CN114317809A - Molecular marker associated with apple tree branch angle related gene and application thereof - Google Patents
Molecular marker associated with apple tree branch angle related gene and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of molecular genetic breeding, in particular to a molecular marker associated with genes related to apple tree branch angles and application thereof. In the invention, an InDel site is screened from an upstream promoter region of a gene of an apple No. 7 chromosome, the InDel site has the same sequence in varieties of small (less than or equal to 30 ℃) and medium (30-60 ℃) tree types of apples, and 6bp deletion exists in varieties of large (more than or equal to 60 ℃) tree types of branches. A pair of specific primers is designed in 370bp upstream and downstream of the InDel locus. The PCR amplification product is detected by polyacrylamide gel electrophoresis to obtain polymorphic fragments for distinguishing the sizes of apple branch angles, and a gene specific InDel molecular marker is developed. The molecular marker and the primer thereof can be used for early screening of the apple branch angle character, and provide an effective molecular marker for apple tree breeding.
Description
Technical Field
The invention relates to the technical field of molecular genetic breeding, in particular to a molecular marker associated with genes related to apple tree branch angles and application thereof.
Background
Apple (Malus x domestica Borkh.) is an important economic fruit tree worldwide, and is the third place for fruit production. China is a big apple producing country, and the cultivation area and the yield both account for more than 50% of the world area and the yield. However, the apple industry development in China is restricted by factors such as low yield per unit area of apples, low grade rate of high-quality fruits, high orchard management cost and the like (Huaishui and Chenside 2018; Chengkong and Deying Zhao 2019). The tree architecture of fruit trees not only affects the planting density, leaf curtain and flowering and fruiting capacity, but also affects the production cost investment such as field management (Hill & Holllender 2019). The breeding of new species with both 'ideal tree type and good fruit quality' is the fundamental solution to the production and cultivation problems. The natural growth tree form of the fruit tree is determined by the branch characteristics of the tree body and is controlled by genetic genes. And (3) excavating branch related genes and molecular markers, and providing theoretical basis and technical support for creating an ideal tree type which is high in production efficiency and suitable for mechanized management.
The plant branch system comprises a main branch and a side branch, and the branch angle (the angle between the side branch and a sprouting branch) of the plant comprises a basal angle (Crotch angle), an Equilibrium angle (Equilibrium angle) or an Inclination angle (Inclination angle) between a main branch and a branch growing direction or a gravity set angle (Geotropic set point angle) and a first terrestrial angle (Geotropic angle) of actively growing side branches. The apple branches (stems) have the characteristic of growing vertically and upwards, the branches with small branch angles have strong vertical advantages, and the crowns grow inwards in an embracing manner, so that the crowns are closed, blossoms and fruits are poor. The opening angle changes the growth direction of branches, moderates the growth vigor, and can promote flowering and fruiting (luzhenzhen et al 2018, Costes et al 2010). In the Chinese fruit tree apple roll, apple varieties can be divided into erect (less than 40 degrees), half-open (40 degrees to 65 degrees), open (66 degrees to 85 degrees) and extreme open or droop (more than 85 degrees) according to branch angles according to tree postures (Jiadingxian et al, 1999). According to the observation of apple perennial fruit trees, the branch angle of the columnar offspring such as 'Wesseck' is 29.22 ° (± 6.52SE), the branch angle of the common variety such as 'golden crown' is 47.29 ° (± 6.81SE), and the branch angle with the weeping character such as Australian green apple is 76.56 ° (± 7.73 SE). In summary, the patent defines apple trees as small (less than or equal to 30 degrees), medium (30-60 degrees) and large (more than or equal to 60 degrees) branch angles.
Tiller Angle Control1(TAC1) is a key gene for regulating the branching Angle of plants, and is firstly cloned (Yu et al, 2007) in cultivated rice, and the gene is identified in arabidopsis thaliana (Jin et al, 2008), corn (Ku et al (2011)), peach (Dardick et al, 2013) and apple (Wang et al, 2018). In the early stage of the subject group, representative varieties of four typical tree types of apple, namely 'Weisaic', 'Xu', 'Fudao braches' and 'Australian green apple', are taken as test materials, and MdTAC1a and MdTAC1b are cloned from apple genome by using a homologous cloning method and are respectively positioned at chr1 and 7. These varieties have only SNP (single nucleotide polymorphism) differences in the CDS region, SNP and InDel (insertion-deletion) in the promoter (2.0kb) sequence, resulting in differences in gene expression among different varieties (Wang et al 2018).
