CN110106270A - The molecular labeling and its application that a kind of and muskmelon yellow seed coat isolates - Google Patents
The molecular labeling and its application that a kind of and muskmelon yellow seed coat isolates Download PDFInfo
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Abstract
The invention discloses a kind of molecular labeling isolated with muskmelon yellow seed coat and its applications.The present invention provides detect in muskmelon genome to be measured whether the substance containing 25-38 nucleotide shown in sequence 3 following 1) -4) at least one of in application: 1) identify or assist whether the kernel seed coat colour for identifying muskmelon to be measured is yellow；2) preparation identifies or assists whether the kernel seed coat colour for identifying muskmelon to be measured is yellow product；3) breeding carries the melon variety of yellow seed coat genotype；4) preparation breeding carries the melon variety product of yellow seed coat genotype.Using the accuracy of molecular markers for identification muskmelon yellow seed coat of the invention up to 100%, it is further useful for muskmelon molecular mark, gene pyramiding breeding etc., it screened, identified in any stage of muskmelon growth, it is high-efficient, specificity is good, accuracy is high, time and cost are greatlyd save, is of great significance to acceleration muskmelon yellow seed coat breeding.
Description
Technical field
The invention belongs to agricultural biological technical fields, and in particular to a kind of molecular labeling isolated with muskmelon yellow seed coat
And its application.
Background technique
Muskmelon (Cucumis melo L.) is Curcurbitaceae (Cucurbitaceae) Cucumis (Cucumis) muskmelon kind 1 year
Raw herbaceous plant, fruit is fragrant and sweet, full of nutrition, is a kind of Important Economic crop planted extensively in world wide.In recent years,
With the increasingly raising of the structural readjustment of rural industry and living standards of the people, the open country and Protectorate cultivation of China's muskmelon are obtained
Tremendous development, the year-round supply for significantly improving muskmelon are horizontal.Because muskmelon has stronger hybrid vigour, muskmelon cenospecies
The production of son, which is cultivated, has huge economic value.Currently, the muskmelon in production belongs to male flower both sexes flower variety mostly, hybridizing
In generation seed production process mainly by the way of artificial pollination, there are larger workload, higher cost and it is easy to mix
Problem, and mostly use field test or molecules inside to mark detection, time-consuming and higher cost the Purity of cenospecies.Such as
Fruit can then greatly save cost when seed production moderate purity identification using macroscopic morphological markers as indication trait.
Muskmelon kernel seed coat colour is divided into the types such as white, yellow and sepia.1936, Hagiwara and Kamimura were for the first time
Muskmelon kernel seed coat colour is analyzed, it is believed that kind skin white be to yellow and sepia it is dominant, by gene Wt (White
Testa it) controls.(1999) such as P é rin have carried out genetic analysis to white kind of a skin character of muskmelon resource PI 414723, show
White kind of skin be to yellow seed coat it is dominant, controlled by a pair of of single dominant gene Wt-2, and by the assignment of genes gene mapping in IV linkage group.?
Prosperous it can wait (2018) that the white gene (WT) for controlling muskmelon kernel seed coat colour is located in the 5th linkage group, both ends linked marker is
HD0520 and HD0519, the genetic distance with linked marker are respectively 13.3cM and 7cM.In muskmelon first generation of hybrid breeding process
In, since yellow seed coat color is easy range estimation identification, seed can be applied to using them as the mark property of hybrids seed purity test
Mechanical admixture, biology hybrid and hybrid seed purity Preliminary Identification range estimation character.But the yellow seed coat of muskmelon is hidden
Property gene control, and planting skin itself is from mother cell development, and character is controlled by maternal gene, the genotype of seed
The character of control will can just be showed in the next generation, typical delayed ingeritance be shown as, therefore, it is necessary to which seed kind is gone down simultaneously
The genotype of previous generation could be judged according to the separation situation of progeny seed.Therefore, conventional hybridization method breeding yellow kind is utilized
Skin kind is bothersome laborious, and the specific molecular marker of exploitation and yellow seed coat gene-correlation carries out the molecule mark of yellow seed coat gene
Remember assisted Selection, efficiency of selection can be improved during progeny selection, breeding cost is reduced, as early as possible by yellow seed coat character application
In Hybrid seed purity test, it is of great significance to promotion muskmelon industrialized development.
Summary of the invention
Molecular labeling that technical problem to be solved by the invention is to provide a kind of to isolate with muskmelon yellow seed coat and its
Using, the invention provides the following technical scheme:
Whether it is an object of the present invention to provide detect in muskmelon genome to be measured containing 25-38 shown in sequence 3
The purposes of the substance of the nucleotide of position.
