CN108796119B - Specific molecular marker PCMI-M001 for rapid identification of male young seedlings of populus - Google Patents

Specific molecular marker PCMI-M001 for rapid identification of male young seedlings of populus Download PDF

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CN108796119B
CN108796119B CN201810690118.XA CN201810690118A CN108796119B CN 108796119 B CN108796119 B CN 108796119B CN 201810690118 A CN201810690118 A CN 201810690118A CN 108796119 B CN108796119 B CN 108796119B
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亢介玉
卢江杰
刘玉洋
刘朋丽
李春阳
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Hangzhou Normal University
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Abstract

The invention discloses a specific molecular marker PCMI-M001 for quickly identifying male seedlings of populus diversifolia. The nucleotide sequence of the upstream primer PCMI-M001-F of the molecular marker PCMI-M001 is shown as SEQ ID No: 1, the nucleotide sequence of the downstream primer PCMI-M001-R is shown as SEQ ID No: 2 is shown in the specification; the nucleotide sequence of the specific band obtained by amplifying the marker in a male individual is shown as SEQ ID No: 3, respectively. The specific molecular marker primer (PCMI-M001-F/R) can be used for quickly identifying the sex of male and female poplars by molecules, is simple, accurate and high in sensitivity, and cannot be achieved by other existing identification methods.

