CN105838800B - A kind of and the operatic circle skin color trait chain molecular labeling and its application - Google Patents
A kind of and the operatic circle skin color trait chain molecular labeling and its application Download PDFInfo
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Abstract
A kind of and the operatic circle skin color trait chain molecular labeling and its application, the molecular labeling is sequence specific amplification area molecular labeling, its marker site is named as ZFRI130-16, at the 922024bp-922149bp of genome assembling Scaffold NW-008988130.1, the molecular labeling site is located at the inside of the sequence specific amplification area molecular labeling amplified production, its upstream primer sequence are as follows: 5'-CACTTTCGAAGCCAACAAAT-3', downstream primer sequence are as follows: 5'-GGGCAAACTGGGAAACTG-3'.And a kind of red skin pears assist-breeding kit comprising above-mentioned primer pair.Molecular labeling of the present invention can be to ' all over the sky red ' × ' all red pears single plants in red crisp-fried ' test group select, and efficiency of selection reaches 100%.Expand verifying discovery, accuracy of the label in ' Yu Luxiang ' × ' all over the sky red ' combination 310 the progeny population of result reaches 91.3% or more, total accuracy in 34 major cliques and 13 kinds reaches 97.87%, the label that can be used as the erythrodermic shape of pears carries out breeding selection application, has a good application prospect.
Description
Technical field
The invention belongs to pears genetic breeding and molecular biology fields, are related to a kind of point chain with the operatic circle skin color trait
Son label and its application.
Background technique
Pears are one of international large fruit and the main fruit in China, and fruits nutrition is abundant, delicious succulence,
It is sweet and dilitious, it is favored by people.China is the main source of east pears kind, the type and variety source very abundant of pears.
For pears, fruit colour is the fruit properties that most people can be attracted to pay attention to, is the important composition of fruit quality and commodity
Part.In recent years, the idea of healthy consumption is increasingly rooted in the hearts of the people, the pears of red peel be considered to have it is higher health value and
Favored by market, the breeding of red peel Pear varieties is also increasingly subject to the attention of pears breeding research mechanism, various countries.
Currently, breeder is in pears breeding work mainly using traditional crossbreeding means, hybrid seed training
After incubation seedling spends virgin age and the result of several years, then later stage evaluation is carried out to all hybrid single plant growth and fruitings, according to
Evaluation result selection leaves and eliminates hybrid single plant.This method breeding cycle is long, takes up a large area, and takes time and effort, breeding efficiency
It is low.Molecular biology is rapidly developed and is combined with Crop Genetics and Breeding section, produces molecular marker assisted selection breeding skill
Art, make it possible to conventional hybridization breeding obtain hybrid single plant selected ahead of time, high efficiency selected.
Red skin/shagreen character of pears is the quantitative character controlled by major gene resistance with minor-polygene regulation, is shown as red
Skin qualitative character similar with shagreen law of segregation, but erythrodermic shape coloring degree range of variation is relatively wide.The exploitation red skin of pears/green
Skin trait molecular marker is simultaneously used for molecular marker assisted selection breeding, can greatly improve red skin pears breeding selection efficiency, save
The cost for hybridizing single-strain planting maintenance and investigation species test, promotes pears breeding in China's to change from conventional breeding to molecular breeding means.
Summary of the invention
The technical issues of solution
In order to solve the problems, such as the length of period present in pears genetic breeding, low efficiency, the present invention provides a kind of and the operatic circles
The chain molecular labeling of skin color trait and application.
Technical solution
A kind of and chain molecular labeling of the operatic circle skin color trait, the molecular labeling are sequence specific amplification area (SCAR)
Molecular labeling, is named as ZFRI130-16, and the molecular labeling site is located at Dangshan pear genome assembling Scaffold NW-
At 008988130.1 922024bp-922149bp, the site is located at the inside of the SCAR molecular labeling amplified production,
Based on the primer pair of the molecular labeling, upstream primer sequence are as follows: 5'-CACTTTCGAAGCCAACAAAT-3'；
Downstream primer sequence are as follows: 5'-GGGCAAACTGGGAAACTG-3'.
Application of the molecular labeling in the operatic circle skin color trait assisted selection.
Application of the molecular labeling in the red skin of pears/shagreen character assisted selection.
Application of the molecular labeling in Dong Fanglihong skin/shagreen character assisted selection.
In addition, further including a kind of red skin pears assist-breeding kit, contain above-mentioned primer pair in the kit.
