CN105802960B - Molecular labeling and its application - Google Patents
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- CN105802960B CN105802960B CN201410834541.4A CN201410834541A CN105802960B CN 105802960 B CN105802960 B CN 105802960B CN 201410834541 A CN201410834541 A CN 201410834541A CN 105802960 B CN105802960 B CN 105802960B
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Abstract
The invention discloses molecular labeling and its applications.Wherein, which shows as the insertion/deletion length polymorphism of nucleotide sequence shown in SEQ ID NO:1.The fruit colour character of molecular labeling and muskmelon of the invention is closely related, can be effective for the molecular mark of muskmelon.
Description
Technical field
The present invention relates to molecular labeling and its applications.In particular it relates to the relevant molecule mark of muskmelon fruit colour
Note, for detecting the primer pair and kit of the molecular labeling, previous molecular label, primer pair, kit are in muskmelon breeding
Purposes, detect muskmelon fruit colour character method and muskmelon auxiliary breeding means.
Background technique
Muskmelon (Cucumis melo L.) is Curcurbitaceae Cucumis, is a kind of important garden crop, quotient with higher
Industry value and economic value.Existing more than the 3000 years melon wilt history in China, is the country for cultivating muskmelon earliest in the world
One, and current melon wilt area in the world is maximum, the highest country of yield.The fruit of muskmelon is sweet and dilitious, is that China is wide
The tradition fresh food fruit that the big both urban and rural residents is generally fond of.With the development of economy with the raising of living standards of the people, the country is to sweet tea
The demand of melon is growing.It is counted according to FAO (Food and Agriculture Organization of the United Nation) (FAO), muskmelon has entered the column of the big important fruit in the world ten.
Since its color has both, while again containing various composition necessary to human nutrition: carbohydrate, protein, fat,
Calcium, phosphorus, iron, carrotene, VC, VB, VB2, Vpp etc., especially VC content are more than more than ten times of the animal foodstuffs such as milk.Muskmelon
Pulp it is sweet in flavor, cold, sliding, there is the effect of quenching the thirst, relieving restlessness heat, diuresis；Its muskmelon pedicel can be used as medicine, name calyx meto.Muskmelon is used
Way extensively, can also be processed into melon dry, melon dried meat, melon sauce etc. other than it can directly eat.In world wide, status is even wanted
Higher than watermelon.
With the improvement of living standards with the diversity of fruit variety, requirement of the domestic consumer to melon and fruit quality be also more next
It is higher.Melon and fruit breeding is more and more diversified, to meet the market requirement.As muskmelon quality have crisp, crisp, soft, soft and succulence, it is soft and
Few juice etc., muskmelon pulp has the colors such as snow-white, orange red, emerald green, can be selected according to the hobby of different regions consumer.
In existing muskmelon breeding practice, to muskmelon Main Agronomic Characters be mostly by phenotype indirectly to genotype into
Row selection, causes breeding cycle long, and low efficiency restricts the process of melon variety improvement.Marker assisted selection, especially with
The associated functional molecular marker of objective trait carries out assisted Selection, traditional Phenotypic Selection can be changed into direct gene
Type selection accelerates genetic improvement so as to which the efficiency of breeding is greatly improved.And it finds and important economical character close linkage
Molecular labeling, be carry out molecular marker assisted selection (also known as molecular breeding) and map based cloning basis.Thus, exploitation has
The molecular labeling of the important character gene of China's characteristic simultaneously carries out assisted selection research, significant.
Size, shape and color are the important indicators of measurement fruit external sort, while can also be according to these indexs pair
Fruit carries out classification processing.Requirement of the different fruits and vegetables to its exterior quality is different.For muskmelon, color is that muskmelon is critically important
Apparent attribute just shows typical fruit colour when muskmelon reaches certain maturity.Outside fructescence, some muskmelons
Pericarp there is also pericarp and cover line (secondary color or the second color) in addition to there is pericarp background color (primary color), general described
Fruit colour refers to pericarp background color.Muskmelon fruit colour has green, white, yellowish green, yellow, orange etc..When general melon fruit maturation, fruit
Skin color all degree are different to change, if any the original graying green of green or yellow；Milky or Huang are become from white
Color；White etc. is become from light green color.The diversity of fruit colour develops muskmelon breeding towards the direction of diversification, to adapt to
Demand of the market to different type muskmelon.Muskmelon fruit colour gene genetic is more stable, not vulnerable to the influence in environmental condition, i.e.,
Fruit colour character is qualitative character.Thus, the DNA marker with muskmelon fruit colour character close linkage is found, it can be effective
Assist the breeding of the muskmelon of specific fruit colour.