Whether contain 25-38 shown in sequence 3 nucleotide in detection provided by the invention muskmelon genome to be measured
Substance following 1) -6) at least one of in application:
1) identify or assist the seed coat color gene type of identification muskmelon to be measured；
2) preparation identification or auxiliary identify the seed coat color gene type of muskmelon to be measured；
3) breeding carries the melon variety of yellow seed coat genotype；
4) preparation breeding carries the product of the melon variety of yellow seed coat genotype；
5) breeding filial generation is the melon variety of non-yellow kind skin；
6) preparation breeding filial generation is the product of the melon variety of non-yellow kind skin；
The seed coat color gene type is yellow seed coat genotype A2A2, non-yellow seed coat gene type A1A1 or non-yellow kind
Skin genotype A1A2；
The yellow seed coat genotype A2A2 is to contain sequence 4 or not in 2 homologues of muskmelon genome
Contain the 25-38 nucleotide of sequence 3；
The non-yellow seed coat gene type A1A1 is to contain sequence 3 or equal in 2 homologues of muskmelon genome
Contain the 25-38 nucleotide of sequence 3；
The non-yellow seed coat gene type A1A2 is to contain sequence 3 in 1 homologue of muskmelon genome or contain
The nucleotide of sequence 3 25-38, containing sequence 4 or without containing sequence 3 25-38 in another article of homologue
Nucleotide.
Whether it is a further object to provide detect in muskmelon genome to be measured containing sequence 3 and/or sequence 4
The purposes of substance.
The present invention provides detect in muskmelon genome to be measured whether the substance containing sequence 3 and/or sequence 4 it is following 1)-
At least one of 6) application in:
1) identify or assist the seed coat color gene type of identification muskmelon to be measured；
2) preparation identification or auxiliary identify the seed coat color gene type of muskmelon to be measured；
3) breeding carries the melon variety of yellow seed coat genotype；
4) preparation breeding carries the product of the melon variety of yellow seed coat genotype；
5) breeding filial generation is the melon variety of non-yellow kind skin；
6) preparation breeding filial generation is the product of the melon variety of non-yellow kind skin；
The seed coat color gene type is yellow seed coat genotype A2A2, non-yellow seed coat gene type A1A1 or non-yellow kind
Skin genotype A1A2；
The yellow seed coat genotype A2A2 is to contain sequence 4 or not in 2 homologues of muskmelon genome
Contain the 25-38 nucleotide of sequence 3；
The non-yellow seed coat gene type A1A1 is to contain sequence 3 or equal in 2 homologues of muskmelon genome
Contain the 25-38 nucleotide of sequence 3；
The non-yellow seed coat gene type A1A2 is to contain sequence 3 in 1 homologue of muskmelon genome or contain
The nucleotide of sequence 3 25-38, containing sequence 4 or without containing sequence 3 25-38 in another article of homologue
Nucleotide.
In above-mentioned application, whether contain 25-38 shown in sequence 3 nucleosides in detection muskmelon genome to be measured
The substance of acid or, in detection muskmelon genome to be measured whether the substance containing sequence 3 and/or sequence 4 be it is following 1) or 2):
1) primer set, primer set single strand dna as shown in sequence 1 in sequence table (YS-F) or its derivative
Single strand dna shown in sequence 2 (YS-R) or derivatives thereof in object sequence table；
2) contain the PCR reagent or kit of the primer set.
The substitution and/or deletion and/or addition of said one or several nucleotide are the substitution no more than 10 nucleotide
And/or deletion and/or addition.
The primer pair meets: carrying out the DNA fragmentation that PCR amplification obtains as template using muskmelon genomic DNA and contains sequence 3
Or sequence shown in sequence 4, wherein the difference of sequence 3 and sequence 4 be whether the 25-38 nucleotide containing sequence 3.
In above-mentioned primer set, the molar ratio of the primer YS-F and the primer YS-R are 1:1.
PCR reagent containing above-mentioned primer pair is also the scope of protection of the invention.
In above-mentioned PCR reagent, the concentration in the PCR reagent of primer YS-F and primer YS-R in the primer pair
It is 0.5 μm of ol/L.
In above-mentioned application, the derivative of single strand dna shown in sequence 1 is that sequence 1 is passed through one in the sequence table
The substitution and/or deletion and/or addition of a or several nucleotide and with the DNA molecular with the same function of sequence 1；
The derivative of single strand dna shown in sequence 2 is that sequence 2 is passed through one or several nucleosides in the sequence table
Acid substitution and/or deletion and/or addition and with the DNA molecular with the same function of sequence 2.
It is a still further object of the present invention to provide a kind of products.
Whether contain 25- shown in sequence 3 in product provided by the invention, including the detection muskmelon genome to be measured
The substance of 38 nucleotide, or, in detection muskmelon genome to be measured whether the substance containing sequence 3 and/or sequence 4；
At least one of the product has the function of following 1) -3):
1) identify or assist the seed coat color gene type of identification muskmelon to be measured；
2) breeding carries the melon variety of yellow seed coat genotype；
3) breeding filial generation is the melon variety of non-yellow kind skin.
