CN110628934A - Populus alba sex determining gene and application thereof in identifying Populus alba sex - Google Patents
Populus alba sex determining gene and application thereof in identifying Populus alba sex Download PDFInfo
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- CN110628934A CN110628934A CN201911012229.6A CN201911012229A CN110628934A CN 110628934 A CN110628934 A CN 110628934A CN 201911012229 A CN201911012229 A CN 201911012229A CN 110628934 A CN110628934 A CN 110628934A
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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Abstract
The invention discloses a poplar sex determining gene and application thereof in identifying the gender of poplar, the poplar sex determining gene is at least one of M1 and M3, the nucleotide sequences are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, M1 and M3 are female specific genes of poplar, therefore, the female can be quickly and conveniently identified by detecting whether the genome of the poplar contains M1 and M3 at any period of the poplar, thereby recovering unnecessary loss and having huge potential application and economic value.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a poplar sex determining gene and application of the poplar sex determining gene in identifying the gender of populus alba.
Background
Populus and salix plants are species of trees of significant ecological and economic value, whether in north america or asia. The poplar is a key species in forest ecosystems of two continental temperate regions and the north, provides an important place for the survival of other animals, plants and microorganisms, and is the basis of the stable development of biological diversification in the ecosystems. Poplar is also an important source for two continents of paper making and wood industry, and more than half of the artificial forests in China are poplar. In addition, insect-repellent chemicals for poplar and willow have extremely important medicinal value, such as aspirin, a modern important drug that has been successfully developed through improvement and is well known and commonly used.
Growth rate and biomass are important targets for tree breeding; however, the cultivated poplar and willow are threatened by gnawing insects, and pollination and fructification (such as catkin and poplar catkin) also become one of important factors of environmental pollution. After the female poplars are fertilized, the female poplars can develop to form poplar catkins, so that the environment and the air quality are seriously polluted; dry and flammable poplar battings also present a fire hazard. In addition, by comparing and analyzing the male and female heteroplant, the male plant is found to have more luxuriant branches and leaves and stronger stems compared with the female plant, and is more suitable for being popularized as economic and environment-friendly forest. However, male and female poplars are difficult to distinguish at the seedling stage, and often require 4 to 5 years of sexual maturity before females can be selectively felled. This results in a significant loss of time and benefit. In view of the above, the method can quickly and conveniently identify the female in any period of the populus alba, thereby recovering unnecessary loss. Therefore, the invention has huge potential application and economic value.
Disclosure of Invention
In view of the above, the present invention is directed to a poplar sex-determining gene; the second purpose of the invention is to provide the application of the poplar sex determining gene in identifying female plants of the populus alba; the third purpose of the invention is to provide a primer pair for amplifying the poplar sex-determining gene; the fourth purpose of the invention is to provide the application of the primer pair in the detection of the gender of the populus alba; the fifth purpose of the invention is to provide a method for identifying the sex of the populus alba.
In order to achieve the purpose, the invention provides the following technical scheme:
1. the poplar sex determining gene is any one of M1 or M3, and the nucleotide sequences are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively.
2. The application of the poplar sex determining gene in identifying female poplar plants.
3. And amplifying the primer pair of the poplar sex determining gene.
Preferably, the primer pair is a primer pair for detecting M1 or M3, and the nucleotide sequence of the primer pair for detecting M1 is shown as SEQ ID NO.5 and SEQ ID NO. 6; the nucleotide sequences of the primer pair for detecting M3 are shown as SEQ ID NO.7 and SEQ ID NO. 8.
4. The primer pair is applied to the detection of the sex of the populus alba.
5. The method for identifying the gender of the populus alba comprises the following steps: using populus alba genome DNA as a template, designing and amplifying a primer pair of M1 or M3 by using the sex-determining gene, carrying out PCR amplification, and if the primer pair for amplifying M1 or M3 amplifies any target gene, determining that the target gene is female; a male is obtained if any band is not amplified by the primer pair of M1 or M3.
Preferably, the nucleotide sequences of the primer pair for amplifying M1 are shown as SEQ ID NO.5 and SEQ ID NO. 6; the nucleotide sequences of the primer pair for amplifying M3 are shown as SEQ ID NO.7 and SEQ ID NO. 8.
The detection efficiency of the method of the invention was found to be 100% by testing parallel lines of male and female 19, respectively.
The invention has the beneficial effects that: the invention provides a poplar sex determining gene which is any one of M1 or M3 respectively, M1 and M3 genes can identify the poplar sex at any growth period, male plants can be selectively transplanted before seedlings are transplanted, thereby saving time and labor cost, expanding economic benefit and providing reliable technical and theoretical support for tree planting and afforestation.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 shows the result of DNA quality determination using ACTIN according to CTAB.