At present, no molecular marker for apple branch angle breeding is reported, and the production screening efficiency can be improved and the breeding process can be accelerated by utilizing the marker for auxiliary selection.
Disclosure of Invention
In view of the above, the invention provides molecular markers associated with genes related to apple tree branch angles and application thereof, and by identifying apples by using the molecular markers, the coincidence rate of polypropylene gel electrophoresis results and expected genotypes is 95.29%.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of InDel molecular markers in identifying, distinguishing and/or predicting apple branch angles. The InDel molecular marker is positioned on a promoter of MdTAC1a gene of chromosome 7 of an apple genome, and has a sequence of GAGAGA.
In some embodiments of the invention, the criteria for identifying, differentiating and/or predicting apple branch angle are:
(ii) if a GAGAGA sequence is present or a 370bp band is present, the apple branch angle is < 30 °, or 30 ° < the apple branch angle is < 60 °;
if the GAGAGAGA sequence is deleted or a 364bp band appears, the apple branch angle is more than or equal to 60 degrees.
The invention also provides a primer group for amplifying the InDel molecular marker.
In some embodiments of the invention, the primer set comprises:
(I) the forward primer has a nucleotide sequence shown as SEQ ID No. 2; and/or
(II) a reverse primer which has a nucleotide sequence shown as SEQ ID No.3 or SEQ ID No. 3; and/or
(III) has a nucleotide sequence obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in (I) or (II), and has the same or similar functions with the nucleotide sequence shown in (I) or (II); and/or
(IV) a nucleotide sequence having at least 80% homology with any of the nucleotide sequences shown in (I) to (III);
the number of the plurality is 2 to 10.
In some embodiments of the invention, the nucleotide sequence of a primer set provided by the invention has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the nucleotide sequence of the primer set.
In some embodiments of the invention, the modification to the nucleotide sequence is a fold amplification.
In some embodiments of the invention, the substitution of the nucleotide sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9 and/or 10 bases.
In some embodiments of the invention, the deletion to the nucleotide sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10 bases.
In some embodiments of the invention, the addition to the nucleotide sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9 and/or 10 bases.
The invention also provides a kit, which comprises the primer group and acceptable auxiliary materials and/or auxiliary agents.
In some embodiments of the invention, the adjuvant or adjuvant comprises 2 × Green Taq Mix, DMSO, TMAC, SSB, formamide, trehalose, betaine, non-ionic detergents, dNTPs, UDG enzyme, Tth enzyme and/or MgCl2。
The invention also provides application of the primer group or the kit in identifying, distinguishing and/or predicting the branching angle of apples and/or selectively breeding.
The invention also provides a method for identifying, distinguishing and/or predicting the apple branch angle, wherein the standard for identifying, distinguishing and/or predicting the apple branch angle is as follows:
(ii) if a GAGAGA sequence is present or a 370bp band is present, the apple branch angle is < 30 °, or 30 ° < the apple branch angle is < 60 °;
if the GAGAGAGA sequence is deleted or a 364bp band appears, the apple branch angle is more than or equal to 60 degrees.
In some embodiments of the invention, the method comprises the steps of:
step (a), primer design: designing and screening to obtain a primer group;
step (b), DNA extraction: extracting total DNA of the apples to be detected;
step (c), PCR amplification: performing PCR amplification by using the total DNA obtained in the step (b) as a template and the primer group obtained in the step (a) to obtain a PCR product;
step (d), electropherogram analysis: and (3) carrying out electrophoresis on the PCR product, and determining the genotype according to the size and the position relation of the bands.
