CN106191276B - Method for rapidly distinguishing columnar apple seedlings by using DNA molecular markers - Google Patents
Method for rapidly distinguishing columnar apple seedlings by using DNA molecular markers Download PDFInfo
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Abstract
The invention relates to a method for rapidly distinguishing columnar apple seedlings by using DNA molecular markers. Taking the DNA of the apple leaves to be detected as a template, taking two pairs of oligodeoxyribonucleotide sequences Co1 and Co2 as a primer pair, simultaneously carrying out PCR (polymerase chain reaction) amplification on DNA molecular markers connected and locked with the columnar genes, and carrying out electrophoresis detection on amplification products, wherein at least 1 pair of primers in the electrophoresis result of the amplification products of the 2 pairs of primers has a DNA specific strip corresponding to the DNA molecular markers, so that the corresponding apple seedlings are the columnar apples; and (3) electrophoresis results of the amplification products of the 2 pairs of primers have no corresponding DNA specific bands, and the apple is a common apple. The method can be used for conveniently, quickly and accurately distinguishing the columnar type in the 1-2-year-old apple nursery stock, greatly improving the breeding efficiency and accelerating the breeding process; meanwhile, true and false cylindrical apple variety seedlings can be simply and quickly identified, and disputes caused by false seedlings are avoided.
Description
The technical field is as follows:
the invention relates to a method for rapidly distinguishing columnar apple seedlings by using DNA molecular markers, and belongs to the technical field of molecular genetic breeding.
Background art:
the column apple (Columnar apple) has the characteristics of short internodes, a large number of short branches formed by axillary buds, little or no lateral growth and new shoot elongation, is in a natural single-stem shape, and is different from a common type, a common short branch type or a dwarfing type. The tree is compact, has extremely light pruning amount, and is very suitable for high-density planting and mechanized management. The popularization of the tree shape can greatly save labor force and production cost and meet the development requirement of labor-saving cultivation of apples. Research shows that the apple columnar character is the quality character controlled by dominant single gene (Co). Therefore, the columnar apple is an ideal genetic resource for the plant type breeding of fruit trees. However, the columnar apple and the common apple are not easy to distinguish in the seedling stage, morphological identification can only wait for the growth of the columnar apple and the common apple to a certain extent, and the columnar apple and the common apple are distinguished by depending on tree shapes and external shapes, so that the time consumption is long, and the time and the labor are wasted.
With the development of genetics and molecular biology, people recognize that the selection efficiency can be greatly improved and the blindness of the breeding process can be reduced by using genetic Markers for Auxiliary Selection (MAS). Marker breeding (Marker breeding) is a technology for tracking and selecting target traits by using genetic markers closely linked with the genes of the target traits. The method utilizes molecular markers closely linked with target character genes to carry out indirect selection, selects the target characters at the molecular level, is not influenced by the environment, and has reliable selection results; when favorable target traits are aggregated, linkage drag is reduced and breeding process is accelerated by genetic background selection in the backcross introgression process. There are four main types of genetic markers associated with breeding: morphological Markers (Morphological Markers), Cytological Markers (Cytological Markers), Biochemical Markers (Biochemical Markers) and molecular Markers (molecular Markers).
In recent years, the development of molecular biology provides a new technical means based on DNA variation, namely a molecular marker technology, for plant genetic breeding. Compared with other markers, the DNA molecular marker has the following main characteristics: firstly, the method can detect individuals, organs and even cells in various development stages, and is not limited by environment and whether gene expression is carried out or not; secondly, the number is extremely large and the whole genome is covered; thirdly, the polymorphism is high, and the analysis of the coverage genome can be completed by utilizing a large amount of primers and probes; fourthly, the expression is neutral, namely the expression of the target character is not influenced, and the target character is not necessarily connected with the undesirable character; fifthly, a plurality of markers are co-dominant, so that a homozygous genotype and a heterozygous genotype can be identified, and complete genetic information is provided.
The invention content is as follows:
the invention aims to overcome the defect that the columnar apple seedlings and the seedling stage of the columnar apple varieties are not easy to distinguish and distinguish morphologically in 1-2 years, and provides a method for quickly distinguishing the columnar apple seedlings by using a DNA molecular marker, namely the DNA molecular marker which is tightly linked with a control apple columnar gene Co and the method for detecting the molecular marker by using a PCR technology.