Beneficial effect
It is all over the sky red " and " red crisp-fried " and be Zhengzhou Fruit-tree Inst., Chinese Agriculture Science Academy's breeding 2 famous red skin pears
It is larger to promote and apply area in China for kind.The two kinds are often used as the parent of red skin pears breeding by breeding units, show
Good parent's genetic characteristics.The present invention is to ' all over the sky red ' × ' red crisp-fried ' cross combination group ZFRI130-16 molecular labeling
It is analyzed, which can select all red skin pears single plants in test group, and efficiency of selection reaches
100%.Expand group and carries out verifying discovery, which exists ' Yu Luxiang ' × the filial generation group of ' all over the sky red ' combination 310 result
Accuracy in body reaches 91.3% or more, and total accuracy in 34 major cliques and 13 kinds reaches 97.87%, can be used as
The label of the erythrodermic shape of pears carries out breeding selection application, has a good application prospect.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
It obtains other drawings based on these drawings, wherein
Banding pattern figure of Fig. 1 molecular labeling ZFRI130-16 in red skin pears and shagreen pears；
Fig. 2 molecular labeling ZFRI130-16 is in ' all over the sky red ' × ' the amplification figure in red crisp-fried ' cross combination；Both sides
Swimming lane is marker, size 100bp；And
Fig. 3 molecular labeling ZFRI130-16 and the red skin of pears/shagreen character site (Rg) linkage map.
Specific embodiment
The present invention is described further combined with specific embodiments below, but the present invention is not by the limit of following embodiment
System.
Embodiment 1
1 material source
Material to be tested is the lifes in 5 years ' all over the sky red ' of Zhengzhou Fruit-tree Inst., Chinese Agriculture Science Academy, proving ground, Xinxiang field planting
× ' red crisp-fried ' cross combination group totally 348 single plants and 2 parts of parent materials.
2 methods
2.1 phenotypic evaluation
Continuous 2 years red skins, shagreen trait phenotypes tune are carried out to ' all over the sky red ' × ' red crisp-fried ' cross combination group single plant
It looks into, investigation result is for the verifying analysis of subsequent molecular labeling.
2.2PCR reaction
It is expanded with DNA of the ZFRI130-16 molecular labeling to extraction, PCR uses the reaction system of 20 μ L, and amplification should
The primer pair of SCAR mark such as table 1, reaction system composition and PCR response procedures are shown in Table 2 and table 3:
Table 1SCAR labeled primer sequence
Table 2PCR reaction system
Table 3PCR response procedures
2.3PCR product electrophoresis
Take 1 μ L amplified production through 8% non-denaturing polyacrylamide gel (PAGE) electrophoresis, electrophoretic buffer be 0.5 ×
TBE, voltage 150V, electrophoresis 2h.After electrophoresis, dyed according to the PAGE silver staining program in this laboratory.Polyacrylamide
After the dyeing of gel silver staining, it is placed on lamp box and takes pictures.Photo is counted for band.
2.4 genetic distances calculate
The genetic distance of character and label is calculated using Joinmap4.0 software, using MapChart draw character with
Linkage map between label.
3 results
The red skin of 3.1 cross combinations/shagreen character investigation result
To the single plant of result carries out red skin/shagreen character in 348 single plants of ' all over the sky red ' × ' red crisp-fried ' combination
Investigation, investigated the fruit of 293 single plants altogether, wherein red skin pears 117, shagreen pears 176, show it is a degree of partially point
From.
3.2SCAR marks ZFRI130-16 and red skin/shagreen character site genetic distance
Phenotypic data using 339 single plants and its genotype data in the site ZFRI130-16 carry out genetic distance point
Analysis, the results showed that, the site ZFRI130-16 and red skin/shagreen character site genetic distance are 2.6cm, and calculated result is used
MapChart draws, as a result as shown in Fig. 3.
3.3SCAR marks ZFRI130-16 to the selective power of red skin hybrid single plant
Count Genotyping situation of the primer ZFRI130-16 in 293 single plants of result, the results showed that, 293
The single plant that red banding pattern is amplified in a single plant has 128, and the single plant for amplifying green banding pattern has 165,117 red skin single plants
Red banding pattern is all showed in ZFRI130-16 primer sites, in addition 11 single plants for showing red banding pattern are green pericarp
(being the first annual bearing).Show that ZFRI130-16 label can all screen the combination red single plant.
4 conclusions
Red skin pears single plant and shagreen pears single plant can be quickly and easily distinguished through the above steps, successfully by molecular labeling
Assist-breeding technology is applied to the breeding of red Pi Liyu shagreen pears in pears breeding, significantly reduces the workload of breeding, saves breeding
Time greatly improves breeding efficiency.
Embodiment 2
1. the spire that acquisition ' Yu Luxiang ' × ' all over the sky red ' 458 single plants of combination have just been sprouted, careful clean are dried, are extracted
DNA。
2. being expanded with DNA of the ZFRI130-16 molecular labeling to extraction, PCR uses the reaction system of 20 μ L, amplification
The primer pair of the molecular labeling is the same as embodiment 1.
3. pair PCR product carries out polyacrylamide gel electrophoresis, and carries out band statistics to electrophoresis result, count red skin/
The banding pattern of shagreen single plant.
4. the result shows that ZFRI130-16 label can be by the accurate area of color of the leather of 283 single plants in 310 result single plant
It branches away, accuracy rate reaches 91.3% or more.
5. can quickly and easily distinguish red Pi Liyu shagreen pears through the above steps, successfully molecular labeling is assisted selecting
Breeding of the technology applied to red Pi Liyu shagreen pears in pears breeding is educated, the workload of breeding is significantly reduced, greatly improves and educate
Kind efficiency.