In some embodiments of the invention, the methods further comprise bioinformatics methods and/or molecular biology methods;
the bioinformatics method includes:
obtaining a sequence on a promoter of MdTAC1a gene of chromosome 7 of the apple genome to be detected, and judging whether a GAGAGA sequence exists according to the sequence so as to conjecture the branching angle of the apple to be detected;
the molecular biological method comprises the following steps:
obtaining nucleic acid with a sequence on a promoter of MdTAC1a gene of chromosome 7 of the apple genome to be detected, and indirectly judging whether the sequence has a GAGAGA sequence through nucleic acid amplification, probe combination or electrophoresis so as to presume the branch angle of the apple to be detected.
In some embodiments of the invention, the concentration of any primer in the primer set in step (c) of the method is no less than 10. mu. mol/L;
the PCR amplification reaction system in the step (c) comprises 2 muL of 100 ng/muL of apple total DNA, 1 muL of forward primer, 1 muL of reverse primer and 2 XGreen Taq Mix 10 mu L, ddH2O 6μL；
The reaction procedure is as follows:
the invention also provides a method for selective breeding, which comprises the step of selectively breeding apples by using the method.
The invention has the following effects:
1. the developed InDel molecular marker can identify, distinguish and/or predict the branching angle of the apples. The InDel molecular marker keeps consistent in the trees with small branching angles (less than or equal to 30 degrees) and medium branching angles (30-60 degrees), and the trees with large branching angles (more than or equal to 60 degrees) have 6bp deletion, and the deletion sequence is GAGAGA.
2. The invention establishes a method for identifying, distinguishing and/or predicting the branch angle of the apple. The method comprises the steps of verifying 23 parts of common apple cultivars and 62 parts of golden bud No.1 and Australian green apple F1 generation hybrid (total 85 parts), detecting PCR amplification products by utilizing polyacrylamide gel electrophoresis, analyzing obtained polymorphic fragments for distinguishing the branch angles of the apples, and determining that the coincidence rate of the result and the expected genotype is 95.29%.
Drawings
In order 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 below.
FIG. 1(A) shows a large branch angle tree type in a parent;
FIG. 1(B) shows a small branch angle tree type in a parent;
FIG. 1(C) shows the branch angle statistics for different varieties;
FIG. 2 shows an InDel site information diagram;
FIG. 3 is a graph showing the result of polyacrylamide gel electrophoresis of the PCR product of the variety; m represents DNA ladder marker; 1 represents mid-autumn king; 2 represents a red star; 3 represents love; 4 represents glow; 5 represents ruby; 6 represents a large wonderful look; 7 represents Venus; 8 represents topaz; 9 represents Qinsiui; 10 represents a rubbing season; 11 represents a dew; 12 represents meisi; 13 represents wish; 14 represents jin red; 15 represents daphne red; 16 represents jinxianghong; 17 represents Huajia; 18 represents wary; 19 represents smoke enriched 10; 20 represents smoke rich 6; 21 represents smoke rich 8;
FIG. 4 shows different branch angles in generation F1; wherein A represents a tree type of medium and small branch angles in F1 generation; b represents a tree type of medium and large branch angles in the F1 generation; c, branch angle statistics; d shows the result of polyacrylamide gel electrophoresis of the PCR product; m represents DNA ladder marker; 1 to 29 in turn represent generations F1-1 to F1-29; 30 represents the parent macadimia green apple; 31 represents parental golden bud No. 1; 32 to 64 in turn represent generations F1-30 to F1-62;
FIG. 5 is a graph showing the sequencing result of sanger.
Detailed Description
The invention discloses a molecular marker associated with a gene related to the branching angle of an apple tree and application thereof, and a person skilled in the art can appropriately improve 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 term "apple" as used herein is intended to mean at the species level, and may refer to a single cell or a multi-cell tissue of the apple species, and may also refer to a root, stem, leaf, flower, fruit or seed of the apple species.
According to the invention, the region related to the branch angle of the fruit tree is detected on chromosome 7 by utilizing the PCR amplification and Sanger sequencing technology, the InDel molecular marker is developed and verified by combining the sequencing information of different branch angle varieties, the primers are designed, and the application is carried out in the hybridization F1 generation, so that a new means can be provided for identifying the characteristics of different branch angles, the improvement process of the characteristic character of the branch angle of the apple is accelerated, and the breeding accuracy and the selection efficiency are improved. The design of the InDel marker as a molecular marker which is closely linked with different branch angles can be realized by utilizing the insertion deletion nucleic acid site, and the InDel marker and the SSR marker have the advantages of stable variation, strong polymorphism and low cost.