FIG. 2 shows the identification of the sex of Populus alba using primer set M1 according to the present invention.
FIG. 3 shows the identification of the sex of Populus alba using primer set M3 according to the present invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The present example is carried out by taking the technical scheme of the present invention as a precursor, and a detailed embodiment and a specific operation process are given, but the protection scope of the present invention is not limited to the following examples. The following examples are given under conditions not specifically mentioned, and the conventional conditions are generally followed. For example, the conditions described in the molecular cloning guidelines (scientific publishers, 2015), or as recommended by the manufacturer.
Example 1 identification of genes characteristic of females
The specific genes of female populus alba are identified according to the genome sequence of the populus alba and are respectively named as M1 and M3, and the nucleotide sequences are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
Example 2 extraction of Populus alba DNA by CTAB method
Taking about 0.5g of leaves of 19 female poplars and male poplars respectively, cleaning, adding liquid nitrogen into a mortar, fully grinding, adding 1mL of CTAB, and transferring into a centrifuge tube; water bath is carried out for 30min at 65 ℃, and then equal volume of chloroform is added to precipitate impurities twice; adding equal volume of isopropanol into the supernatant to settle DNA at-20 ℃, then washing twice by using 75% ethanol in volume fraction, collecting precipitate, and adding 50 mu L of precipitation buffer into the precipitate after air drying; take 5. mu.L for electrophoresis detection.
Example 3 identification of DNA quality
The extracted DNA is used as a template, and a specific primer designed according to the poplar ACTIN sequence is used for amplification:
ACTIN-F：5’-cactcgtttgcgataatgga-3’(SEQ ID NO.3)；
ACTIN-R：5’-ctcgttgtagaaggtatgat-3’(SEQ ID NO.4)；
then, Primer start high fidelity DNA polymerase of TaKaRa is used for amplification, and PCR amplification conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 20s, and circulating for 30 times; extending for 5min at 72 ℃, and detecting the amplification quality of the target band by nucleic acid electrophoresis, wherein the result is shown in figure 1. The result shows that the extracted DNA can amplify ACTIN target band with good quality, and the next step of sex identification can be carried out.
Example 4 sex identification of Populus alba
The detected normal DNA is taken as a template, and a specific primer designed according to the sequences of M1 and M3 is used for amplification:
M1-F：5’-tactaatgaaaacattcagt-3’(SEQ ID NO.5)；
M1-R：5’-ttgactagtcaagacttgta-3’(SEQ ID NO.6)；
M3-F：5’-tagataaccacataagatc-3’(SEQ ID NO.7)；
M3-R：5’-gtaattacctgctcagcaatt-3’(SEQ ID NO.8)；
then using Primer start high fidelity DNA polymerase of TaKaRa company to amplify, wherein the PCR amplification condition is pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 20s, and circulating for 30 times; stretching for 5min at 72 ℃. Detecting the amplification condition of the target band by nucleic acid electrophoresis, if a certain DNA can be amplified to obtain any one or two 300bp bands by M1 or M3 primers, the DNA is female; males if neither M1 nor M3 primers amplified any band.
The results of the detection of the 19 male and female parallel lines are shown in FIGS. 2 and 3, and the detection efficiency of the method is 100%.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Sequence listing
<110> Sichuan university
<120> sex-determining gene of Populus alba and application thereof in identifying Populus alba sex
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 426
<212> DNA
<213> Populus alba L
<400> 1
tactaatgaa aacattcagt caataatacc gccagaatta caaacaaaaa aaatcctgct 60
tttatttaac aaaataatgg cgacctcaaa ttatcaacag aagtattcca tcagtaaatt 120
tgttgatatt aaataaattt ctgatagtga aatcaattta taatcttgat ttgattttat 180
ttaacaaatt ttgttttggt tcccaatatt tataaaaaca tcattaaaaa ctatgaaaca 240
tgaaattaaa tcataatgac tcatttattc ccatgttttt atttttttta tcattacatt 300
ccaattgtaa gttatttggt ctcgcaaagt ctattgttag ttgtcccatg tttttatttt 360
tagctaatgt tgaattagga aatatgaggc ttggtttttg gtcaactaca agtcttgact 420
agtcaa 426
<210> 2
<211> 408
<212> DNA
<213> Populus alba L
<400> 2
tagataacca cataagatct gcaaaaggga tgaaacagtg aaatgcataa cccacataag 60
ttaaacaact tctattgatt gcagtagccc tagaattacg cctccaattc aacattttcc 120
ccttcttgtc tctctttctg atatctttta tgcatgttcg tcttctcctg caaagccccc 180
ttgtaatcga ttaattattc agctttttta tatggcacct cagctttttc gcatctgata 240
gctgaagagg cttcagcaag aattcttgag ctcctccctc catgcacctg tttaggtgga 300
aaatatttaa caaatcaatg atttcttttc ctctatccaa ctaaatagac aaaagacaaa 360
aaatcaacag agattattca agactgaaat tgctgagcag gtaattac 408
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence ((Artificial Sequence)
<400> 3
cactcgtttg cgataatgga 20
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ctcgttgtag aaggtatgat 20
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tactaatgaa aacattcagt 20
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ttgactagtc aagacttgta 20
<210> 7
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tagataacca cataagatc 19
<210> 8
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gtaattacct gctcagcaat t 21
Claims (7)
1. Populus alba sex-determining gene, characterized in that: the populus alba gender determining gene is any one of M1 or M3, and the nucleotide sequences are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