In order to achieve the above object, the effectiveness of the verification marker is performed on the columnar type and common type individuals and the columnar type and common type apple varieties in the segregation population of the filial generation of the columnar type apples by using the 2 pairs of primers of the invention. PCR amplification is carried out on Co1 by using a primer pair, so that a 585bp DNA specific fragment can be amplified in the columnar apple; PCR amplification is carried out by using a Co2 primer pair, and a 319bp DNA specific fragment can be amplified in the columnar apple; no band was amplified in any of the common apples by the primer pair Co1 and the primer pair Co 2. The molecular marker is used for detecting the columnar apple characters, so that 1-year-old seedlings of filial generations are purposefully selected, non-target characters are eliminated as soon as possible, the breeding efficiency is greatly improved, the accuracy of identifying the columnar apple varieties in the seedling stage can be improved by using the molecular marker, and technical guidance is provided for production and sale of columnar apple seedlings.
The molecular marker primer pairs Co1, Co2, Co1 and Co2 provided by the invention can at least amplify 1 specific band in the columnar apple and can not amplify 2 pairs of primers in the common apple, wherein the deoxyribonucleotide sequence of the primer pair Co1 is as follows:
the upstream primer Co 1-F: GATGCGAGAATTAACTAGCACAC (SEQ ID NO: 3)
The downstream primer Co 1-R: GAATTGTTGTATGCGTTTTTCC (SEQ ID NO: 4);
the primer is used for PCR amplification, only one 585bp DNA specific fragment can be amplified in the columnar apple, and the deoxyribonucleic acid sequence of the fragment is shown as the following SEQ ID NO: 1:
GATGCGAGAATTAACTAGCACACAAATTAAACCCTCTTTTTGTCAATTGTAGTATAAGTATAAGTAGGGTATCGTTCTAGGCCGGGGATTAGGAGGGCTTGCTAAAACCTCTTAAAAACATAAAAACAAAGTTAAAAATATTAAACAAGACTCAAGGACACAAAACTAGGCTAAAAACTCTAATAATTCGAAACACACTTAAAATGACTCAAAATAATAAAAACAATCAAAATAGACACTAGGAATTGAATGGACGGAAATTAAATTAAAAGACTAACAATAAAGAAAACTAACTAAATAATATAATTTAATAATGGGTGGGTGTTTGGTTTGATGAAAAGTAAATTAAACTTAATTAAATTACAGAATTGACAAAAACATAAAATTAAGGTGAAAGGATAAATGACGGACTAGCTAGAGGGTTCTTCTCCACACATGACACATATGCAACCTAAATTGATTTTCAGTTGTTCTTTCAATAAATTGTGAATCTCAATACTCCAGATTAACCGTGAACAGCACTTTTTTAATCTTCAAGTTTTCCTTAAGTTATTGAATTGGACGGAAAAACGCATACAACAATTC
the deoxyribonucleotide sequence of the primer pair Co2 is as follows:
Co2-F：TCTACTCCTCTTTTGCCTTGG(SEQ ID NO：5)；
Co2-R:ACTTCGAATTCACTCGTCTTT(SEQ ID NO：6)；
the primer is used for PCR amplification, only a 319bp DNA specific fragment can be amplified in the columnar apple, and the deoxyribonucleic acid sequence of the fragment is shown as the following SEQ ID NO: 2:
TCTACTCCTCTTTTGCCTTGGATTCTTTCCTTTCTTTGTCCAACTCTTTCTTTCCACCGTTTTCTTTCTTTTTTTTTTCTACAAAACAAAACCATCATGATGATGTTTCAAACATCATCACGACTTATCATTATTAAATATAATTTTTAATTTAATTTAAAAGCTGATCTACACAGACAGTTTTGACGAATTTATTACATAAAATTTCACTTGTTCTATTTTTATTTTCTTTGCATAACAAATCCTATAAACACAAAAATAACGTAAATAGCTCAAAAATATAAGGAACTAACTAAGAAAAGACGAGTGAATTCGAAGT
the method of the invention is operated according to the following steps:
extraction of apple genome DNA by CTAB method
1. Taking 2ml of a centrifuge tube, adding 1.2ml of CTAB (cetyl trimethyl ammonium bromide) and 50 mu L of beta-mercaptoethanol, preheating in a 65 ℃ water bath kettle, taking about 0.5g of young and tender apple leaves, adding liquid nitrogen, quickly grinding into fine powder, transferring into the centrifuge tube filled with CTAB, shaking, uniformly mixing, placing in a 65 ℃ water bath kettle, carrying out water bath for 30min, and reversing and uniformly mixing for many times;