The present invention also provides a kind of biomaterials comprising 25-38 nucleotide shown in sequence 3 or including sequence
DNA molecular shown in DNA molecular shown in 3 and/or sequence 4；
Or, the present invention also provides the biomaterials following 1) -3) at least one of in application:
1) identify or assist the seed coat color gene type of identification muskmelon to be measured；
2) breeding carries the melon variety of yellow seed coat genotype；
3) breeding filial generation is the melon variety of non-yellow kind skin.
The present invention also provides a kind of methods that the seed coat color gene type of muskmelon to be measured is identified in identification or auxiliary, including such as
Lower step: it detects in the genome of muskmelon to be measured whether containing 25-38 shown in sequence 3 nucleotide, if described to be measured
25-38 shown in sequence 3 nucleotide is not contained in the genome of muskmelon, then the seed coat color gene of the muskmelon to be measured
Type is yellow seed coat genotype A2A2；If containing 25-38 shown in sequence 3 nucleosides in the genome of the muskmelon to be measured
Acid, then the seed coat color gene type of the muskmelon to be measured is non-yellow seed coat gene type A1A1 or non-yellow seed coat gene type
A1A2；
The yellow seed coat genotype A2A2 is to contain sequence 4 or not in 2 homologues of muskmelon genome
Contain the 25-38 nucleotide of sequence 3；
The non-yellow seed coat gene type A1A1 is to contain sequence 3 or equal in 2 homologues of muskmelon genome
Contain the 25-38 nucleotide of sequence 3；
The non-yellow seed coat gene type A1A2 is to contain sequence 3 in 1 homologue of muskmelon genome or contain
The nucleotide of sequence 3 25-38, containing sequence 4 or without containing sequence 3 25-38 in another article of homologue
Nucleotide.
The present invention also provides a kind of method that the filial generation kernel seed coat colour of muskmelon to be measured is identified in identification or auxiliary, including it is as follows
Step:
It detects in the genome of muskmelon to be measured whether containing 25-38 shown in sequence 3 nucleotide, if described to be measured
25-38 shown in sequence 3 nucleotide is not contained in the genome of muskmelon, then the filial generation kind skin face of the muskmelon to be measured
Color is or candidate is yellow；If containing 25-38 shown in sequence 3 nucleotide in the genome of the muskmelon to be measured,
The muskmelon seed to be measured for kernel seed coat colour is not or candidate is not yellow.
The melon variety of yellow seed coat genotype is carried the present invention also provides a kind of breeding or breeding filial generation is yellow kind
The method of the melon variety of skin is the muskmelon to be measured that the above-mentioned seed coat color gene type of breeding is yellow seed coat genotype A2A2.
The melon variety of non-yellow seed coat gene type is carried the present invention also provides a kind of breeding or breeding filial generation is non-Huang
It is non-yellow seed coat gene type A1A1 or A1A2 that the method for the melon variety of color kind skin, which is the above-mentioned seed coat color gene type of breeding,
Muskmelon to be measured.
In the above method, whether contain 25-38 shown in sequence 3 nucleosides in detection muskmelon genome to be measured
The method of acid is as follows:
1) direct Sequencing；
2) muskmelon to be measured is expanded with the primer set, detects amplified production；
If the amplified production does not contain sequence 3 only containing the segment of 117bp in the genome of the muskmelon to be measured
Shown in 25-38 nucleotide；
If the amplified production contains the segment of 131bp, contain shown in sequence 3 in the genome of the muskmelon to be measured
25-38 nucleotide.
PCR amplification is carried out using the primer pair, following reaction system specifically can be used and carry out PCR amplification: 0.8 μ L
Described in Buffer, 0.8 μ L dNTPs (concentration of four kinds of every kind of dNTP is 2mmol/L), the YS-F and the YS-R, 1 μ L
Muskmelon genomic DNA (50ng/ μ L) to be measured, 0.1 μ LDNA Polymerase for PAGE, ddH2O is mended to 10 μ
L.Buffer andDNA Polymerase for PAGE can produce for Beijing Quanshijin Biotechnology Co., Ltd
Product.
Annealing temperature when carrying out PCR amplification using the primer pair can be 59 DEG C.PCR expansion is carried out using the primer pair
Following reaction condition: 94 DEG C of initial denaturation 4min specifically can be used in increasing；94 DEG C of denaturation 30s, 59 DEG C of annealing 30s, 72 DEG C of extension 30s,
35 circulations；72 DEG C of extension 5min.
The experiment proves that present invention discover that muskmelon yellow seed coat molecular labeling (YS) and muskmelon yellow seed coat
Correlation, two articles of chromosomes are free of A2A2 genotype muskmelon offspring's kind of 25-38 nucleotide of sequence 3 in ordered list
Son is yellow seed coat.Whether there is yellow seed coat genotype using muskmelon yellow seed coat molecular markers for identification muskmelon of the invention
For accuracy up to 100%, muskmelon yellow seed coat molecular labeling of the invention is further useful for muskmelon molecular mark,
Screened, identified in any stage of muskmelon growth, it is high-efficient, specificity is good, accuracy is high, greatly save the time and at
This, is of great significance to the acceleration short climing breeding of muskmelon.
