results are shown in FIG. 1: agarose gel electrophoresis results show that 397bp bands and 251bp specific bands appear in male plant genomes, 397bp products are only amplified in female plant genomes, sequencing results of the female plant genome amplification products are shown as SEQ ID NO.4 in a sequence table, and sequencing results of the male plant genome amplification products are respectively shown as SEQ ID NO.4(397bp) and SEQ ID NO.5(251bp) in the sequence table.
The amplified PCR product is detected by using polyacrylamide, and only 397bp single band is amplified in female plants, and 251bp band is amplified in male plants.
The result completely accords with the actual result, and the detection accuracy can reach 100 percent.
Example 2 application of the marker I1-10 in identification of marijuana hologynic species and hermaphrodite species
1. Extraction of DNA of all-female variety plant and male-female variant plant
Extracting DNA of all-female variety plants and male-female variant plants to be detected by adopting CTAB method
2. Application of I1-10 marker in identification of marijuana hologynic variety and hermaphrodite variety
Utilizes the primer sequences of SEQ ID NO.2 and SEQ ID NO.3 to amplify the DNA genomes of the hemp full-female variety and the male-female heterostrain variety,
the PCR reaction system comprises: 50 μ M dNTPs,0.2 μ M primer, 0.5U Taq polymerase (TaKaRa, Mass.) and 30ng of DNA template;
PCR amplification procedure: 5min at 95 ℃; 30s at 94 ℃ (Tm value) for 1min, 40s at 72 ℃, and 35 cycles; extending at 72 deg.C for 5min, and storing at 10 deg.C for 10 min.
The results are shown in FIG. 2: agarose gel electrophoresis results show that only 397bp bands appear in the genome of all plants of the all-female variety, and 251bp specific bands appear in partial plants of the male and female variety.
The polyacrylamide amplification PCR product is used for detection, only 397bp single bands are amplified in the whole female, and 251bp bands are amplified in the partial plants of the male and female variant strains.
The result completely accords with the actual result, and the detection accuracy can reach 100 percent.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Sequence listing
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<120> DNA sequence marker for identifying hemp plant sex and detection primer thereof
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tatgcataag gtaaatatgt ccatatctag agtaatcgtc aatgaaagtg acaaagtact 60
cataaccacc tcgagctttt acattcaaag gtccgcagac atcagaatgc actaacccta 120
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acctaacgat ttccaaaagc gtgcgattcc gtctttttgc aactccattt tgctgtggag 60
tgctaggggc ggttaattgg gattcaattc caagttcaat taaataatct ttgaactgca 120
tatccatata ttctccaccc ttatcagttc gcaagatctt taatgattta cctaattggt 180
tttgagccaa tgcgtgaaat tcctgaaact ttgcaaatgt ttcagatttc ttatgcataa 240
ggtaaatatg tccatatcta gagtaatcgt caatgaaagt gacaaagtac tcataaccac 300
ctcgagcttt tacattcaaa ggtccgcaga catcagaatg cactaaccct aggggttgtt 360
tggcacgctc tccctttgca aagaaagaac gtttggt 397
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acctaacgat ttccaaaagc gtgcgatttc ttctttctgc aattacattc tactatggag 60
tgctaggggc agttaattga gattaaatgc caggttcgat caaaagatct ttgaactgca 120
tatccatata ttctccaccc ctatcaattc gcaagatctt taaagtttta cccaattagt 180
tttgagctaa tgcaggctat tcctgaaact tttgaaacgt ttcagtggtc cgcaaagaaa 240
gaacgtttgg t 251
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