Specifically, Tiller Angle Control1a (TAC1a) is a member of the IGT family, expressed mainly in axillary bud tissue. The invention screens an InDel locus on the promoter of MdTAC1a gene of apple genome No. 7 chromosome. At the InDel site, the trees with small branching angle (less than or equal to 30 degrees) and medium branching angle (30-60 degrees) are consistent, while the trees with large branching angle (more than or equal to 60 degrees) have 6bp deletion, and the deletion sequence is GAGAGA. A pair of labeled primers designed in 370bp upstream and downstream of InDel provides a basis for identification and auxiliary molecular breeding of different branch angles. Further, an InDel molecular marker is developed, 23 parts of common apple cultivars and 62 parts of golden bud No.1 and Australian green apple F1 generation hybrid (total 85 parts) are verified, and the molecular marker can classify different branch angles. The molecular marker provided by the invention can be used for breeding apple branch angles, and the production screening efficiency can be improved and the breeding process can be accelerated by utilizing the marker for auxiliary selection.
Unless otherwise specified, the reagents and other materials used in the present invention can be synthesized and purchased by reagent companies, and different apple varieties and hybrid F1 generations are planted at Shanzhuang laboratory sites of Chinese agricultural university.
The invention is further illustrated by the following examples:
example 1: site identification and development of InDel molecular marker
1. Material treatment
In the present example, 23 parts of common apple cultivars and 62 parts of golden bud No.1 and Australian green apple F1 hybrid (total 85 parts) were identified. The 85 materials were planted at the Shang test base of Chinese agricultural university. The branch angle statistics (see fig. 1, fig. 3, table 1, table 2), leaf tissue sampling, and the samples were rapidly stored at-80 ℃ during the growth phase.
TABLE 1 Branch Angle statistics
| Variety of (IV) C | Mean angle (°) | Error of label |
| Mid-autumn king | 54.00 | 5.83 |
| Red star | 40.33 | 6.45 |
| Love concubine | 41.00 | 6.88 |
| Glorious | 77.00 | 3.56 |
| Hongyu incense | 58.00 | 3.56 |
| Large and luxury | 39.67 | 5.31 |
| Venus | 50.33 | 4.64 |
| Huang Yuxiang | 70.00 | 4.08 |
| Qin crisp | 73.33 | 5.68 |
| Tuo season | 53.67 | 5.31 |
| Dew bead | 84.73 | 8.19 |
| Meisi food | 76.33 | 3.86 |
| Hope for | 51.33 | 4.64 |
| Jinzhou red wine | 50.33 | 4.64 |
| Daixianhong tea | 38.00 | 4.40 |
| Jinxianghong tea | 49.00 | 3.74 |
| Hua Jia | 75.33 | 4.27 |
| Huarui brand | 78.00 | 3.56 |
| Rich in tobacco | 74.33 | 4.42 |
| Cigarette riches six | 74.67 | 4.64 |
| Smoke rich eight | 70.87 | 3.90 |
TABLE 2 Branch Angle statistics
| Variety of (IV) C | Gold bud No.1 | Australia green apple | F1 generation small angle | Large angle of generation F1 |
| Mean angle (°) | 32.69 | 88.08 | 35.77 | 87.69 |
| Standard error of | 2.49 | 3.12 | 4.74 | 4.65 |
The CTAB method is adopted to extract the whole genome DNA of each material leaf. The specific method comprises the following steps:
1) put 1g of leaf into mortar and grind fully, move to 2mL precooled centrifuge tube rapidly, add 1mL 2% CTAB, mix fully.
2) Water bath at 65 deg.C for 1h (shaking once every 15 min); centrifuge at 12000rpm for 5 min.
3) The supernatant was taken into a new 2mL centrifuge tube, and an equal volume of chloroform-isoamyl alcohol (volume ratio 24: 1) mix by gentle inversion for 5min, and let stand for 5 min.
4) Centrifuge at 12000rpm for 10min, and take the supernatant in a new tube.
5) Repeating the steps 3 and 4 once.