Detailed description of the invention
Fig. 1 is melon variety east honey No.1 (white kind of skin) and beautiful (yellow seed coat) phenotype of yellow interest.
Fig. 2 is the genotyping for marking YS to parent and offspring；Wherein P1: east honey No.1；P2: yellow interest is beautiful；F1:
East honey No.1 makees the hybridization F that maternal and yellow interest jade makees male parent preparation1；1-36 is yellow seed coat F2For single plant；M:100bp
DNA ladder；.
Fig. 3 is label YS to the testing result in 12 melon varieties；Wherein 1-12 is respectively east honey No.1, selected flower
Crisp, the red-way side of honey, especially big white sand honey, U.S. logical sequence ride on Bus No. 11, avenge beautiful crisp No.1, beautiful, the yellow seed Jinxiangyu of yellow interest, emperor's interest, glad spring,
Clear monarch No. nine, early spring honey, the beautiful crisp, Rui Jin of snow；M:100bp DNA ladder.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Only the present invention will be further described for following embodiment, is not intended to limit the present invention.In embodiment unless otherwise specified,
Used experimental method is conventional method；Reagent etc. used, can be obtained by commercial sources.
Test material as used in the following examples, wherein white kind of a skin material east honey No.1 is recorded in: " Zhang Qun, king
Asia, the east the Wang Ming Shanghai honey No.1 high-yield culture technique [J] vegetables, 2016 (1): the text of 77-78 " one is inventor from Xinjiang
Hao Le seed Co., Ltd buys, and the public can obtain from applicant, the biomaterial only attach most importance to duplicate invention related experiment
It is used, it not can be used as other purposes and use；Yellow seed coat material Huang interest jade is recorded in " Dai Zuyun, Zhao Hui, Xia Chengdong, Yang Pei
Column, breeding [J] .2006 (1): 23-25 " one text of the precocious resistance to dense Calusena lansium melon variety Huang interest jade of storage of Liu Yongzhong, is inventor
From Anhui, Yangze river and Huai river gardening Zhong Ye Science and Technology Co., Ltd. is bought, and the public can obtain from applicant, which only attaches most importance to
Used in the related experiment of duplicate invention, it not can be used as other purposes and use.Remaining material used can be obtained through commercial channels.
Embodiment 1, the screening with muskmelon kernel seed coat colour related molecular marker
1, the building and genetic analysis of segregating population
A white kind of skin thick-skinned melon variety east honey No.1, a yellow kind are screened from the melon variety of collection
The thin osculant melon variety Huang interest of skin depth is beautiful (Fig. 1).Make maternal, yellow interest jade using east honey No.1 and makees male parent preparing hybrid
Combination, obtained F1Seed kind skin is white, makees male parent, the maternal preparing hybrid combination of yellow interest jade work using east honey No.1,
Obtained F1Seed kind skin is yellow, shows that kernel seed coat colour shows delayed ingeritance effect, and F1Seed kind skin is sent out by mother cell
It educates.
East honey No.1 is made into maternal, yellow interest jade and makees the obtained F of paternal hybrid1The F obtained after selfing2Seed total 470
Strain divides single-strain planting in field, and each single plant is selfed respectively, and the maturity period harvests seed, that is, F3In generation, investigates each single plant after seed dries
Seed phenotypes, to obtain F2For plant genotype.Data statistic analysis shows F2In generation, bears seeds (F3Generation) kernel seed coat colour point
Be white from ratio: yellow=358:112 shows the Mendelian inheritance segregation ratio (χ of 3:12=0.28 < 3.84, p value=
0.59), these are statistics indicate that yellow seed coat character is controlled by single stealthy karyogene.
2, the exploitation and screening of molecular labeling
Genome sequence (https: //www.melonomics.net/) is referred to according to muskmelon, utilizes online software
Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi? LINK_
LOC=BlastHome specific primer) is designed.
Extracting parent east honey No.1, yellow interest jade, F1 respectively using CTAB method, (yellow interest jade is male parent, east honey
No.1 is the filial generation that hybridization of female parent obtains) and yellow seed coat F2The genomic DNA of single plant.Choose 15 yellow seed coat F2Single plant
Genomic DNA mixed in equal amounts establishes mixed pond, in addition totally three samples sieve 120 pairs of muskmelon primers of synthesis to two parents
Choosing.The reaction system of PCR amplification is as follows: 0.8 μ L Buffer, 0.8 μ L dNTPs (2mmol/L each), 1 μ L Primers, 1
μ L genomic DNA (50ng/ μ L), 0.1 μ LDNA Polymerase for PAGE is (purchased from the full formula gold biology in Beijing
Technology Co., Ltd.) and 6.3 μ L ddH2O.The response procedures of PCR amplification are 94 DEG C of initial denaturation 4min；94 DEG C of denaturation 30s, 59 DEG C
Anneal 30s, and 72 DEG C of extension 30s run 35 circulations；72 DEG C of extension 5min.It is detected using 8% polyacrylamide gel electrophoresis
Pcr amplification product.