However, the molecular labeling with muskmelon fruit colour gene close linkage at this stage, still has to be excavated.
Summary of the invention
The present invention is directed at least solve one of the technical problems existing in the prior art.For this purpose, one object of the present invention
It is to propose that one kind is related to muskmelon fruit colour character, it can be effective for the molecular labeling of muskmelon breeding.
It should be noted that the present invention is the following work based on inventor and completes: inventor is with muskmelon " gold honey six
Number " it is research object, by constructing breeding population, the deep gene informations such as RAD sequencing, Genotyping are carried out to group's single plant
Analysis, in conjunction with phenotypic evaluation information, using biology information technology association important character and genome sequence (based on current muskmelon
Genome sequencing is completed, and inventor has selected to instruct opening for SNP marker using muskmelon whole genome sequence information
Hair), it quickly located gene/QTL site of control fruit colour character, and successfully develop the molecule mark with its close linkage
Note, so as to provide theoretical foundation for the molecular mark of muskmelon.
According to an aspect of the present invention, the present invention provides a kind of relevant molecular labelings of muskmelon fruit colour.According to
The embodiment of the present invention, the insertion/deletion length that the molecular labeling shows as nucleotide sequence shown in SEQ ID NO:1 are polymorphic
Property.According to an embodiment of the invention, nucleotide sequence shown in SEQ ID NO:1 is following (445bp):
AGAAATCTGTATATGAGCGGGAGCTCATGGCTATTGTCTTATCGATGGAAAAGTGGCGACATTACATA
TTAGGACAGTGTATACTGACCAGAAAGCTCTTCACCACATTCTTGAGCAAAGAGAAGTCCCTTCTGACGTGCAAAA
ATGGGTGACAATATTAATAGAGTTTGACTTTGAAATTTGTTATAGATCAAGAGCAGAAAATAAGCCAGCGGATGCT
CTCTCGCATGCCTGAGGAGGGATATCAGTGCTGGTGATCATGGATTGCCTTAGCAAATACGCTCATTTTTTGATCT
TGGGACACCCTTTTTTGAGGACAGTTGACATGATTTTCATTCAAGAAGTGGTAAGAGTCCATGGATACCCACAATC
CATTGTCTTAGATCGTGGTAGGGTTTCTCTGAGTCACTTTTGGATGGATTTATTACGATTGGAAGAAACTCAA(SEQ
ID NO:1).Wherein, the genetic linkage distance of molecular labeling of the invention and muskmelon fruit colour (background color) gene is 0.11cM.
According to an embodiment of the invention, having nucleotide sequence shown in SEQ ID NO:1 and in the molecular labeling site
Homozygous individual, shows as pericarp yellow green；Lack nucleotide sequence shown in SEQ ID NO:1 and in the molecular labeling site
Homozygous individual, shows as fruit yellow；It is with nucleotide sequence shown in SEQ ID NO:1 and miscellaneous in the molecular labeling site
The individual of conjunction shows as pericarp yellow green.
It should be noted that term " fruit colour " used in herein refers to the pericarp background color of muskmelon, the present invention
Fruit colour character detected is the pericarp background color character of muskmelon, and the statement above-mentioned to fruit colour each means pericarp bottom
Color, such as expression way " showing as pericarp yellow green " refer to that showing as pericarp background color is yellow green.