Embodiment 3
1. acquisition from ' good fortune water ' × ' red crisp-fried ', ' early U.S. crisp ' × ' red crisp-fried ', ' Zhongli 1 ' × ' red crisp-fried ' with
And the cross combinations such as ' new century ' × ' red crisp-fried ' amount to 34 major cliques and ' early whitish honey ', ' early U.S. is crisp ', ' Zhongli 1 ', ' in
Pears 2 ', ' middle pears 3 ', ' middle pears 4 ', ' July is crisp ', ' August is crisp ', ' goose pears ', ' early crisp ', ' the wide pears of duck ', ' Dangshan is crisp ',
The spire that 13 kinds such as ' golden pear ' have just been sprouted, careful clean are dried, and DNA is extracted.
2. being expanded with DNA of the ZFRI130-16 molecular labeling to extraction, PCR uses the reaction system of 20 μ L, amplification
The primer pair of the molecular labeling is the same as embodiment 1.
3. pair PCR product carries out polyacrylamide gel electrophoresis, and carries out band statistics to electrophoresis result, count red skin/
The banding pattern of shagreen single plant.
4. the result shows that ZFRI130-16 label can be accurate with the color of the leather of all 13 kinds by 33 in 34 major cliques
It distinguishes, accuracy rate reaches 97.87%.
5. can quickly and easily distinguish red Pi Liyu shagreen pears through the above steps, successfully molecular labeling is assisted selecting
Breeding of the technology applied to red Pi Liyu shagreen pears in pears breeding is educated, the workload of breeding is significantly reduced, greatly improves and educate
Kind efficiency.
The above, only the preferred embodiment of the present invention, anyone skilled in the art is in this hair
In the technical scope of bright disclosure, the simple change or equivalence replacement carry out to technical solution of the present invention each falls within protection of the invention
Range.
Claims (4)
1. a kind of application of molecular labeling in the operatic circle skin color trait assisted selection, which is characterized in that the molecule mark
It is denoted as sequence specific amplification area molecular labeling, the molecular labeling site is named as ZFRI130-16, is located at genome and assembles
At the 922024bp-922149bp of Scaffold NW_008988130.1, the molecular labeling site is located at the sequence specific
The inside of property amplification region molecular labeling amplified production, the upstream primer sequence of the molecular labeling are as follows: 5'-
CACTTTCGAAGCCAACAAAT-3', downstream primer sequence are as follows: 5'-GGGCAAACTGGGAAACTG-3'.
2. a kind of application of molecular labeling in the red skin of pears/shagreen character assisted selection, which is characterized in that the molecule mark
It is denoted as sequence specific amplification area molecular labeling, the molecular labeling site is named as ZFRI130-16, is located at genome and assembles
At the 922024bp-922149bp of Scaffold NW_008988130.1, the molecular labeling site is located at the sequence specific
The inside of property amplification region molecular labeling amplified production, the upstream primer sequence of the molecular labeling are as follows: 5'-
CACTTTCGAAGCCAACAAAT-3', downstream primer sequence are as follows: 5'-GGGCAAACTGGGAAACTG-3'.
3. a kind of application of molecular labeling in Dong Fanglihong skin/shagreen character assisted selection, which is characterized in that described point
Son label is area's molecular labeling, and the molecular labeling site is named as ZFRI130-16, is located at genome
At the 922024bp-922149bp for assembling Scaffold NW_008988130.1, the molecular labeling site is located at the sequence
The inside of specific amplification area molecular labeling amplified production, the upstream primer sequence of the molecular labeling are as follows: 5'-
CACTTTCGAAGCCAACAAAT-3', downstream primer sequence are as follows: 5'-GGGCAAACTGGGAAACTG-3'.
4. a kind of red skin pears assist-breeding kit, which is characterized in that contain in the kit and draw as described in claim 1
Object pair.
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| CN113481320B (en) * | 2021-08-25 | 2022-06-03 | 中国农业科学院果树研究所 | Site linked with pear peel red character, molecular marker and application thereof |
| CN115011723B (en) * | 2022-06-14 | 2022-12-09 | 中国农业科学院果树研究所 | Application of molecular marker linked with pear peel red character in pear peel red/green character auxiliary selective breeding |
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| CN105296472A (en) * | 2015-08-12 | 2016-02-03 | 浙江省农业科学院 | Molecular marker of peel full-brown trait gene locus of Chinese 'Qingxiang'-variety pears and screening method of molecular marker |
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| KR101258694B1 (en) * | 2010-12-20 | 2013-04-29 | 대한민국 | SCAR marker for cultivar discrimination in pear and usage thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105296472A (en) * | 2015-08-12 | 2016-02-03 | 浙江省农业科学院 | Molecular marker of peel full-brown trait gene locus of Chinese 'Qingxiang'-variety pears and screening method of molecular marker |
Non-Patent Citations (1)
| Title |
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| 赵广.红皮梨色泽变异系的分子标记鉴定及遗传差异分析.《中国优秀硕士学位论文全文数据库•农业科技辑》.2015,(第3期),对比文件1第一章和第二章. * |
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