Description

Specific molecular marker PCMI-M001 for rapid identification of male young seedlings of populus
Technical Field
The invention belongs to the technical field of molecular identification of economic forest trees, and particularly relates to a specific molecular marker PCMI-M001 for quickly identifying male seedlings of populus tremuloides.
Background
The Populus (Populus cathayana) is a male and female heterotropous plant of Populus of Salicaceae, is used as a common economic and ecological restoration tree species in China, is mainly distributed in China, such as North China, northwest China, Liaoning China, Sichuan China and the like, has the advantages of drought resistance, fast growth, strong applicability, easiness in propagation and the like, is light and flexible in wood, has linear wood texture and fine and smooth structure, is suitable for economic materials such as furniture, wooden boxes and buildings, windbreak, sand fixation, greening environment and the like, and has important significance in the aspects of economic development and ecological environment. At present, researchers find that female poplars and female poplars grow adjacently, the root systems of plants grow greatly but branches are few; the root system of the male and female adjacent growth is small but has more branches. That is, the adjacent growth of male and female populus japonica has no influence on male, but is beneficial to the growth of male and female plants and enhances the growth vigor of the female plants. Therefore, if the reasonable planning of male and female seedlings can be carried out when the forestation is yes, the forestation efficiency is improved, and the method is based on the early identification of the female sex of the populus seedlings. The traditional identification method mainly aims at the detection of adult plants through morphological observation, physiological and biochemical detection, amino acid content difference detection and the like, and human factors have great influence on the detection result, so that the detection result is inaccurate. The invention researches the male and female plants of the young populus diversifolia seedlings on a molecular level and develops the rapid detection primers for the male and female plants of the young populus diversifolia seedlings. Thereby improving the breeding efficiency, shortening the breeding period, adjusting the sex mode of breeding and having great application potential and better economic value.
Disclosure of Invention
An object of the present invention is to improve the above problems by providing a specific molecular marker PCMI-M001 for rapid identification of female young plants of populus diversifolia.
The invention discloses a specific molecular marker primer for identifying the seedling sex of populus diversifolia, which is characterized by comprising the following nucleotide sequences:
the upstream primer PCMI-M001-F: 5'-TACCACTGCGGGGAAGGAAA-3', as shown in SEQ ID No: 1 is shown in the specification;
the downstream primer PCMI-M001-R: 5'-CACTGCGCAAGAAGCAGTAA-3', as shown in SEQ ID No: 2, respectively.
The specific molecular marker primer pair is characterized in that a common PCR technology is adopted, target associated target polymorphic DNA (SCoT) is utilized to mark a universal primer to amplify the DNA of a female poplar sample and a male poplar sample, the amplified products are subjected to electrophoresis, female and male single-plant specific bands are screened for sequencing, then female and male differential band sequences obtained by sequencing are compared, and a specific primer (PCMI-M001-F/R) is designed according to the sequence. The primer pair has extremely high specificity, and only male plants can obtain 626bp DNA bands while female plants do not; the nucleotide sequence of the specific fragment obtained by amplification of the male strain is shown as SEQ ID No: 3, respectively.
The invention also aims to provide a method for quickly identifying the male and female of different populus diversifolia seedlings by using the specific molecular marker primer.
The invention adopts the specific molecular marker primer (PCMI-M001-F/R) as a specific amplification primer, takes the genomic DNA of a populus diversifolia sample to be detected as a template, performs PCR amplification, performs electrophoresis detection on an amplification product, and if a DNA strip of 626bp appears in an electrophoresis result, the sample to be detected is a male individual; if a 626bp DNA band does not appear in the electrophoresis result, the sample to be detected is a female individual.
It should be noted that the specific molecular marker primer and the detection method of the invention are only suitable for the identification of male and female populus diversifolia individuals, that is, the sample to be detected is limited to the range of the populus diversifolia sample, and the method of the invention can be used by ordinary persons in the art to rapidly identify the female sex of the populus diversifolia individuals, and the method is simple, accurate and rapid, and cannot be realized by other existing identification methods.
The method comprises the following specific steps:
(1) taking 0.2-0.5g of fresh leaves of a populus sample to be detected, adding liquid nitrogen for grinding, and extracting the genomic DNA of the populus by using a plant genomic DNA extraction kit;
(2) performing common PCR amplification by taking the genomic DNA obtained in the step (1) as a template and the specific molecular marker primer (PCMI-M001-F/R) as an amplification primer:
the PCR reaction system (total volume 10. mu.l) consisted of:
Figure BDA0001712263740000021
Figure BDA0001712263740000031
the PCR reaction program is: pre-denaturation at 94 ℃ for 5 min; 32 cycles (denaturation at 94 ℃ for 30 seconds, annealing at 56 ℃ for 40 seconds, and extension at 72 ℃ for 90 seconds); final extension at 72 ℃ for 10 min.
(3) Detecting the PCR amplification product obtained in the step (2) by using 1% agarose gel electrophoresis, and carrying out imaging photographing by using a gel imaging system, wherein if a 626bp DNA band appears in an electrophoresis result, the sample to be detected is a male individual; if a 626bp DNA band does not appear in the electrophoresis result, the sample to be detected is a female individual.
The invention has the following beneficial effects: the specific molecular marker primer (PCMI-M001-F/R) can be used for quickly identifying the sex of male and female poplars by molecules, is simple, accurate and high in sensitivity, and cannot be achieved by other existing identification methods. The specific molecular marker PCMI-M001 can be used for early identification and detection of male and female sex of the populus at the seedling stage, is beneficial to developing reasonable planning of male and female seedlings during afforestation, promotes root growth of male and female populus, and further avoids the problems of uneven growth, poor disease and insect resistance, poor stress resistance and the like of the populus due to single sex; in addition, the specific molecular marker can be used for male and female sex marker assisted selection, improves the breeding efficiency, shortens the breeding period, regulates the sex mode of breeding, and has great application potential and better economic value.
Drawings
FIG. 1 is an electrophoresis diagram of female and male populus diversifolia individuals subjected to PCR amplification by using the specific molecular marker of the invention; wherein the channel L is DNA standard molecular weight Marker DL 2000; channels M1-M12: male populus (M1-M4 are from Qinghai Xining, M5-M8 are from Shandong Guangxian, and M9-M12 are from Sichuan Chengdu); passages F1 to F12: female populus (F1-F4 are from Qinghai Xining, F5-F8 are from Shandong Guangxian, and F9-F12 are from Sichuan Chengdu).
Detailed Description
The following examples are provided to further illustrate the embodiments of the present invention.
Example 1.
1. Genomic DNA extraction of populus diversifolia sample to be detected
Taking 0.2-0.5g of fresh leaves of a populus diversifolia sample to be detected, adding liquid nitrogen, grinding the leaves into powder in a mortar, extracting genomic DNA of the populus diversifolia sample to be detected by using an Ezup column type plant genomic DNA extraction kit of Shanghai bioengineering technology Limited, performing electrophoresis on the obtained DNA by using 1% agarose gel to detect purity, detecting the concentration of the DNA by using a NanoDrop TM 1000 type ultramicro ultraviolet spectrophotometer of American Saimer Feishel, diluting to 20-50 ng/mu L, and placing in a 4-degree refrigerator for later use.
2. Synthesis of specific molecular marker primers
Designing specific molecular marker primers based on male and female sex difference sequences of populus,
the upstream primer PCMI-F001-F: 5'-TACCACTGCGGGGAAGGAAA-3', respectively;
the downstream primer PCMI-F001-R: 5'-CACTGCGCAAGAAGCAGTAA-3', respectively;
synthesized by Shanghai bioengineering technology, Inc.
3. PCR amplification of specific molecular marker primers (PCMI-F001-F/R)
PCR amplification System (Total volume 10. mu.L): 10 XPCR Buffer (containing Mg)2+) mu.L of 1. mu.L, 0.3. mu.L of 10mmol/L dNTPs, 0.5. mu.L of 10. mu.mol/L upstream primer, 0.5. mu.L of 10. mu.mol/L downstream primer, 0.2. mu.L of 5U/. mu.L Taq enzyme, 1. mu.L DNA template (50 ng/. mu.L), ddH2O 6.5μL。
The PCR reaction program is: pre-denaturation at 94 ℃ for 5 min; 32 cycles (denaturation at 94 ℃ for 30 seconds, annealing at 56 ℃ for 40 seconds, and extension at 72 ℃ for 90 seconds); final extension at 72 ℃ for 10 min.
4. Electrophoretic detection
Taking 5-10 mu L of the PCR amplification product in the step (3), and carrying out electrophoresis detection by using 1% agarose gel, wherein the electrophoresis result is shown in figure 1.
According to the method, detecting male and female populus diversifolia individuals by using a specific molecular marker PCMI-M001, wherein an electrophoresis chart is shown in figure 1, male populus diversifolia individuals with the numbers of M1-M12 are amplified to form a DNA strip of 626 bp; no 626bp DNA band was amplified from any of the female populus individuals numbered F1-F12. This shows that the specific molecular marker PCMI-M001 of the invention has extremely high specificity, and therefore, can be used for the early rapid identification of male and female sex of young populus diversifolia seedlings.
Sequence listing
<110> university of teachers in Hangzhou
<120> specific molecular marker PCMI-M001 for rapid identification of male seedlings of populus tremuloides
<130> 1
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence (Unknown)
<400> 1
taccactgcg gggaaggaaa 20
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence (Unknown)
<400> 2
cactgcgcaa gaagcagtaa 20
<210> 3
<211> 626
<212> DNA
<213> Artificial sequence (Unknown)
<400> 3
taccactgcg gggaaggaaa ggagagggtc atgaatcatg attttaagct aaaacagagg 60
ggatattgtt tgaggcgagg aaaagaggga aggtggcttt gttccttctt ctctttttta 120
ggaaaaatgg tgcaggggtt tacttttggg ttctctcccc cctctttttt ttcaatagat 180
ttcaccacta tttataaggg ttgaaagaag agtatgaaat gtttatctta catgaatctt 240
gagccttgat tcaaatagaa aggatctcaa tcgttggcta aaagccacca tcataagctg 300
tgaaattggg ttgcacaggc taattgggtt agtatctttg gggcggtggc gtggtagata 360
cagaggttac caccttgtga aaatcatatt ggggcatggt acaatgttgg tccatgatag 420
tgacgcctag tttgtcctgt ttgatggaga gataaggcat taaggtagcg tttgtttagg 480
aggttgcgtc cgtgttttgt ttaaaatgca aatgcaacct gtttggtatt gaaaaaaaag 540
tagtttactg ttcatgggct atactgattt tagcgtttga aacgcaaggg aaaggaaatc 600
agctccttac tgcttcttgc gcagtg 626

