CN109722489A - A kind of efficient homozygous method for genetic of muskmelon breeding material objective trait - Google Patents
A kind of efficient homozygous method for genetic of muskmelon breeding material objective trait Download PDFInfo
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Abstract
The invention discloses a kind of efficient homozygous method for genetic of muskmelon breeding material objective trait, include the following steps: that (1) germplasm collection and DNA are extracted；(2) PCR amplification；(3) electrophoresis；(4) silver staining；(5) data are analyzed；(6) acquisition of heterozygosis Offspring F1；(7) selection of homozygous offspring.The DNA molecular marker of the present invention application RAPD, ISSR, SSR are scanned muskmelon group, and combine the table data of muskmelon, the molecular labeling of structure division, excavation and objective trait close linkage is carried out to group on molecular level, pass through hybridization technique means, can quick homozygous muskmelon objective trait, excellent basis, great marketing application value have been established for the breeding of muskmelon excellent variety.
Description
Technical field
The invention belongs to breeding technology fields, and in particular to a kind of efficient homozygosis side of muskmelon breeding material objective trait
Method.
Background technique
Muskmelon belongs to Curcurbitaceae Cucumis herbaceous plant.The delicious sweetness of melon fruit, it is full of nutrition, in China's cultivated area
Extensively.In melon variety Breeding Process, how to formulate, filter out the family's based material for stablizing homozygosis, is to influence crossbreeding process
Key.When carrying out the initiative of muskmelon idioplasm material, some heterozygosis offsprings can be obtained, these heterozygosis offspring is subjected to objective trait
Homozygosis needs to carry out inbreeding of more generation, and whether the field phenotype by observing self progeny's family shows unanimously, to determine that character is
No homozygosis, heavy workload, period are long.
DNA molecular marker is the genetic marker based on the polymorphism of DNA, can stablize heredity, and mode of inheritance letter
It is single.Oneself has some molecular marking techniques to establish in recent years, and is widely used in field of crop genetic breeding rapidly.Pass through application
The DNA molecular markers such as RAPD, SSR are scanned group, can further on molecular level to group carry out structure division,
Excavate the molecular labeling with objective trait close linkage.
Summary of the invention
The purpose of the present invention is being directed to existing problem, a kind of efficient homozygosis side of muskmelon breeding material objective trait is provided
Method.
The present invention is achieved by the following technical solutions:
A kind of efficient homozygous method for genetic of muskmelon breeding material objective trait, includes the following steps:
(1) germplasm collection and DNA are extracted:
Muskmelon idioplasm material to be collected, the muskmelon idioplasm material of collection is sowed, after germinating to seedling, sampling carries out DNA extraction,
It is spare；
(2) PCR amplification:
Result chooses RAPD, ISSR, SSR label primer according to previous studies, by the labeled primer with polymorphism to step (1)
The DNA of middle extraction carries out PCR amplification, and it is spare to obtain amplified production；
(3) electrophoresis:
Amplified production obtained in step (2) is subjected to gel electrophoresis；
(4) silver staining:
Gel after electrophoresis in step (3) is subjected to silver staining, colour developing is spare；
(5) data are analyzed:
A. according to the PCR product electrophoretic band position developed the color in step (4), polymorphism analysis is carried out, it is standby to obtain molecular data
With；
B. molecular data obtained in application operating a carries out group structure analysis by structure software, obtains group structure
Data are spare；
C. the phenotype of field test record Germplasm Resources of Cucumis Melo L, it is spare to obtain phenotypic data；
D. molecular data obtained in application operating a, phenotype obtained in group structure data, operation c obtained in operation b
Data are associated, thus the molecular labeling of identification and each character close linkage, and further the special equipotential of analysis mining becomes
It is different；
(6) acquisition of heterozygosis Offspring F1:
According to breeding objective, in conjunction with Muskmelon Planting resource phenotypic data, selection has the material of objective trait, carry out hybridization or from
It hands over, obtains heterozygosis Offspring F1；
(7) selection of homozygous offspring:
A. heterozygosis Offspring F1 obtained in step (6) being subjected to selfing and obtains F2 for seed, F2 nurses young plants in hothouses for seed, and 2