2. taking out, cooling to room temperature, adding chloroform, slightly reversing, mixing, standing, centrifuging at 4 ℃ and 12000rpm, and taking 1000 mu L of supernate into a new 2ml centrifuge tube;
3. adding 1/2 volumes of Tris (Tris) saturated phenol, reversing and mixing, standing, adding 1/2 volumes of chloroform, reversing and mixing, standing, centrifuging at 4 ℃ and 12000rpm, and taking 800 mu L of supernatant into a new 2ml centrifuge tube;
4. adding chloroform, reversing, mixing, standing, centrifuging at 4 deg.C and 12000rpm, and collecting 600 μ L supernatant to a new 1.5ml centrifuge tube;
5. adding anhydrous ethanol of the supernatant 1/10 and 1/20 sodium acetate, standing at-20 deg.C for 30min, centrifuging at 4 deg.C and 12000rpm, and transferring the supernatant into a new 1.5ml centrifuge tube;
6. adding isopropanol with the same volume, standing at-20 deg.C for 2 hr, centrifuging at 4 deg.C and 12000rpm, removing supernatant, and keeping precipitate;
7. adding 1ml of 75% anhydrous ethanol, washing the precipitate once, washing with anhydrous ethanol once, evaporating the ethanol to dryness, and adding 50 μ L of sterile double distilled water (Distiltation-Distiltation H)2O，ddH2O), dissolving, taking 5 mu LDNA, and detecting the concentration and the quality of the DNA by using 1% agarose gel electrophoresis;
second, PCR amplification
Firstly, carrying out PCR amplification by using an apple housekeeping gene MdActin primer and taking the extracted apple DNA as a template to detect the quality of the DNA, eliminating test errors caused by the DNA, and then carrying out PCR amplification by using a primer pair Co1 and Co2 and taking the extracted apple DNA as the template, wherein the primer sequence of the used apple MdActin gene is as follows:
MdActin-F:AAGATTTGGCATCACACGTTC(SEQ ID NO：7)
MdActin-R:TGGATGGCAACATACATAGCA(SEQ ID NO：8)
the PCR reaction used a 12. mu.L system:
PCR reaction procedure:
1) 5 minutes at 94 DEG C
2)94 ℃ for 30 seconds
3) 30 seconds at 58 DEG C
4)72 ℃ for 30 seconds
Repeating 2) to 4)30 cycles
5) 10 minutes at 72 DEG C
Thirdly, carrying out electrophoresis detection on the amplification product
After the PCR reaction is finished, carrying out 1.5% agarose gel electrophoresis on the product, and checking DNA bands generated in the PCR reaction of different samples to be detected;
wherein the deoxyribonucleotide sequence of the primer pair Co1 is as follows:
the upstream primer Co 1-F: GATGCGAGAATTAACTAGCACAC (SEQ ID NO: 3)
The downstream primer Co 1-R: GAATTGTTGTATGCGTTTTTCC (SEQ ID NO: 4)
The primer is used for PCR amplification, only one 585bp DNA specific fragment can be amplified in the columnar apple, and the deoxyribonucleic acid sequence of the fragment is shown as the following SEQ ID NO: 1:
GATGCGAGAATTAACTAGCACACAAATTAAACCCTCTTTTTGTCAATTGTAGTATAAGTATAAGTAGGGTATCGTTCTAGGCCGGGGATTAGGAGGGCTTGCTAAAACCTCTTAAAAACATAAAAACAAAGTTAAAAATATTAAACAAGACTCAAGGACACAAAACTAGGCTAAAAACTCTAATAATTCGAAACACACTTAAAATGACTCAAAATAATAAAAACAATCAAAATAGACACTAGGAATTGAATGGACGGAAATTAAATTAAAAGACTAACAATAAAGAAAACTAACTAAATAATATAATTTAATAATGGGTGGGTGTTTGGTTTGATGAAAAGTAAATTAAACTTAATTAAATTACAGAATTGACAAAAACATAAAATTAAGGTGAAAGGATAAATGACGGACTAGCTAGAGGGTTCTTCTCCACACATGACACATATGCAACCTAAATTGATTTTCAGTTGTTCTTTCAATAAATTGTGAATCTCAATACTCCAGATTAACCGTGAACAGCACTTTTTTAATCTTCAAGTTTTCCTTAAGTTATTGAATTGGACGGAAAAACGCATACAACAATTC
the deoxyribonucleotide sequence of the primer pair Co2 is as follows:
Co2-F：TCTACTCCTCTTTTGCCTTGG(SEQ ID NO：5)
Co2-R:ACTTCGAATTCACTCGTCTTT(SEQ ID NO：6)