6) 2 times volume of precooled absolute ethyl alcohol is added into the supernatant to precipitate DNA, and the DNA is placed at the temperature of minus 20 ℃ for 2 hours.
7) Centrifuge at 12000rpm for 10min, and pour off the supernatant.
8) Adding 500 μ L75% ethanol, washing for 2 times, and removing supernatant; and (5) drying at room temperature.
9) After air drying, 100. mu.L of RNase was added: ddH2The mixture of O (1: 100, V: V) dissolves DNA.
DNA sample concentration, quality and integrity were determined by 1% agarose gel electrophoresis and Thermo's NanoDrop 1000.
2. Development of InDel molecular marker
As shown in FIG. 2, an InDel site was selected from the promoter of MdTAC1a gene of chromosome 7 of apple genome. At the InDel site, the branch angles are small (less than or equal to 30 degrees) and medium (30-60 degrees) in the tree type and are consistent, and a deletion of 6bp exists in a large branch angle (60-90 degrees), and the deletion sequence is GAGAGA.
The promoter 2000bp sequence (SEQ ID No.1) is as follows:
TCTCCCTTTCATTTTGTCTTTTTCCTTTTTTTTTTAAATTAATAAAAAGTTGCTAGTTTGATTTTTCATTTTATTATTGATTAACTTTTGCCAAGCTGTTGGGCATGAGCCCTTGGTACCTTGGAGAATAGCAGGAATTGAGGACACACGTTCCCATTATATTGTGCAATGTATTAACCTTTTTGGAGAAATTTTATTTGATGTGATTGAAGTTTTTGCGAATTCTCCACCGAAAGGCAAAACAGTTAAAAACTTTTGTCACATAGATAGCTATATGTTTTGTCAGTGATTCAATATTTTGTGTTTAGATTAGTTGGTTTTTCCAATAAAAAAAGATTAGTTGGTTTTTTACTATTAGTTTTGATAATCGGTTGTATACTCATATTCAAACGTAAAGTTATAGTATTTGGCTCAAGACATATATGTTAATTTGTGGTGAGTTTAGTGTTTTCATGCAATATTCAAAACACAAAATAGGTAATGAAACCCCTCTAGTTAGTCTTTTGTTCAATTAATTTCGAATTGAATCATCACATTCTAATCGTCTTTTAAACTAATTACATAGAAAAATTATTGTGAGATGAAACCATGAATCATGTATGTACATTTGCTTTAGAACTTTACAGTATTATATAGGTTGATCCAAAAGTTGATTACTTCTGGTTGGTTATTTTTCTTGTAGGACTTTTTTTTCTTGGATCTGATAAATTACAAAGATATGGATTTGTGAATGAAACCTATGTGTTGCATTTGTGGGTGTTTGAAATTGTGTTTTTGTTTCATGCATATCACACTACTTATGAAACTACAACAAAATTACACGCTATTTCTGAAACTGAACCAAAATATTCTACATTATTTCTGAAACTACAACAAAATAAACAACATTCTTCTATTTTCATGCCTTCTCTTCCTTCTATCATTTTCTTTTTCTCCTCTTCTCAGTCTACCATAGCAGATGGAAATTTTGATAAGTTTTGATAAAATTTGAAGTATCTTAAAACCTACAGCAGCAACTTGAGTTACATGATAAGTCTAAAAGAATTTGGTTGCTTTGTCAAATTTACAAACTTTAACTTTTTTCCCAGAAAACTAACCAAGATTCCATGCTTCAAATTCCATATATCGAAAAAAAACCAGCAACATGATAATCCTTGTATGCAACCTCTTGATTGTTGGGCATGAAAAACAAAATTGCTCTAAATCTGTGTAATAATTTTCAATAAAAGAAAACAAATTGGTGACAAGTTTATGCAGATTCATGGAAAAGAAGAGAGGGTTGGAGAAGAAAACTCAAAAAAAGAGAGAAATAGAGGGTGTAAAAGTCAAGGTAGACATTAATGCTTTGTCATGGAAAAAGCAAATGGAATTTAATTTTCAATCACAGTTGGATTTGAATAAGAAAACGGGTTTGGTTGGATTCCGTAAGGGGCAAGTTTGGAAAAAAATATTTACTGTTGATATGGGCTTTTAATGCAGTCCATGAGTTGTTATGTTTGTAGAGCTTGATACATGAATAATTGTGTCATTATGATTAAATTTTAAGTCCTTATGATTAAATAATAGTTTATGATGATTTTTGGTTAAATTAAGGTTAGCCCAATTCATTTTCATTAAAGCTTTTAAATTTTAGAGATCATTGTTAAAGTTTACTCTTACACAAACTTAGAACTTCATTGACAGGCGGCGGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGTAATTAATTAAACACATCTTATCAAGAATTTCGGCACATCGATCATGCCACAGAGACCCAACATCGTGGCTCGTCAAAGAAGCCAATGCACCCACAAACACAGGTGCATTTTAGGGCCCCGCTCTGGACGCGTGTCGTTTTCAGACTCTGCAACCCACTTGGCCCCTGCCCTTTTATTCTGGTACTCATAATTATTCCTCTTCAGTTTGTGTTTCAGTCGCTGGGGCTTTGCTCTGATAATTGGCAACTTTGCTCAGAAAAAT