DNA is extracted in Zhou Hou, sampling, using F2 for seedling DNA as template, with obtain associated point of objective trait in step (5) operation d
Sub- labeled primer carries out PCR amplification and gel electrophoresis analysis；
B. the gel electrophoresis strip position according to obtained in operation a, the offspring that selection carries multiple synergy allelic variations carry out field
Between cultivate, and combine actual observation, carry out Single-plant selection or carrying out mixing selection to the consistent family of phenotype, obtain F2 single plant or strain
System；
C. each F2 for operating primary election in b is selfed respectively for single plant or strain, obtains F3 for seed, and operate a according to step (7)
In method F3 is nursed young plants in hothouses for seed, extract DNA, is carried out using with the associated molecular labeling primer of objective trait
PCR amplification and gel electrophoresis analysis；
D. the gel electrophoresis strip position according to obtained in operation a, the offspring that selection carries multiple synergy allelic variations carry out field
Between cultivate, and combine actual observation, carry out Single-plant selection or carrying out mixing selection to the consistent family of phenotype, obtain F3 single plant or strain
System；
E. the different F3 selected in d will be operated to be selfed respectively for single plant or strain, F4 generation is obtained, therefrom select relatively homozygous and table
Existing type meets the breeding material of breeding objective
Further, what is taken in the step (1) is the same area of Muskmelon Plants homolog.
Further, in the step (3) RAPD and ISSR molecular labeling PCR product in 1 × TAE buffer with 2% fine jade
Electrophoresis on sepharose, SSR marker PCR product carry out electrophoresis on 12% non-denaturing polyacrylamide gel.
Further, phenotypic data includes begin flower section, blade profile, section of bearing fruit, fruit shape in step (5) the operation c
Shape, fruit colour, pulp colour, the hardness of fruit, fruit center refractive power pol, fruit development period, the time of infertility, single fruit weight,
Per mu yield.
The present invention has the advantage that compared with prior art
The DNA molecular marker of the present invention application RAPD, ISSR, SSR are scanned muskmelon group, and combine the Phenotype Number of muskmelon
According to the molecular labeling of structure division, excavation and objective trait close linkage is carried out to group on molecular level, and analyzes acquisition
It is closely related specifically to wait with character and does not make a variation, these molecular labelings scans heterozygosis offspring by application, detect and carry in offspring
The relevant allelic variation situation of objective trait, the offspring that selection carries corresponding allelic variation are used further to the selfing of next step, can
Quickly and effectively to carry out the screening of homozygous family, be established for the breeding of muskmelon excellent variety in terms of molecule and phenotype two
Excellent basis, great marketing application value.
Specific embodiment
Embodiment 1
A kind of efficient homozygous method for genetic of muskmelon breeding material objective trait, includes the following steps:
(1) germplasm collection and DNA are extracted:
Muskmelon idioplasm material to be collected, the muskmelon idioplasm material of collection is sowed, after germinating to seedling, sampling carries out DNA extraction,
It is spare；
(2) PCR amplification:
Result chooses RAPD, ISSR, SSR label primer according to previous studies, by the labeled primer with polymorphism to step (1)
The DNA of middle extraction carries out PCR amplification, and it is spare to obtain amplified production；
(3) electrophoresis:
Amplified production obtained in step (2) is subjected to gel electrophoresis；
(4) silver staining:
Gel after electrophoresis in step (3) is subjected to silver staining, colour developing is spare；
(5) data are analyzed:
A. according to the PCR product electrophoretic band position developed the color in step (4), polymorphism analysis is carried out, it is standby to obtain molecular data
With；
B. molecular data obtained in application operating a carries out group structure analysis by structure software, obtains group structure
Data are spare；
C. the phenotype of field test record Germplasm Resources of Cucumis Melo L, it is spare to obtain phenotypic data；
D. molecular data obtained in application operating a, phenotype obtained in group structure data, operation c obtained in operation b
Data are associated, thus the molecular labeling of identification and each character close linkage, and further the special equipotential of analysis mining becomes
It is different；
(6) acquisition of heterozygosis Offspring F1:
According to breeding objective, in conjunction with Muskmelon Planting resource phenotypic data, selection has the material of objective trait, carry out hybridization or from
It hands over, obtains heterozygosis Offspring F1；
(7) selection of homozygous offspring:
A. heterozygosis Offspring F1 obtained in step (6) being subjected to selfing and obtains F2 for seed, F2 nurses young plants in hothouses for seed, and 2
DNA is extracted in Zhou Hou, sampling, using F2 for seedling DNA as template, with obtain associated point of objective trait in step (5) operation d
Sub- labeled primer carries out PCR amplification and gel electrophoresis analysis；
B. the gel electrophoresis strip position according to obtained in operation a, the offspring that selection carries multiple synergy allelic variations carry out field
Between cultivate, and combine actual observation, carry out Single-plant selection or carrying out mixing selection to the consistent family of phenotype, obtain F2 single plant or strain
System；
C. each F2 for operating primary election in b is selfed respectively for single plant or strain, obtains F3 for seed, and operate a according to step (7)
In method F3 is nursed young plants in hothouses for seed, extract DNA, is carried out using with the associated molecular labeling primer of objective trait
PCR amplification and gel electrophoresis analysis；
D. the gel electrophoresis strip position according to obtained in operation a, the offspring that selection carries multiple synergy allelic variations carry out field
Between cultivate, and combine actual observation, carry out Single-plant selection or carrying out mixing selection to the consistent family of phenotype, obtain F3 single plant or strain
System；
E. the different F3 selected in d will be operated to be selfed respectively for single plant or strain, F4 generation is obtained, therefrom select relatively homozygous and table
Existing type meets the breeding material of breeding objective
Further, what is taken in the step (1) is the same area of Muskmelon Plants homolog.
Further, in the step (3) RAPD and ISSR molecular labeling PCR product in 1 × TAE buffer with 2% fine jade
Electrophoresis on sepharose, SSR marker PCR product carry out electrophoresis on 12% non-denaturing polyacrylamide gel.
Further, phenotypic data includes begin flower section, blade profile, section of bearing fruit, fruit shape in step (5) the operation c
Shape, fruit colour, pulp colour, the hardness of fruit, fruit center refractive power pol, fruit development period, the time of infertility, single fruit weight,
Per mu yield.
In order to further compare effect of the present invention, to the 104 parts of muskmelon idioplasm materials compiled as experimental subjects, press
According to the method homozygosis objective trait of embodiment 1.Under specific method and result:
One, method:
(1) germplasm collection and DNA are extracted:
Compile 104 points of muskmelon idioplasm materials are sowed, and samples and carries out DNA extraction, it is spare；(2) PCR amplification:
Result chooses RAPD, ISSR, SSR label primer according to previous studies, by the labeled primer with polymorphism to step (1)
The DNA of middle extraction carries out PCR amplification, and it is spare to obtain amplified production；
(3) electrophoresis:
It is slow in 1 × TAE that amplified production obtained in step (2) is subjected to gel electrophoresis, RAPD and ISSR molecular labeling PCR product
With electrophoresis on 2% Ago-Gel in fliud flushing, SSR marker PCR product carries out electrophoresis on 12% non-denaturing polyacrylamide gel；
(4) silver staining:
Gel after electrophoresis in step (3) is subjected to silver staining, colour developing is spare；
(5) data are analyzed:
A. according to the PCR product electrophoretic band position developed the color in step (4), polymorphism analysis is carried out, it is spare to obtain molecular data
(table 1)；
1 polymorphism analysis result of table
B. molecular data obtained in application operating a carries out group structure analysis by structure software, obtains group structure
Data are spare；
C. the phenotype of field test record Muskmelon Planting resource, it is spare to obtain phenotypic data；
D. Phenotype Number obtained in molecular data obtained in a, group structure data, operation c obtained in operation b will be operated
According to being associated, thus identification molecular labeling (table 2) associated with each character；
E. acquisition and the significantly associated molecular labeling site of phenotypic character in operation d, on this basis further analyze it
Excavate special allelic variation and its carrier material table 3)；
2 characters of table-label association results
Identify 2 SSR markers (SSRC, SSR08) and center refractive power sugar by means of the present invention it can be seen from upper table 1
Degree is related, identifies 3 (RAPD49, RAPD11, ISSR02) and is closely related with single melon weight.
