CN107996168B - Anvil bud configuration method for high grafting and crown changing of hollow plum - Google Patents
Anvil bud configuration method for high grafting and crown changing of hollow plum Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 22
- 230000002068 genetic effect Effects 0.000 claims abstract description 17
- 235000013399 edible fruits Nutrition 0.000 claims abstract description 15
- 240000005049 Prunus salicina Species 0.000 claims abstract description 10
- 235000012904 Prunus salicina Nutrition 0.000 claims abstract description 10
- 235000003681 Prunus ussuriensis Nutrition 0.000 claims abstract description 10
- 239000003550 marker Substances 0.000 claims abstract description 10
- 235000021018 plums Nutrition 0.000 claims abstract description 10
- 238000012408 PCR amplification Methods 0.000 claims abstract description 8
- 238000012216 screening Methods 0.000 claims description 5
- 238000003559 RNA-seq method Methods 0.000 claims description 4
- 108700028369 Alleles Proteins 0.000 claims description 3
- 230000003321 amplification Effects 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 claims description 2
- 241000196324 Embryophyta Species 0.000 abstract description 6
- 230000034303 cell budding Effects 0.000 abstract description 3
- 230000004083 survival effect Effects 0.000 description 6
- 238000013461 design Methods 0.000 description 4
- 235000004789 Rosa xanthina Nutrition 0.000 description 2
- 241000220222 Rosaceae Species 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000004576 sand Substances 0.000 description 2
- 244000144730 Amygdalus persica Species 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 235000011432 Prunus Nutrition 0.000 description 1
- 241001631271 Prunus fasciculata Species 0.000 description 1
- 244000018795 Prunus mume Species 0.000 description 1
- 235000011158 Prunus mume Nutrition 0.000 description 1
- 235000006040 Prunus persica var persica Nutrition 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000021022 fresh fruits Nutrition 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002985 plastic film Substances 0.000 description 1
- 229920006255 plastic film Polymers 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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Abstract
The invention relates to a stock bud configuration method for high grafting and crown changing of hollow plum, and belongs to the technical field of plant grafting cultivation. The main technical scheme is as follows: respectively extracting leaf genome DNAs of different germplasm Prunus salicina and stock seedlings, wherein initial scions of the stock seedlings are all from the same mother tree; carrying out PCR amplification on the genome DNA of the hollow plum and the leaf of the stock seedling by utilizing a polymorphic hollow plum SSR marker primer; calculating the genetic distance between the hollow plums with different germplasms and the stock seedlings according to the PCR amplification strip result; selecting hollow plums with genetic distance between 0 and 0.0081 and excellent fruit quality and character, and designing a grafting combination; adopting a budding method with xylem to perform top grafting crown replacement on the hollow plum. Aiming at low yield and poor fruit quality root tiller seedlings and cutting seedlings, the invention realizes high grafting and crown changing of high-quality germplasm of the hollow plum by using an inter-germplasm molecular genetic distance recognition technology and a xylem budding method.
Description
Technical Field
The invention relates to the technical field of plant grafting cultivation, in particular to a stock bud configuration method for high grafting and crown changing of hollow plums.
Background
Plum is perennial arbor of Prunoideae (Prunoideae) of Rosaceae (Rosaceae), Prunoideae (Prunus L.) sand hollow plum is named because the kernel and the pulp are naturally split when mature, has been cultivated for over 130 years so far, has yellow and white pulp, moderate hardness, crisp and tender, more and more tasty and refreshing juice, fragrant and thick sweet, and has the reputation of 'fresh fruit between people and Japanese apricot tree' in 2006.
In the patent 'grafting method of plum' (application No. 201610117517.8), a plum seedling of 50cm is taken as a stock, and a middle-aged branch is grafted at a position 20cm away from the ground. The patent does not relate to an anvil bud configuration technique using superior germ cell identification. At present, in many areas, particularly along river counties, plum is rarely used as a grafting stock, and sand hollow plum seeds along the river counties are often immature, so that plum stock seedlings are difficult to obtain.
Disclosure of Invention
In order to make up the defects of the prior art, the invention aims at the root-tillering seedlings and the cutting seedlings with low yield and poor fruit quality, and utilizes the inter-germplasm molecular genetic distance recognition technology and the xylem bud grafting method to realize the high grafting and crown changing of the high-quality germplasm of the hollow plum.