The result shows that: mixed pond DNA, yellow interest jade and No. 5 chromosomal marker YS show linkage relationship.
Primer R shown in label YS primers F shown in sequence 1 and sequence 2 is formed,
Primers F: CTTATCCGCCTCCGACACC (sequence 1)；
Primer R:AGTTAACGCACCCGAACCC (sequence 2).
Therefore, label YS can be used for identifying muskmelon kernel seed coat colour to be measured.
Embodiment 2, molecular labeling YS identify the seed coat color gene type of muskmelon
Utilize the primer pair parent east honey No.1 of the YS label of the acquisition of embodiment 1, yellow interest jade, F1(east honey No.1
Make maternal, yellow interest jade and makees the obtained F of paternal hybrid1) and above-mentioned 470 F2For single plant (white 358 plants of kind of skin single plant, yellow kind
112 plants of skin single plant) genotyping is carried out, specific as follows:
The single plant genomic DNA extracted using CTAB method as template, with the primers F of above-mentioned label YS and primer R into
Row PCR amplification, obtains pcr amplification product.
The reaction system of above-mentioned PCR amplification is as follows: 0.8 μ L Buffer, 0.8 μ L dNTPs (2mmol/L each), 1 μ L
Primers (10 μm of ol/L, F+R (the reaction final concentration of every primer is 0.5 μm of ol/L), 1 μ L genomic DNA (50ng/ μ
L)、0.1μLDNA Polymerase for PAGE (being purchased from Beijing Quanshijin Biotechnology Co., Ltd) and 6.3
μL ddH2O。
The response procedures of above-mentioned PCR amplification are 94 DEG C of initial denaturation 4min；94 DEG C of denaturation 30s, 59 DEG C of annealing 30s, 72 DEG C are prolonged
30s is stretched, 35 circulations are run；72 DEG C of extension 5min.
8% polyacrylamide gel electrophoresis detects pcr amplification product.
Partial results are as shown in Fig. 2, mark YS to parent and part F2It is detected for single plant pcr amplification product；P1: east honey
No.1；P2: yellow interest is beautiful；1-36: yellow seed coat F2Single plant；M:100bp DNA ladder；As can be seen that PCR in two parents
Amplified production is range in 100-200bp and segment of different sizes, and F1 generation is containing there are two pcr amplification products in parent；
By sequencing, from P1The nucleotides sequence of the target fragment amplified in (east honey No.1) is classified as sequence 3, and size is
131bp, the genotype represented are named as A1A1 (containing sequence 3 in 2 homologues)；
From P2The nucleotides sequence of the target fragment amplified in (yellow interest is beautiful) is classified as sequence 4, size 117bp, by it
The genotype of representative is named as A2A2 (containing sequence 4 in 2 homologues)；
F1It is white kind of skin for kernel seed coat colour, simultaneously containing nucleotide sequence shown in sequence 3 and sequence 4, genotype is
A1A2 (one contains sequence 3 in 2 homologues, and one contains sequence 4)；
470 F2For in single plant, white 358 plants of kind of skin (contains comprising 120 plants of A1A1 genotype in 2 homologues
Have sequence 3), 238 plants of A1A2 heterozygous genotypes (one contains sequence 3 in 2 homologues, and one contains sequence 4)；Yellow
Kind 112 pnca gene type of skin is all A2A2 (containing sequence 4 in 2 homologues).
It is in Fig. 2 the result shows that, 1-36 yellow seed coat F2Single plant and parent P2(yellow interest is beautiful) banding pattern unanimously shows to be divided into
From.
By sequence alignment, the difference of 4 segment of sequence 3 and sequence is, the 25-38 institutes of sequence 3 are not contained in sequence 4
The nucleotide shown.
Above the experimental results showed that, the kind skin of the Product Sequence 3 or sequence 4 and muskmelon of label YS of the invention and its amplification
Color gene type is related, can be used to detect the seed coat color gene type of muskmelon.