The special allelic variation in 3 part of table and carrier material
By
Upper table 3 in as can be seen that be filled with admiration the synergy of light pol the most it is apparent that SSRC-A174 and SSR08-A156, carries the two
The germplasm materials number of allelic variation is respectively 13 and 18.
(6) heterozygosis germplasm materials prepare:
According to high pol breeding objective, in conjunction with sugared content data and other economical characters in Muskmelon Planting resource phenotypic data, choosing
The S19 muskmelon idioplasm for carrying high sugared allelic variation SSRC-A174 is selected as material, which is Hybrids F1, needs to carry out high sugar
Character is homozygous；
(7) selection of filial generation:
A. the F1 screened in step (6) is subjected to selfing and obtains F2 for seed, F2 nurses young plants in hothouses for seed, after 2 weeks, adopts
Sample extracts DNA, using F2 for seedling DNA as template, with obtained in step (5) operation d the relevant molecular labeling SSRC of height sugar,
SSR08 primer carries out PCR amplification and gel electrophoresis analysis；
B. the resulting gel electrophoresis strip position of PCR amplification is carried out in each seedling DNA of F2 according to SSRC, SSR08 primer in operation a
It sets, the offspring that selection carries the synergy allelic variations such as SSRC-A174, SSR08-A156 carries out field cultivation, and combines field real
Border observation and sugared content measurement result obtain sugar F2 offspring (F2-1, F2-2, F2-3, F2-4, F2-5, F2- 6 high of primary election
6)；
C. each F2 for operating primary election in b is selfed respectively, obtains F3 for seed, and operate the method in a for F3 according to step (7)
It nursed young plants in hothouses for seed, extract DNA, with height sugar relevant molecular labeling SSRC, the SSR08 obtained in step (5) operation d
Primer carries out PCR amplification and gel electrophoresis analysis；
D. the resulting gel electrophoresis strip position of PCR amplification is carried out in each seedling DNA of F3 according to SSRC, SSR08 primer in operation a
It sets, the offspring that selection carries the synergy allelic variations such as SSRC-A174, SSR08-A156 carries out field cultivation, and combines field real
Border observation and sugared content measurement result select sugar F3 offspring (F3-1, F3-2, F3-3, F3- 4 high in F2-2 self progeny
4)；
E. the different F3 that select in d will be operated to be selfed respectively for single plant or strain, obtain F4 for seed, wherein F3-1 self-fertilization family (sff)
Offspring showed in terms of this character of high sugar content it is almost the same, through SSRC, SSR08 augmentation detection, and carry SSRC-A174
Allelic variation.
(8) through this method, when the F4 for selecting F3-1 selfing to obtain is used for breeding for high sugar content target, parent's material of crossbreeding
Material.Obtain relatively homozygous breeding for high sugar content material.