the primer is used for PCR amplification, only a 319bp DNA specific fragment can be amplified in the columnar apple, and the deoxyribonucleic acid sequence of the fragment is shown as the following SEQ ID NO: 2:
TCTACTCCTCTTTTGCCTTGGATTCTTTCCTTTCTTTGTCCAACTCTTTCTTTCCACCGTTTTCTTTCTTTTTTTTTTCTACAAAACAAAACCATCATGATGATGTTTCAAACATCATCACGACTTATCATTATTAAATATAATTTTTAATTTAATTTAAAAGCTGATCTACACAGACAGTTTTGACGAATTTATTACATAAAATTTCACTTGTTCTATTTTTATTTTCTTTGCATAACAAATCCTATAAACACAAAAATAACGTAAATAGCTCAAAAATATAAGGAACTAACTAAGAAAAGACGAGTGAATTCGAAGT
the PCR results show that: the primer pair Co1 and Co2 amplified DNA specific bands in column apples, but not in general apples.
The method comprises the steps of taking the DNA of apple leaves to be detected as a template, taking two pairs of oligodeoxyribonucleotide sequences Co1 and Co2 as a primer pair, carrying out PCR (polymerase chain reaction) amplification on DNA molecular markers linked with a columnar gene, and carrying out electrophoresis detection on an amplification product, wherein at least 1 pair of primers in the electrophoresis result of the amplification product of 2 pairs of primers has a DNA specific strip corresponding to the DNA molecular markers, so that the corresponding apple seedlings are columnar apples; and (3) electrophoresis results of the amplification products of the 2 pairs of primers have no corresponding DNA specific bands, and the apple is a common apple.
The identification rate of the columnar apple material by the method can reach 88 percent; the identification rate of common apple materials reaches 100 percent.
By using the DNA molecular marker, early auxiliary selection can be effectively carried out in the seedling stage, and non-target individuals can be eliminated as early as possible. According to the method, the apple leaves are taken as test materials, and molecular marker primers disclosed by the invention can be used for carrying out molecular marker-assisted selection on hybrid offspring apple seedlings in a seedling stage, so that the defect that the morphology is difficult to identify in the seedling stage is avoided, the breeding efficiency can be greatly improved, and the breeding process is accelerated. Meanwhile, the columnar type and the common apple seedlings can be distinguished simply and quickly, the columnar type in the 1-2-year-old apple seedlings can be distinguished conveniently, quickly and accurately, and disputes caused by false seedlings are avoided.
Description of the drawings:
FIG. 1 is a characteristic diagram of bands of amplification products of 56 samples using MdActin primer pairs;
FIG. 2 is a characteristic diagram of bands of amplification products of the Co1 and Co2 primer pairs in 25 columnar apples;
FIG. 3 is a characteristic diagram of bands of amplification products of the Co1 and Co2 primer pairs in 31 common apples.
The specific implementation mode is as follows:
the method of the invention is further illustrated with reference to the accompanying drawings and specific examples.
Examples 1,
In the present example, 25 columnar apple types and 31 general apple types stored in the national agricultural science and technology demonstration garden of the Qingdao university of agriculture are used as test materials, and the 25 columnar types include 23 columnar hybrid individuals of a population of 'Fuji' apple and columnar apple hybrid F1 generation, 2 columnar apple varieties 'Lujia No. 4' and 'Weisaike Xue'; the 31 common apple types included 29 common hybrid individuals of the 'Fuji' apple and columnar apple cross F1 generation population, 2 common varieties 'Fuji' and 'Xue'.