according to a Primer design principle, the application designs a pair of InDel labeled primers by using Primer 5 in 370bp upstream and downstream of the InDel locus, wherein the pair of InDel labeled primers comprises a forward Primer sequence and a reverse Primer, and the Primer sequences are as follows: f: GGTTAAATTAAGGTTAGCCCAATTC (SEQ ID No.2), R: AATTATCAGAGCAAAGCCCCAG (SEQ ID No. 3).
Example 2: application verification of InDel labeled primer
The sample whole genome DNA is used as a template, the developed InDel labeled primer PCR amplification reaction system and the amplification program are utilized to carry out PCR amplification, and the PCR product is subjected to polypropylene gel electrophoresis, so that different allelic variation can be detected and distinguished. 23 parts of common apple cultivars and 62 parts of golden bud No.1 and Australian green apple F1 generation hybrid (total 85 parts) were verified. The total volume of the PCR amplification reaction system is 20. mu.L, and the PCR amplification reaction system consists of 6mL ddH2O, 10. mu.L 2 × Green Taq Mix, 1. mu.L 10. mu.M forward primer, 1. mu.L 10. mu.M reverse primer and 2. mu.L whole genome DNA. The PCR amplification reaction program is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 98 ℃ for 10s, annealing at 54.5 ℃ for 15s, extension at 72 ℃ for 30s, and 35 cycles; extension at 72 ℃ for 10 min.
The polyacrylamide gel electrophoresis results are shown in fig. 3 and 4: in FIG. 3, "M" represents DNA ladder marker, 1. mid-autumn king, 2. Hongxing, 3. Imperial concubine, 4. brilliance, 5. ruby, 6. Huashuo, 7. Venus, 8. topaz, 9. Qin crisp, 10. Tuo season, 11. dewdrop, 12. Meisi, 13. hope, 14. Jinzhou red, 15. daphne red, 16. brocade red, 17. Huajia, 18. Huarui, 19. Nipaofu 10, 20. Nipaofu 6, 21. Nipaofu 8, in FIG. 4P 1 represents Jinlei No.1, P2 represents Australian Qingzhou, B represents broad crown F1 generation, and N represents narrow crown F1 generation, which are combined, that there is 6bp deletion in large branching angle (. gtoreq.60) tree type, and there is no such deletion in large branching angle (. gtoreq.30 ℃ to medium (30 ℃ to 60) tree type. The polyacrylamide gel electrophoresis result and the expected genotype coincidence rate are 95.29%.
In addition, Sanger sequencing was performed on the 85 PCR products, and the results are shown in fig. 5: the Sanger sequencing result shows that 6bp deletion exists in a large branching angle (more than or equal to 60 degrees), the deletion sequence is GAGAGA, and no deletion phenomenon exists at the site in the tree types with small branching angle (less than or equal to 30 degrees) and medium branching angle (30 degrees to 60 degrees). The InDel molecular marker is further proved to be capable of distinguishing the branching angles of apple trees.
In conclusion, the InDel molecular marker can identify different branch angles, select apple tree types with different branch angles, and can be used for auxiliary selection to quickly select and breed.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> university of agriculture in China
<120> molecular marker associated with apple tree branch angle related gene and application thereof
<130> MP21037511
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2000
<212> DNA
<213> apple (Malus X domestica Borkh.)