Claims (4)
1. a kind of efficient homozygous method for genetic of muskmelon breeding material objective trait, which comprises the steps of:
(1) germplasm collection and DNA are extracted:
Muskmelon idioplasm material to be collected, the muskmelon idioplasm material of collection is sowed, after germinating to seedling, sampling carries out DNA extraction,
It is spare；
(2) PCR amplification:
Result chooses RAPD, ISSR, SSR label primer according to previous studies, by the labeled primer with polymorphism to step (1)
The DNA of middle extraction carries out PCR amplification, and it is spare to obtain amplified production；
(3) electrophoresis:
Amplified production obtained in step (2) is subjected to gel electrophoresis；
(4) silver staining:
Gel after electrophoresis in step (3) is subjected to silver staining, colour developing is spare；
(5) data are analyzed:
A. according to the PCR product electrophoretic band position developed the color in step (4), polymorphism analysis is carried out, it is standby to obtain molecular data
With；
B. molecular data obtained in application operating a carries out group structure analysis by structure software, obtains group structure
Data are spare；
C. the phenotype of field test record Germplasm Resources of Cucumis Melo L, it is spare to obtain phenotypic data；
D. molecular data obtained in application operating a, phenotype obtained in group structure data, operation c obtained in operation b
Data are associated, thus the molecular labeling of identification and each character close linkage, and further the special equipotential of analysis mining becomes
It is different；
(6) acquisition of heterozygosis Offspring F1:
According to breeding objective, in conjunction with Muskmelon Planting resource phenotypic data, selection has the material of objective trait, carry out hybridization or from
It hands over, obtains heterozygosis Offspring F1；
(7) selection of homozygous offspring:
A. heterozygosis Offspring F1 obtained in step (6) being subjected to selfing and obtains F2 for seed, F2 nurses young plants in hothouses for seed, and 2
DNA is extracted in Zhou Hou, sampling, using F2 for seedling DNA as template, with obtain associated point of objective trait in step (5) operation d
Sub- labeled primer carries out PCR amplification and gel electrophoresis analysis；
B. the gel electrophoresis strip position according to obtained in operation a, the offspring that selection carries multiple synergy allelic variations carry out field
Between cultivate, and combine actual observation, carry out Single-plant selection or carrying out mixing selection to the consistent family of phenotype, obtain F2 single plant or strain
System；
C. each F2 for operating primary election in b is selfed respectively for single plant or strain, obtains F3 for seed, and operate a according to step (7)
In method F3 is nursed young plants in hothouses for seed, extract DNA, is carried out using with the associated molecular labeling primer of objective trait
PCR amplification and gel electrophoresis analysis；
D. the gel electrophoresis strip position according to obtained in operation a, the offspring that selection carries multiple synergy allelic variations carry out field
Between cultivate, and combine actual observation, carry out Single-plant selection or carrying out mixing selection to the consistent family of phenotype, obtain F3 single plant or strain
System；
E. the different F3 selected in d will be operated to be selfed respectively for single plant or strain, F4 generation is obtained, therefrom select relatively homozygous and table
Existing type meets the breeding material of breeding objective.
2. a kind of efficient homozygous method for genetic of muskmelon breeding material objective trait according to claim 1, which is characterized in that the step
Suddenly what is taken in (1) is the same area of Muskmelon Plants homolog.
3. a kind of efficient homozygous method for genetic of muskmelon breeding material objective trait according to claim 1, which is characterized in that the step
Suddenly in (3) RAPD and ISSR molecular labeling PCR product in 1 × TAE buffer with electrophoresis, SSR marker on 2% Ago-Gel
PCR product carries out electrophoresis on 12% non-denaturing polyacrylamide gel.
4. a kind of efficient homozygous method for genetic of muskmelon breeding material objective trait according to claim 1, which is characterized in that the step
Suddenly phenotypic data includes begin flower section, blade profile, section of bearing fruit, fruit shapes, fruit colour, pulp colour, fruit in (5) operation c
Real hardness, fruit center refractive power pol, fruit development period, the time of infertility, single fruit weight, per mu yield.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113981135A (en) * | 2021-12-16 | 2022-01-28 | 宁波市农业科学研究院 | Molecular marker for rapidly identifying and purifying parent P149 of melon |
| CN115961068A (en) * | 2022-07-29 | 2023-04-14 | 黑龙江八一农垦大学 | Molecular marker closely linked with melon single-fruit-weight major QTL-sfw2.2 and application thereof |
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| US20140059712A1 (en) * | 2012-08-23 | 2014-02-27 | Seminis Vegetable Seeds, Inc. | Multiple-virus-resistant melon |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113981135A (en) * | 2021-12-16 | 2022-01-28 | 宁波市农业科学研究院 | Molecular marker for rapidly identifying and purifying parent P149 of melon |
| CN113981135B (en) * | 2021-12-16 | 2023-12-29 | 宁波市农业科学研究院 | Molecular marker for rapidly identifying and purifying muskmelon parent P149 |
| CN115961068A (en) * | 2022-07-29 | 2023-04-14 | 黑龙江八一农垦大学 | Molecular marker closely linked with melon single-fruit-weight major QTL-sfw2.2 and application thereof |
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