The specific operation steps are as follows:
extraction of apple genome DNA by CTAB method
1. Adding 1.2ml of CTAB and 50 mu L of beta-mercaptoethanol into a 2ml centrifuge tube, preheating in a 65 ℃ water bath kettle, adding liquid nitrogen into about 0.5g of young and tender apple leaves, quickly grinding into fine powder, transferring into the centrifuge tube filled with CTAB, shaking, mixing uniformly, placing in a 65 ℃ water bath kettle, carrying out water bath for 30min, and reversing and mixing uniformly for many times;
2. taking out, cooling to room temperature, adding 700 μ L chloroform, slightly reversing, mixing, standing for 5min, centrifuging at 4 deg.C and 12000rpm for 15min, and taking 1000 μ L supernatant into a new 2ml centrifuge tube;
3. adding 1/2 volumes of Tris saturated phenol into the supernatant, reversing and mixing the mixture evenly, standing the mixture for 5min, adding 1/2 volumes of chloroform into the supernatant, reversing and mixing the mixture evenly, standing the mixture for 5min, centrifuging the mixture for 15min at 4 ℃ and 12000rpm, and taking 800 mu L of the supernatant into a new 2ml centrifuge tube;
4. adding 800 μ L chloroform, mixing, standing for 5min, centrifuging at 4 deg.C and 12000rpm for 15min, and collecting 600 μ L supernatant to a new 1.5ml centrifuge tube;
5. adding anhydrous ethanol of the supernatant 1/10 and 1/20 sodium acetate, standing at-20 deg.C for 30min, centrifuging at 4 deg.C and 12000rpm for 15min, and transferring the supernatant into a new 1.5ml centrifuge tube;
6. adding isopropanol with the same volume, standing at-20 deg.C for 2 hr, centrifuging at 4 deg.C and 12000rpm for 15min, removing supernatant, and retaining precipitate;
7. adding 1ml of 75% absolute ethanol, washing the precipitate once, washing with absolute ethanol once, after the ethanol is evaporated, adding 50 μ L of sterile ddH2And dissolving the O. Taking 5 mu LDNA, and detecting the concentration and the quality of the DNA by using 1% agarose gel electrophoresis;
secondly, PCR amplification:
firstly, carrying out PCR amplification by using an apple housekeeping gene MdActin primer and taking the extracted apple DNA as a template to detect the quality of the DNA, eliminating test errors caused by the DNA, and then carrying out PCR amplification by using a primer pair Co1 and Co2 and taking the extracted apple DNA as the template, wherein the primer sequence of the used apple MdActin gene is as follows:
MdActin-F:AAGATTTGGCATCACACGTTC(SEQ ID NO：7)
MdActin-R:TGGATGGCAACATACATAGCA(SEQ ID NO：8)
the PCR reaction used a 12. mu.L system:
PCR reaction procedure:
1) 5 minutes at 94 DEG C
2)94 ℃ for 30 seconds
3) 30 seconds at 58 DEG C
4)72 ℃ for 30 seconds
Repeating 2) to 4)30 cycles
5) 10 minutes at 72 DEG C
Thirdly, carrying out electrophoresis detection on the amplification product
After the PCR reaction is finished, the product is subjected to 1.5% agarose gel electrophoresis, and DNA bands generated in the PCR reaction of different samples to be detected are checked.
Wherein the deoxyribonucleotide sequence of the primer pair Co1 is as follows:
the upstream primer Co 1-F: GATGCGAGAATTAACTAGCACAC (SEQ ID NO: 3)
The downstream primer Co 1-R: GAATTGTTGTATGCGTTTTTCC (SEQ ID NO: 4)
The primer is used for PCR amplification, only one 585bp DNA specific fragment can be amplified in the columnar apple, and the deoxyribonucleic acid sequence of the fragment is shown as the following SEQ ID NO: 1:
GATGCGAGAATTAACTAGCACACAAATTAAACCCTCTTTTTGTCAATTGTAGTATAAGTATAAGTAGGGTATCGTTCTAGGCCGGGGATTAGGAGGGCTTGCTAAAACCTCTTAAAAACATAAAAACAAAGTTAAAAATATTAAACAAGACTCAAGGACACAAAACTAGGCTAAAAACTCTAATAATTCGAAACACACTTAAAATGACTCAAAATAATAAAAACAATCAAAATAGACACTAGGAATTGAATGGACGGAAATTAAATTAAAAGACTAACAATAAAGAAAACTAACTAAATAATATAATTTAATAATGGGTGGGTGTTTGGTTTGATGAAAAGTAAATTAAACTTAATTAAATTACAGAATTGACAAAAACATAAAATTAAGGTGAAAGGATAAATGACGGACTAGCTAGAGGGTTCTTCTCCACACATGACACATATGCAACCTAAATTGATTTTCAGTTGTTCTTTCAATAAATTGTGAATCTCAATACTCCAGATTAACCGTGAACAGCACTTTTTTAATCTTCAAGTTTTCCTTAAGTTATTGAATTGGACGGAAAAACGCATACAACAATTC
the deoxyribonucleotide sequence of the primer pair Co2 is as follows:
Co2-F：TCTACTCCTCTTTTGCCTTGG(SEQ ID NO：5)
Co2-R:ACTTCGAATTCACTCGTCTTT(SEQ ID NO：6)
the primer is used for PCR amplification, only a 319bp DNA specific fragment can be amplified in the columnar apple, and the deoxyribonucleic acid sequence of the fragment is shown as the following SEQ ID NO: 2:
TCTACTCCTCTTTTGCCTTGGATTCTTTCCTTTCTTTGTCCAACTCTTTCTTTCCACCGTTTTCTTTCTTTTTTTTTTCTACAAAACAAAACCATCATGATGATGTTTCAAACATCATCACGACTTATCATTATTAAATATAATTTTTAATTTAATTTAAAAGCTGATCTACACAGACAGTTTTGACGAATTTATTACATAAAATTTCACTTGTTCTATTTTTATTTTCTTTGCATAACAAATCCTATAAACACAAAAATAACGTAAATAGCTCAAAAATATAAGGAACTAACTAAGAAAAGACGAGTGAATTCGAAGT
according to the method, firstly, 56 test materials are subjected to PCR amplification by using the apple housekeeping gene MdActin primer, and the result shows that all the test materials can amplify target bands, so that the extracted DNA can be used for subsequent experiments, as shown in figure 1, M: DNA marker; 1-23: hybrid F1 generation cygon apple; 24: column apple 'Lujia No. 4', 25 column apple 'Weisaike' and Xue; common apples in the 26-54 hybrid F1 generation; 55, common apple 'Fuji'; 56 common apple 'Xu'; then, 56 samples were subjected to PCR amplification using the primer pairs Co1 and Co 2. If the lane of the DNA to be detected has a DNA specificity band of 585bp of a Co1 primer pair or 319bp of a Co2 primer pair, the sample can be judged to belong to a columnar apple, and if the lane of the DNA to be detected does not have the DNA specificity bands corresponding to the Co1 and Co2 primer pairs, the sample can be judged to belong to a common apple. The results showed that only 3 of the 23 columnar hybrids F1 generated material failed to amplify the corresponding specific bands using the Co1 and Co2 primer pairs, as shown in fig. 2, where M: DNA marker; 1-23: hybrid F1 generation cygon apple; 24: column apple 'Lujia No. 4', 25 column apple 'Weisaike' and Xue; a is an amplification product band of a primer pair Co 1; b is the band of the amplification product of the primer pair Co 2. None of the 29 common hybrid generations F1 was able to amplify the corresponding specific bands using the Co1 and Co2 primer pairs, as shown in fig. 3, where M: DNA marker; 1-29: common apples in hybrid F1 generation; 30: common apple 'fuji'; 31: apple of the general type 'Xu'.
In this example, the pillar apple varieties ` Weisaike `, ` Lujia IV `, and the general apple varieties ` Xu ` and ` Fuji `werealso tested, and the PCR results showed that: DNA-specific bands were amplified in both column apple ` Welcack `and ` Lujia IV ` with the Co1 and Co2 primer pairs, as shown in FIG. 2; however, DNA-specific bands were not amplified in the general apple types ` Asahi ` and ` Fuji ` as shown in FIG. 3.
The method can distinguish 22 column-shaped apple materials with the identification rate of 88 percent; 31 common apple materials can be distinguished, and the distinguishing rate reaches 100%.