The technical scheme of the invention is as follows: a method for configuring stock buds of hollow plum by high grafting and crown changing comprises the following steps:
(1) respectively extracting leaf genome DNAs of different germplasm Prunus salicina and stock seedlings, wherein initial scions of the stock seedlings are all from the same mother tree; at present, most of commercial seedlings planted by farmers are obtained by taking annual wild peaches as rootstocks and hollow plums as scions and utilizing a low grafting method. The purpose of using the same hollow plum tree as the scion is to ensure that the genetic backgrounds are the same when the hollow plum tree is used as the stock for the second time as much as possible in the follow-up test;
(2) carrying out PCR amplification on the hollow plum obtained in the step (1) and the stock seedling leaf genome DNA by utilizing a polymorphic hollow plum SSR marker primer;
(3) calculating the genetic distance between the hollow plums with different germplasms and the stock seedlings according to the PCR amplification strip result in the step (2);
(4) selecting hollow plums with genetic distance between 0 and 0.0081 and excellent fruit quality and character, and designing a grafting combination;
(5) and (4) combining the grafting designed in the step (4), and performing top grafting crown replacement on the hollow plum by adopting a bud grafting method with xylem.
Further, the screening method of the polymorphic Prunus salicina SSR marker primer in the step (2) comprises the following steps:
1) developing SSR markers based on RNA-Seq technology;
2) and screening the polymorphic hollow plum SSR marker primer by utilizing a polyacrylamide gel electrophoresis result.
The invention has the following beneficial effects:
(1) the invention develops SSR marker primers of the hollow plums based on an RNA-Seq technology, utilizes the polymorphic SSR marker primers to detect and obtain genetic distances among different hollow plum germplasms, configures stock bud combinations by combining closely related germplasms and high-quality traits of fruits, adopts a budding method with xylem to perform top grafting crown changing, achieves the grafting survival rate of more than 80 percent, and provides a top grafting crown changing grafting design mode for improving the quality and the efficiency of the hollow plums.
(2) The invention utilizes the allele locus method to count the SSR primer amplification bands, and can more accurately calculate the genetic distance between various germplasms compared with the statistical methods of 0 and 1.
(3) According to the invention, by combining the genetic relationship between germplasms and the evaluation of fruit quality, the grafting survival rate is improved, seedlings with good fruit quality are obtained, and the bottleneck of industrial development is broken: in the production, the stock of the improved variety of the hollow plum is lacked, farmers often adopt the hollow plum root tiller seedling and the hollow plum grafted peach seedling to build a garden, and in recent years, the fruit quality of the plum trees is degraded year by year.
Detailed Description
The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.
Example 1 extraction of genomic DNA of Prunus salicina leaf
21 hollow plum germplasms M1, M2, M3, M4, M6, M7, M8, YH1, YH2, YH3, YH4, YH5, YH6, YH7, YH8, YH9, YH10, YH11, YH30, YH31, YH32 and 1 rootstock seedling ZM5 along the river county are used as materials, leaf genomic DNAs of different hollow plum germplasms and rootstock seedlings are extracted according to a method recommended by a novel Tiangen plant genomic DNA extraction kit (DP320), the initial scion of the rootstock seedlings is derived from the same mother tree, and the genomic DNA concentration of each germplasm is 20-40 ng/. mu L and is preserved at-20 ℃.
Example 2 development of an SSR marker of Prunus salicina
Taking hollow plum leaves and fruits as materials, and extracting and purifying total RNA by referring to a recommended method of a columnar plant total RNA extraction and purification kit of Shanghai's chemical company. Performing transcriptome high-throughput sequencing by adopting an RNA-Seq technology to obtain SSR loci and design primers, selecting 71 pairs of primers from the SSR loci, and performing PCR amplification by taking 4 different hollow plum germplasm DNAs as templates, wherein the reaction system is as follows:
the reaction procedure is as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; extending for 10min at 72 ℃; storing at 4 ℃. And detecting the PCR result of 71 pairs of SSR primers by agarose gel electrophoresis, and screening to obtain 42 pairs of SSR labeled primers suitable for the hollow plums, wherein the result is shown in the following table 1.