Claims (5)

1. The specific molecular marker PCMI-M001 for fast identifying male young seedlings of populus diversifolia is characterized in that an amplification sequence for amplifying the molecular marker is as follows:
the upstream primer PCMI-M001-F: 5'-TACCACTGCGGGGAAGGAAA-3', as shown in SEQ ID No: 1 is shown in the specification;
the downstream primer PCMI-M001-R: 5'-CACTGCGCAAGAAGCAGTAA-3', as shown in SEQ ID No: 2, respectively.
2. The use of the specific molecular marker PCMI-M001 for the rapid identification of male seedlings of populus diversifolia as claimed in claim 1 in the identification of male and female molecules of populus diversifolia seedlings.
3. A method for identifying male and female molecules of young populus seedlings by using the specific molecular marker of claim 1, which is characterized in that genomic DNA of a single plant sample of male and female populus seedlings to be detected is extracted as a template, and PCR amplification is carried out by using the specific molecular marker PCMI-M001 of claim 1 to obtain an amplification product; carrying out electrophoresis detection on the obtained amplification product, wherein if a 626bp DNA band appears in the electrophoresis result, the sample to be detected is a male populus diversifolia seedling individual; if the electrophoresis result does not have a DNA band of 626bp, the sample to be detected is a female populus diversifolia seedling individual.
4. The identification method according to claim 3, wherein the PCR amplification system: containing Mg2+10 XPCR Buffer 1 uL, 10mmol/L dNTPs 0.3 uL, 10 umol/L upstream primer 0.5 uL, 10 umol/L downstream primer 0.5 uL, 5U/uL Taq enzyme 0.2 uL, 50 ng/uL DNA template 1 uL, ddH2O6.5. mu.L, 10. mu.L total.
5. The identification method according to claim 3, wherein the PCR reaction procedure is: pre-denaturation at 94 ℃ for 5 min; 32 cycles, each cycle comprising denaturation at 94 ℃ for 30 seconds, annealing at 57 ℃ for 40 seconds, and extension at 72 ℃ for 90 seconds; final extension at 72 ℃ for 10 min.
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