Sequence listing
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<213> Artificial Sequence (Artificial Sequence)
<400> 8
tggatggcaa catacatagc a 21
Claims (1)
1. A method for rapidly distinguishing columnar apple seedlings by using DNA molecular markers is characterized by comprising the following steps:
firstly, extracting apple genome DNA by a CTAB method: adding 1.2ml of Cetyl Trimethyl Ammonium Bromide (CTAB) and 50 mu L of beta-mercaptoethanol into a 2ml centrifuge tube, preheating the centrifuge tube in a 65 ℃ water bath kettle, adding liquid nitrogen into about 0.5g of young and tender apple leaves, quickly grinding the apple leaves into fine powder, transferring the apple leaves into the centrifuge tube filled with the CTAB, uniformly mixing the apple leaves by shaking, putting the apple leaves into the 65 ℃ water bath kettle in a water bath for 30min, and reversely and uniformly mixing the apple leaves; taking out, cooling to room temperature, adding chloroform, slightly reversing, mixing, standing, centrifuging at 4 ℃ and 12000rpm, and taking 1000 mu L of supernate into a new 2ml centrifuge tube; adding 1/2 volumes of trihydroxymethyl aminomethane saturated phenol into the supernatant, reversing and mixing the mixture evenly, standing the mixture, adding 1/2 volumes of chloroform into the supernatant, reversing and mixing the mixture evenly, standing the mixture, centrifuging the mixture at 4 ℃ and 12000rpm, and taking 800 mu L of supernatant into a new 2ml centrifuge tube; adding chloroform, reversing, mixing, standing, centrifuging at 4 deg.C and 12000rpm, and collecting 600 μ L supernatant to a new 1.5ml centrifuge tube; adding anhydrous ethanol of the supernatant 1/10 and 1/20 sodium acetate, standing at-20 deg.C for 30min, centrifuging at 4 deg.C and 12000rpm, and transferring the supernatant into a new 1.5ml centrifuge tube; adding isopropanol with the same volume, standing at-20 deg.C for 2 hr, centrifuging at 4 deg.C and 12000rpm, removing supernatant, and keeping precipitate; adding 1ml of 75% absolute ethyl alcohol to wash the precipitate once, and then using the absolute ethyl alcohol to cleanWashing once, after the alcohol is evaporated to dryness, adding 50 μ L sterile double distilled water ddH2Dissolving O, taking 5 mu LDNA, and detecting the concentration and the quality of the DNA by using 1% agarose gel electrophoresis;
step two, PCR amplification: firstly, carrying out PCR amplification by using an apple housekeeping gene MdActin primer and taking the extracted apple DNA as a template to detect the quality of the DNA, eliminating test errors caused by the DNA, and then carrying out PCR amplification by using a primer pair Co1 and Co2 and taking the extracted apple DNA as the template, wherein the primer sequence of the used apple MdActin gene is as follows:
MdActin-F:AAGATTTGGCATCACACGTTC，
MdActin-R:TGGATGGCAACATACATAGCA；
the PCR reaction used a 12. mu.L system:
PCR reaction procedure:
thirdly, carrying out electrophoresis detection on the amplification product: after the PCR reaction is finished, carrying out 1.5% agarose gel electrophoresis on the product, and checking DNA bands generated in the PCR reaction of different samples to be detected; wherein the deoxyribonucleotide sequence of the primer pair Co1 is as follows:
the upstream primer Co 1-F: GATGCGAGAATTAACTAGCACAC the flow of the air in the air conditioner,
the downstream primer Co 1-R: GAATTGTTGTATGCGTTTTTCC, respectively;
the primer is used for PCR amplification, only one 585bp DNA specific fragment can be amplified in the columnar apple, and the deoxyribonucleic acid sequence of the fragment is shown as SEQ ID NO. 1 in a sequence table;
the deoxyribonucleotide sequence of the primer pair Co2 is as follows:
Co2-F：TCTACTCCTCTTTTGCCTTGG，
Co2-R:ACTTCGAATTCACTCGTCTTT；
the primer is used for PCR amplification, only a 319bp DNA specific fragment can be amplified in the columnar apple, and the deoxyribonucleic acid sequence of the fragment is shown as SEQ ID NO. 2 in a sequence table; wherein, at least 1 pair of primers in the electrophoresis result of the amplification products of the 2 pairs of primers has DNA specific strips corresponding to the DNA molecular markers, and the corresponding apple seedlings are columnar apples; the electrophoresis results of the amplification products of the 2 pairs of primers have no corresponding DNA specific bands, and the apple is a common apple; the identification rate of the columnar apple material reaches 88 percent; the identification rate of common apple materials reaches 100 percent.
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| CN104531844A (en) * | 2014-12-05 | 2015-04-22 | 中国农业科学院郑州果树研究所 | SSR genotype-based fruit tree variety distinguishing and characteristic fingerprint displaying method |
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| CN104531844A (en) * | 2014-12-05 | 2015-04-22 | 中国农业科学院郑州果树研究所 | SSR genotype-based fruit tree variety distinguishing and characteristic fingerprint displaying method |
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