<400> 1
tctccctttc attttgtctt tttccttttt tttttaaatt aataaaaagt tgctagtttg 60
atttttcatt ttattattga ttaacttttg ccaagctgtt gggcatgagc ccttggtacc 120
ttggagaata gcaggaattg aggacacacg ttcccattat attgtgcaat gtattaacct 180
ttttggagaa attttatttg atgtgattga agtttttgcg aattctccac cgaaaggcaa 240
aacagttaaa aacttttgtc acatagatag ctatatgttt tgtcagtgat tcaatatttt 300
gtgtttagat tagttggttt ttccaataaa aaaagattag ttggtttttt actattagtt 360
ttgataatcg gttgtatact catattcaaa cgtaaagtta tagtatttgg ctcaagacat 420
atatgttaat ttgtggtgag tttagtgttt tcatgcaata ttcaaaacac aaaataggta 480
atgaaacccc tctagttagt cttttgttca attaatttcg aattgaatca tcacattcta 540
atcgtctttt aaactaatta catagaaaaa ttattgtgag atgaaaccat gaatcatgta 600
tgtacatttg ctttagaact ttacagtatt atataggttg atccaaaagt tgattacttc 660
tggttggtta tttttcttgt aggacttttt tttcttggat ctgataaatt acaaagatat 720
ggatttgtga atgaaaccta tgtgttgcat ttgtgggtgt ttgaaattgt gtttttgttt 780
catgcatatc acactactta tgaaactaca acaaaattac acgctatttc tgaaactgaa 840
ccaaaatatt ctacattatt tctgaaacta caacaaaata aacaacattc ttctattttc 900
atgccttctc ttccttctat cattttcttt ttctcctctt ctcagtctac catagcagat 960
ggaaattttg ataagttttg ataaaatttg aagtatctta aaacctacag cagcaacttg 1020
agttacatga taagtctaaa agaatttggt tgctttgtca aatttacaaa ctttaacttt 1080
tttcccagaa aactaaccaa gattccatgc ttcaaattcc atatatcgaa aaaaaaccag 1140
caacatgata atccttgtat gcaacctctt gattgttggg catgaaaaac aaaattgctc 1200
taaatctgtg taataatttt caataaaaga aaacaaattg gtgacaagtt tatgcagatt 1260
catggaaaag aagagagggt tggagaagaa aactcaaaaa aagagagaaa tagagggtgt 1320
aaaagtcaag gtagacatta atgctttgtc atggaaaaag caaatggaat ttaattttca 1380
atcacagttg gatttgaata agaaaacggg tttggttgga ttccgtaagg ggcaagtttg 1440
gaaaaaaata tttactgttg atatgggctt ttaatgcagt ccatgagttg ttatgtttgt 1500
agagcttgat acatgaataa ttgtgtcatt atgattaaat tttaagtcct tatgattaaa 1560
taatagttta tgatgatttt tggttaaatt aaggttagcc caattcattt tcattaaagc 1620
ttttaaattt tagagatcat tgttaaagtt tactcttaca caaacttaga acttcattga 1680
caggcggcgg gagagagaga gagagagaga gagagagaga gagagagaga gagagagtaa 1740
ttaattaaac acatcttatc aagaatttcg gcacatcgat catgccacag agacccaaca 1800
tcgtggctcg tcaaagaagc caatgcaccc acaaacacag gtgcatttta gggccccgct 1860
ctggacgcgt gtcgttttca gactctgcaa cccacttggc ccctgccctt ttattctggt 1920
actcataatt attcctcttc agtttgtgtt tcagtcgctg gggctttgct ctgataattg 1980
gcaactttgc tcagaaaaat 2000
<210> 2
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ggttaaatta aggttagccc aattc 25
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
aattatcaga gcaaagcccc ag 22
Claims (10)
- The application of the InDel molecular marker in identifying, distinguishing and/or predicting the branch angle of the apple;the InDel molecular marker is positioned on a promoter of MdTAC1a gene of chromosome 7 of an apple genome, and has a sequence of GAGAGA.