Therefore, whether contain 25-38 shown in sequence 3 Nucleotide identities by detecting in muskmelon genome to be measured
Or auxiliary identifies whether the seed coat color gene type of muskmelon to be measured or the filial generation seed kind skin of muskmelon to be measured are yellow, specifically such as
Under:
It detects in the genome of muskmelon to be measured whether containing 25-38 shown in sequence 3 nucleotide, if without orderly
25-38 nucleotide shown in column 3, then the filial generation kernel seed coat colour of muskmelon to be measured is yellow, then the kind skin face of muskmelon to be measured
Color genotype is yellow seed coat genotype A2A2；If containing 25-38 shown in sequence 3 nucleotide, muskmelon to be measured
Filial generation kernel seed coat colour is not yellow, then the seed coat color gene type of muskmelon to be measured is non-yellow seed coat gene type A1A1 or non-yellow
Seed coat gene type A1A2；
Yellow seed coat genotype A2A2 is to contain sequence 4 in 2 homologues of muskmelon genome to be measured；
Non-yellow seed coat gene type A1A1 is to contain sequence 3 in 2 homologues of muskmelon genome to be measured；
Non-yellow seed coat gene type A1A2 is to contain sequence 3 in 1 homologue of muskmelon genome to be measured, another
Contain sequence 4 in homologue；
Or whether identify or assist containing sequence 3 and/or sequence 4 in muskmelon genome to be measured to identify sweet tea to be measured by detecting
Whether the seed coat color gene type of melon or the filial generation seed kind skin of muskmelon to be measured are Huang, specific as follows:
It whether detects in the genome of muskmelon to be measured containing shown in sequence 3 and/or DNA molecular shown in sequence 4,
If without containing sequence 3 and containing sequence 4 in the genome of muskmelon to be measured, the filial generation kernel seed coat colour of muskmelon to be measured is
Or candidate is yellow, then the seed coat color gene type of muskmelon to be measured is yellow seed coat genotype A2A2；
If having contained only sequence 3 in the genome of muskmelon to be measured and without containing sequence 4, the filial generation kernel seed coat colour of muskmelon to be measured
It is not or candidate is not yellow, then the seed coat color gene type of muskmelon to be measured is non-yellow seed coat gene type A1A1；
If containing sequence 3 and sequence 4 in the genome of muskmelon to be measured, the filial generation kernel seed coat colour of muskmelon to be measured is not or waits
Choosing is not yellow, then the seed coat color gene type of muskmelon to be measured is non-yellow seed coat gene type A1A2.
Whether the method containing 25-38 nucleotide shown in sequence 3 is in above-mentioned detection muskmelon genome to be measured
It is as follows:
1) direct Sequencing；
2) with the primer pair amplifies muskmelon to be measured of label YS, amplified production is detected；
If amplified production only contains 117bp, 25-38 are not contained shown in sequence 3 in the genome of muskmelon to be measured
Nucleotide；
If amplified production contains the segment (the only segment containing 131bp or the segment containing 117bp and 131bp) of 131bp,
Then contain 25-38 shown in sequence 3 nucleotide in the genome of muskmelon to be measured.
Whether containing shown in sequence 3 and/or DNA molecular shown in sequence 4 in the genome of above-mentioned detection muskmelon to be measured
Method is as follows:
1) direct Sequencing；
2) with the primer pair amplifies muskmelon to be measured of label YS, amplified production is detected；
If amplified production size is only 117bp, containing sequence 4 and without containing sequence 3 in the genome of muskmelon to be measured；
If amplified production size is only 131bp, containing sequence 3 and without containing sequence 4 in the genome of muskmelon to be measured；
If amplified production size is 131bp and 117bp, contain sequence 3 and sequence 4 in the genome of muskmelon to be measured.
The application of embodiment 3, label YS in muskmelon yellow seed coat molecular marker assisted selection kind
In order to carry out the molecular marker assisted selection breeding of yellow seed coat gene, and verification mark YS is in kernel seed coat colour
Accuracy, according to the method in embodiment 2, using YS label to other different heredity in addition to east honey No.1, yellow interest are beautiful
12 parts of melon varieties of background carried out it is actually detected, altogether identify table 1 14 parts of melon varieties.It is specific as follows:
Table 1 is 14 parts of melon variety information
The genomic DNA that every part of material is extracted using CTAB method, according to the method in embodiment 2 using label YS to table
14 parts of muskmelon materials in 1 have carried out PCR amplification, obtain pcr amplification product.
If amplified production only contains 117bp, 25-38 are not contained shown in sequence 3 in the genome of muskmelon to be measured
Nucleotide；
If amplified production contains the segment (the only segment containing 131bp or the segment containing 117bp and 131bp) of 131bp,
Then contain 25-38 shown in sequence 3 nucleotide in the genome of muskmelon to be measured.
If without containing sequence 3 and containing sequence 4 in the genome of muskmelon to be measured, the filial generation kernel seed coat colour of muskmelon to be measured is
Or candidate is yellow, then the seed coat color gene type of muskmelon to be measured is yellow seed coat genotype A2A2；
If having contained only sequence 3 in the genome of muskmelon to be measured and without containing sequence 4, the filial generation kernel seed coat colour of muskmelon to be measured
It is not or candidate is not yellow, then the seed coat color gene type of muskmelon to be measured is non-yellow seed coat gene type A1A1；
If containing sequence 3 and sequence 4 in the genome of muskmelon to be measured, the filial generation kernel seed coat colour of muskmelon to be measured is not or waits
Choosing is not yellow, then the seed coat color gene type of muskmelon to be measured is non-yellow seed coat gene type A1A2.