2. Use of a poplar sex-determining gene according to claim 1 for identifying female plants of poplar.
3. A primer set for amplifying the poplar sex-determining gene of claim 1.
4. The primer pair according to claim 3, wherein: the primer pair is used for detecting M1 or M3, and the nucleotide sequence of the primer pair for detecting M1 is shown as SEQ ID NO.5 and SEQ ID NO. 6; the nucleotide sequences of the primer pair for detecting M3 are shown as SEQ ID NO.7 and SEQ ID NO. 8.
5. Use of a primer pair according to claim 3 or claim 4 for the detection of the sex of Populus alba.
6. The method for identifying the gender of the populus alba is characterized by comprising the following steps: performing PCR amplification by using genomic DNA of Populus alba as a template and using the sex-determining gene of claim 1 to design and amplify a primer pair M1 or M3, wherein the amplified primer pair M1 or M3 is female if any target gene is amplified; a male is obtained if any band is not amplified by the primer pair of M1 or M3.
7. The method of claim 6, wherein: the nucleotide sequences of the primer pair for amplifying M1 are shown as SEQ ID NO.5 and SEQ ID NO. 6; the nucleotide sequences of the primer pair for amplifying M3 are shown as SEQ ID NO.7 and SEQ ID NO. 8.
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| CN201911012229.6A CN110628934A (en) | 2019-10-23 | 2019-10-23 | Populus alba sex determining gene and application thereof in identifying Populus alba sex |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110777153A (en) * | 2019-12-17 | 2020-02-11 | 南京林业大学 | Populus deltoides androgenesis gene MmS and application thereof |
| CN111235177A (en) * | 2020-02-07 | 2020-06-05 | 中国林业科学研究院 | Populus alba PDS gene knocked out by CRISPR/Cas9 system and application thereof |
| CN111269974A (en) * | 2020-03-09 | 2020-06-12 | 南京林业大学 | Specific genomic DNA sequence of male Populus microphylla strain and application thereof |
| CN115109867A (en) * | 2022-08-04 | 2022-09-27 | 北京林业大学 | Specific PCR primer for identifying FERR and FERR-R genes of poplar and application thereof in identifying gender of poplar |
| CN116064593A (en) * | 2023-02-09 | 2023-05-05 | 四川大学 | PGAG gene of populus tomentosa and application thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110777153A (en) * | 2019-12-17 | 2020-02-11 | 南京林业大学 | Populus deltoides androgenesis gene MmS and application thereof |
| CN111235177A (en) * | 2020-02-07 | 2020-06-05 | 中国林业科学研究院 | Populus alba PDS gene knocked out by CRISPR/Cas9 system and application thereof |
| CN111235177B (en) * | 2020-02-07 | 2022-03-29 | 中国林业科学研究院 | Populus alba PDS gene knocked out by CRISPR/Cas9 system and application thereof |
| CN111269974A (en) * | 2020-03-09 | 2020-06-12 | 南京林业大学 | Specific genomic DNA sequence of male Populus microphylla strain and application thereof |
| CN111269974B (en) * | 2020-03-09 | 2020-12-04 | 南京林业大学 | Specific genomic DNA sequence of male Populus microphylla strain and application thereof |
| CN115109867A (en) * | 2022-08-04 | 2022-09-27 | 北京林业大学 | Specific PCR primer for identifying FERR and FERR-R genes of poplar and application thereof in identifying gender of poplar |
| CN115109867B (en) * | 2022-08-04 | 2023-09-26 | 北京林业大学 | Specific PCR primer for identifying poplar FERR and FERR-R genes and application thereof in sex identification of poplar |
| CN116064593A (en) * | 2023-02-09 | 2023-05-05 | 四川大学 | PGAG gene of populus tomentosa and application thereof |
| CN116064593B (en) * | 2023-02-09 | 2024-05-14 | 四川大学 | PGAG gene of populus tomentosa and application thereof |
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