TABLE 142 pairs of Prunus salicina SSR primers
The 42 pairs of SSR primers of the prunus salicina are used for carrying out PCR amplification on the germplasm DNA of 22 prunus salicina in example 1, the reaction system and conditions are as described above, and the amplification products are detected by 8% polyacrylamide gel vertical plate electrophoresis. And (3) marking the allele loci of the detection result bands of the templates, such as AA, AB, BC and the like, and calculating the genetic distance among the germplasms by using MEGA software.
Example 3 selecting scions with good fruit quality and close to rootstock seedlings, designing hollow plum high grafting combination
Based on the genetic distance between each germplasm obtained in example 2 and the stock seedling ZM5 and the fruit quality traits of the other 21 hollow plum germplasm, a high scion was selected as shown in table 2 below. Selecting current-year branches of germplasm with high sugar-acid ratio (M1, YH4, YH8 and YH9) and average single fruit weight (YH30, YH31 and YH32) as scions, and smoothly cutting 2 cm-long full buds slightly containing xylem from top to bottom; selecting 3-4 years branches on the stock seedlings, and cutting the branches into smooth sections of 2-2.5cm from top to bottom, wherein the depth is suitable for cutting off a few xylems; the bud is close to the cutting position of the stock, and the joint is tightly bound by a plastic film. Each grafting combination designs 15 plants with the same tree age and growth potential, each plant is grafted with 5 buds, the survival rate is investigated after 60 days of grafting, and as shown in table 3 below, when the genetic distance is below 0.0081, the grafting survival rate reaches more than 80%.
Partial fruit quality traits of 221 empty plum germplasms in table
TABLE 37 grafting survival rates of high-quality hollow plum scions
According to the invention, different high grafting combinations are designed according to the genetic distance between hollow plum germplasms and the fruit quality characteristics, and when the genetic distance is less than 0.0081, the grafting survival rate reaches more than 80%. The invention provides a high grafting and crown changing grafting design mode for improving quality and efficiency of hollow plum root tillering seedlings and grafted seedlings.
Claims (2)
1. A method for configuring anvil buds of hollow plum for high grafting and crown changing is characterized by comprising the following steps:
(1) respectively extracting leaf genome DNAs of different germplasm Prunus salicina and stock seedlings, wherein initial scions of the stock seedlings are all from the same mother tree;
(2) carrying out PCR amplification on the hollow plum obtained in the step (1) and the stock seedling leaf genome DNA by utilizing a polymorphic hollow plum SSR marker primer;
(3) according to the PCR amplification strip result in the step (2), marking the allele sites of the amplification result strips of each template, and calculating the genetic distance between different germplasm Prunus salicina and the stock seedling by using MEGA software;
(4) selecting hollow plums with genetic distance between 0 and 0.0081 and excellent fruit quality and character, and designing a grafting combination;
(5) and (4) combining the grafting designed in the step (4), and performing top grafting crown replacement on the hollow plum by adopting a bud grafting method with xylem.
2. The method for preparing anvil buds of hollow plum for top grafting and crown changing according to claim 1,
the screening method of the polymorphic hollow plum SSR marker primer in the step (2) comprises the following steps:
1) developing SSR markers based on RNA-Seq technology;
2) and screening the polymorphic hollow plum SSR marker primer by utilizing a polyacrylamide gel electrophoresis result.
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| CN113481319B (en) * | 2021-08-12 | 2024-02-09 | 四川省农业科学院园艺研究所 | SSR molecular marker primer for identifying green and crisp plum varieties and application thereof |
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| CN104285684A (en) * | 2014-09-15 | 2015-01-21 | 安吉中科园林绿化工程有限公司 | Method for grafting prunus cerasifera onto wild peach stock |
| CN105075850B (en) * | 2015-08-18 | 2017-11-24 | 大连民族大学 | A kind of molecular design method of new camellia oleifera lam high-yield culturing |
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| CN106854677B (en) * | 2017-01-19 | 2020-08-25 | 浙江省农业科学院 | Method for screening waxberry scions grafted by taking waxberry as rootstock based on SSR (simple sequence repeat) markers |
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