As a result as shown in figure 3,1-6,7-12,13 and 14 respectively correspond 14 kinds in table 1；Wherein 5 parts of white kind skins
Kind (selected nectar is crisp, red-way side, especially big white sand honey, U.S. logical sequence ride on Bus No. 11, avenges crisp No.1), genotype consistent with east honey No.1
For A1A1；Two parts of white kind skin kind (the beautiful crisp, Rui Jin of snow) genotype are heterozygosis A1A2；(yellow seed gold is fragrant for 5 parts of yellow seed coat varieties
Beautiful, emperor's interest jade, glad spring, clear monarch No. nine, early spring honey) it is beautiful unanimously with yellow interest, genotype A2A2 identifies that accuracy rate is
100%, illustrate to mark YS can Rapid identification muskmelon kernel seed coat colour, and distinguish A1A1, A1A2 and A2A2 genotype.
Based on the above results, using yellow seed coat material and white kind skin material using conventional sexual hybridization and backcross breeding
Method, be aided with label YS carry out molecular marker assisted selection, can quickly breeding go out carry yellow seed coat genotype muskmelon new product
Kind, greatly improve breeding efficiency.
Above-described embodiment is only the illustrative description of the present invention, but embodiments of the present invention are not by above-described embodiment
The limitation of kind or material, it is other it is any do not violate the change made under spirit of the invention and principle, modification, substitution,
Combination simplifies, and should be equivalent substitute mode, falls within the scope of protection of the present invention.
Sequence table
<110>Beijing City Agriculture and Forestry Institute
<120>a kind of molecular labeling isolated with muskmelon yellow seed coat and its application
<160> 4
<170> PatentIn version 3.5
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<211> 19
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<213> Artificial sequence
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cttatccgcc tccgacacc 19
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<213> Artificial sequence
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agttaacgca cccgaaccc 19
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<212> DNA
<213> Artificial sequence
<400> 3
cttatccgcc tccgacaccc tccgtcgccc cacctccgcc gctctctccc ccgaggactt 60
gactgagact gagtggttct atttgttgtg tctctctttc tcttttcctc cggggttcgg 120
gtgcgttaac t 131
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cttatccgcc tccgacaccc tccgccgctc tctcccccga ggacttgact gagactgagt 60
ggttctattt gttgtgtctc tctttctctt ttcctccggg gttcgggtgc gttaact 117
Claims (10)
1. detect in muskmelon genome to be measured whether the substance containing 25-38 nucleotide shown in sequence 3 following 1) -6)
At least one of in application:
1) identify or assist the seed coat color gene type of identification muskmelon to be measured；
2) preparation identification or auxiliary identify the seed coat color gene type of muskmelon to be measured；
3) breeding carries the melon variety of yellow seed coat genotype；
4) preparation breeding carries the product of the melon variety of yellow seed coat genotype；
5) breeding filial generation is the melon variety of non-yellow kind skin；
6) preparation breeding filial generation is the product of the melon variety of non-yellow kind skin；
The seed coat color gene type is yellow seed coat genotype A2A2, non-yellow seed coat gene type A1A1 or non-yellow kind scytoblastema
Because of type A1A2；
The yellow seed coat genotype A2A2 is to contain sequence 4 in 2 homologues of muskmelon genome or do not contain
The nucleotide of sequence 3 25-38；
The non-yellow seed coat gene type A1A1 is to contain sequence 3 in 2 homologues of muskmelon genome or contain
The nucleotide of sequence 3 25-38；
The non-yellow seed coat gene type A1A2 be muskmelon genome 1 homologue in containing sequence 3 or contain sequence 3
25-38 nucleotide, containing sequence 4 or without containing sequence 3 25-38 nucleosides in another article of homologue
Acid.
2. detect in muskmelon genome to be measured whether the substance containing sequence 3 and/or sequence 4 following 1) -6) at least one of in
Application:
1) identify or assist the seed coat color gene type of identification muskmelon to be measured；
2) preparation identification or auxiliary identify the seed coat color gene type of muskmelon to be measured；
3) breeding carries the melon variety of yellow seed coat genotype；
4) preparation breeding carries the product of the melon variety of yellow seed coat genotype；
5) breeding filial generation is the melon variety of non-yellow kind skin；
6) preparation breeding filial generation is the product of the melon variety of non-yellow kind skin；
The seed coat color gene type is yellow seed coat genotype A2A2, non-yellow seed coat gene type A1A1 or non-yellow kind scytoblastema
Because of type A1A2；
The yellow seed coat genotype A2A2 is to contain sequence 4 in 2 homologues of muskmelon genome or do not contain
The nucleotide of sequence 3 25-38；
The non-yellow seed coat gene type A1A1 is to contain sequence 3 in 2 homologues of muskmelon genome or contain
The nucleotide of sequence 3 25-38；
The non-yellow seed coat gene type A1A2 be muskmelon genome 1 homologue in containing sequence 3 or contain sequence 3
25-38 nucleotide, containing sequence 4 or without containing sequence 3 25-38 nucleosides in another article of homologue
Acid.