- 2. The use of claim 1, wherein the criteria for identifying, differentiating and/or predicting apple branch angles are:(ii) if a GAGAGA sequence is present or a 370bp band is present, the apple branch angle is < 30 °, or 30 ° < the apple branch angle is < 60 °;if the GAGAGAGA sequence is deleted or a 364bp band appears, the apple branch angle is more than or equal to 60 degrees.
- 3. A primer set for amplifying the InDel molecular marker for use according to claim 1 or 2.
- 4. The primer set of claim 3, comprising:(I) the forward primer has a nucleotide sequence shown as SEQ ID No. 2; and/or(II) a reverse primer which has a nucleotide sequence shown as SEQ ID No.3 or SEQ ID No. 3; and/or(III) has a nucleotide sequence obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in (I) or (II), and has the same or similar functions with the nucleotide sequence shown in (I) or (II); and/or(IV) a nucleotide sequence having at least 80% homology with any of the nucleotide sequences shown in (I) to (III);the number of the plurality is 2 to 10.
- 5. Kit comprising a primer set according to claim 3 or 4 and acceptable adjuvants and/or adjuvants.
- 6. Use of the primer set according to claim 3 or 4 or the kit according to claim 5 for identifying, differentiating and/or predicting apple branch angle and/or for selective breeding.
- 7. Method for identifying, differentiating and/or predicting apple branch angles, characterized in that the criteria for identifying, differentiating and/or predicting apple branch angles are:(ii) if a GAGAGA sequence is present or a 370bp band is present, the apple branch angle is < 30 °, or 30 ° < the apple branch angle is < 60 °;if the GAGAGAGA sequence is deleted or a 364bp band appears, the apple branch angle is more than or equal to 60 degrees.
- 8. The method of claim 7, comprising the steps of:step (a), primer design: designing and screening to obtain a primer group;step (b), DNA extraction: extracting total DNA of the apples to be detected;step (c), PCR amplification: taking the total apple DNA obtained in the step (b) as a template, and performing PCR amplification by adopting the primer group obtained in the step (a) to obtain a PCR product;step (d), electropherogram analysis: and (3) carrying out electrophoresis on the PCR product, and determining the genotype according to the size and the position relation of the bands.
- 9. The method of claim 8, wherein the concentration of any primer in the primer set in step (c) is no less than 10 μmol/L;the PCR amplification reaction system in the step (c) comprises 2 muL of 100 ng/muL of apple total DNA, 1 muL of forward primer, 1 muL of reverse primer and 2 XGreen Taq Mix 10 mu L, ddH2O 6μL；The reaction procedure is as follows:
- 10. a method of selective breeding of apples according to any of claims 7 to 9.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014165650A1 (en) * | 2013-04-04 | 2014-10-09 | The United States Of America, As Represented By The Secretary Of Agriculture | Novel ppetac1 gene and method to manipulate tree architecture |
| CN110903367A (en) * | 2019-12-05 | 2020-03-24 | 西北农林科技大学 | Application of transcription factor in increasing opening angle of apple branches |
| CN112126699A (en) * | 2020-09-15 | 2020-12-25 | 中国农业大学 | Malus plant complete genome InDel marker genotype database and application thereof in germplasm resource specificity identification |
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2022
- 2022-01-19 CN CN202210060680.0A patent/CN114317809A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014165650A1 (en) * | 2013-04-04 | 2014-10-09 | The United States Of America, As Represented By The Secretary Of Agriculture | Novel ppetac1 gene and method to manipulate tree architecture |
| CN110903367A (en) * | 2019-12-05 | 2020-03-24 | 西北农林科技大学 | Application of transcription factor in increasing opening angle of apple branches |
| CN112126699A (en) * | 2020-09-15 | 2020-12-25 | 中国农业大学 | Malus plant complete genome InDel marker genotype database and application thereof in germplasm resource specificity identification |
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| Title |
|---|
| LIMIN WANG等: "The isolation of the IGT family genes in Malus × domestica and their expressions in four idiotype apple cultivars", 《TREE GENETICS & GENOMES》 * |
| 徐兴兴等: "苹果栽培品种的微卫星标记鉴定", 《中国农学通报》 * |
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