3. applying according to claim 1 or shown in 2, it is characterised in that:
Whether the substance containing 25-38 nucleotide shown in sequence 3 is or, described in the detection muskmelon genome to be measured
Detect in muskmelon genome to be measured whether the substance containing sequence 3 and/or sequence 4 be it is following 1) or 2):
1) primer set, primer set single strand dna as shown in sequence 1 in sequence table or derivatives thereof sequence table
Single strand dna shown in middle sequence 2 or derivatives thereof；
2) contain the PCR reagent or kit of the primer set.
4. application according to claim 3, it is characterised in that:
The derivative of single strand dna shown in sequence 1 is by sequence 1 by one or several nucleotide in the sequence table
Replace and/or deletion and/or addition and with the DNA molecular with the same function of sequence 1；
The derivative of single strand dna shown in sequence 2 is by sequence 2 by one or several nucleotide in the sequence table
Replace and/or deletion and/or addition and with the DNA molecular with the same function of sequence 2.
5. whether containing shown in sequence 3 in a kind of product, including detection muskmelon genome to be measured described in claim 3 or 4
The substance of 25-38 nucleotide, or, whether containing sequence in detection muskmelon genome to be measured described in claim 3 or 4
3 and/or sequence 4 substance；
At least one of the product has the function of following 1) -3):
1) identify or assist the seed coat color gene type of identification muskmelon to be measured；
2) breeding carries the melon variety of yellow seed coat genotype；
3) breeding filial generation is the melon variety of non-yellow kind skin；
Or, biomaterial comprising 25-38 nucleotide shown in sequence 3 or including DNA molecular shown in sequence 3 and/
Or DNA molecular shown in sequence 4；
Or, the biomaterial is following 1) -3) at least one of in application:
1) identify or assist the seed coat color gene type of identification muskmelon to be measured；
2) breeding carries the melon variety of yellow seed coat genotype；
3) breeding filial generation is the melon variety of non-yellow kind skin.
6. a kind of method that identification or auxiliary identify the seed coat color gene type of muskmelon to be measured, includes the following steps: to detect to be measured
Whether containing 25-38 shown in sequence 3 nucleotide in the genome of muskmelon, if in the genome of the muskmelon to be measured not
Containing 25-38 shown in sequence 3 nucleotide, then the seed coat color gene type of the muskmelon to be measured is yellow seed coat gene
Type A2A2；If containing 25-38 shown in sequence 3 nucleotide, the sweet tea to be measured in the genome of the muskmelon to be measured
The seed coat color gene type of melon is non-yellow seed coat gene type A1A1 or non-yellow seed coat gene type A1A2；
The yellow seed coat genotype A2A2 is to contain sequence 4 in 2 homologues of muskmelon genome or do not contain
The nucleotide of sequence 3 25-38；
The non-yellow seed coat gene type A1A1 is to contain sequence 3 in 2 homologues of muskmelon genome or contain
The nucleotide of sequence 3 25-38；
The non-yellow seed coat gene type A1A2 be muskmelon genome 1 homologue in containing sequence 3 or contain sequence 3
25-38 nucleotide, containing sequence 4 or without containing sequence 3 25-38 nucleosides in another article of homologue
Acid.
7. a kind of method that identification or auxiliary identify the filial generation kernel seed coat colour of muskmelon to be measured, includes the following steps:
It detects in the genome of muskmelon to be measured whether containing 25-38 shown in sequence 3 nucleotide, if the muskmelon to be measured
Genome in without containing 25-38 shown in sequence 3 nucleotide, then the filial generation kernel seed coat colour of the muskmelon to be measured is
Or candidate is yellow；If described containing 25-38 shown in sequence 3 nucleotide in the genome of the muskmelon to be measured
Muskmelon seed to be measured for kernel seed coat colour is not or candidate is not yellow.
8. a kind of breeding carries melon variety or the breeding filial generation of yellow seed coat genotype as the side of the melon variety of yellow seed coat
Method is the muskmelon to be measured that seed coat color gene type described in breeding claim 6 is yellow seed coat genotype A2A2.
9. melon variety or breeding filial generation that a kind of breeding carries non-yellow seed coat gene type are the melon variety of non-yellow kind skin
Method, be seed coat color gene type described in breeding claim 6 be the to be measured of non-yellow seed coat gene type A1A1 or A1A2
Muskmelon.
10. method according to claim 6 or 7, it is characterised in that: whether contain in the detection muskmelon genome to be measured
The method of 25-38 nucleotide is as follows shown in sequence 3:
1) direct Sequencing；
2) primer set described in claim 3 or 4 expands the muskmelon to be measured, detects amplified production；
If the amplified production is only containing the segment of 117bp, without containing shown in sequence 3 in the genome of the muskmelon to be measured
25-38 nucleotide；
If the amplified production contains the segment of 131bp, shown in sequence 3 the is contained in the genome of the muskmelon to be measured
25-38 nucleotide.
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| CN114763579A (en) * | 2021-01-13 | 2022-07-19 | 北京市农林科学院 | Molecular marker related to melon fruit shape and method and application thereof |
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