Inventors have found that by detection muskmelon genomic DNA (its source is unrestricted, such as can be from the kind of muskmelon
Son, plant, pulp etc. extract) in whether have above-mentioned molecular labeling, its fruit colour character can be effectively determined.Specifically
Ground, as previously mentioned, the melon fruit for not having the molecular labeling is fruit yellow, the melon fruit with the molecular labeling is
Pericarp yellow green, thus, when detecting that muskmelon does not have the molecular labeling, then it can determine muskmelon fruit yellow to be measured, and work as
When muskmelon has the molecular labeling, then it can determine muskmelon pericarp yellow green to be measured.Further, when using for the molecular labeling
Specific primer PCR amplification is carried out to muskmelon genomic DNA sample to be measured and when detected through gel electrophoresis, passes through purpose band
Item number and length can effectively determine the fruit colour character of muskmelon to be measured, specifically, when purpose band is one, and institute
When stating purpose band and being about 886bp, muskmelon pericarp yellow green to be measured can be determined；When purpose band is one, and the purpose
When band is about 441bp, muskmelon fruit yellow to be measured can be determined；When purpose band is two, and the length of the purpose band
When degree is respectively about 441bp and 886bp, muskmelon pericarp yellow green to be measured can be determined.Inventor determines as a result, of the invention
The fruit colour character of molecular labeling and muskmelon is closely related, can be effective for the molecular mark of muskmelon.In turn
The requirement of fruit colour can further effectively improve muskmelon breeding material progress Seedling selection according in practical breeding
The efficiency and accuracy of breeding improve the genetic level of muskmelon reproductive population, excellent so as to accurately and efficiently select muskmelon
Non-defective unit kind.For example, being expanded using the specific primer of the molecular labeling to muskmelon genomic DNA to be measured, gel electrophoresis, select
It selects amplified production purpose band to be one and be about the sample to be tested of 886bp, can easily screen and obtain pericarp yellow green
Muskmelon, so as to directly apply to molecular breeding practice.
In addition, according to some embodiments of the present invention, carrying out muskmelon molecular labeling auxiliary using molecular labeling of the invention
Breeding, have the advantages that early screening, save the time, be low in cost, accuracy it is high.
According to another aspect of the present invention, the present invention also provides a kind of for detecting mentioned-above molecule of the invention
The primer pair of label.According to an embodiment of the invention, the primer pair has nucleotide sequence shown in SEQ ID NO:2-3.
Specifically, the sequence of primer pair of the invention is as follows:
5 '-GCTATGGTAACATTGCTGGT-3 ' (SEQ ID NO:2)；
5 '-ATGGTATCAGAGCAAGTGGT-3 ' (SEQ ID NO:3).
According to an embodiment of the invention, carrying out PCR amplification to muskmelon genomic DNA to be measured using the primer pair, and examine
The length for surveying amplified fragments, as the long 886bp of the amplified fragments, the muskmelon to be measured has nucleosides shown in SEQ ID NO:1
The insertion of acid sequence shows as pericarp yellow green；As the long 441bp of the amplified fragments, the muskmelon to be measured lacks SEQ ID
Nucleotide sequence shown in NO:1, shows as fruit yellow；It is described to be measured when the amplified fragments are 441bp and two kinds of 886bp
The molecular labeling site heterozygosis of muskmelon, shows as pericarp yellow green.
According to an embodiment of the invention, detecting the amplified fragments using the preferred agarose gel electrophoresis of gel electrophoresis
Length.
It is surprisingly found by the inventors that can be effectively to the above-mentioned and pericarp face of muskmelon to be measured using primer pair of the invention
Segment where the relevant molecular labeling of color character carries out PCR amplification, and then can effectively be realized to this by sequencing or electrophoresis
The detection of molecular labeling, determines whether muskmelon to be measured has the molecular labeling.For example, being produced when passing through electrophoresis detection according to amplification
The quantity and length of object purpose band can effectively determine the fruit colour character of muskmelon to be measured.Specifically, drawn using above-mentioned
Muskmelon Plants to be measured are carried out PCR amplification and when detected through gel electrophoresis by object pair, when purpose band is one, and the purpose item
When band is about 886bp, muskmelon pericarp yellow green to be measured can be determined；When purpose band is one, and the purpose band is about
When 441bp, muskmelon fruit yellow to be measured can be determined；When purpose band is two, and the length of the purpose band is respectively
When about 441bp and 886bp, muskmelon pericarp yellow green to be measured can be determined.And the amplification of the amplified production and 886bp of 441bp produces
Object is molecular labeling of the invention compared to the sequence of missing, thus molecule labelled series of the invention are located at the amplification of 886bp
In product.It, can be effective for the molecule of muskmelon as a result, for detecting the primer pair of mentioned-above molecular labeling of the invention
Marker-assisted breeding, and then early stage can be assisted to realize short time, low cost, high accuracy ground breeding muskmelon excellent variety.
It should be noted that those skilled in the art are known, the sequence shown in above-mentioned SEQ ID NO:2 and SEQ ID NO:3
In column, 1~10 base can be increased separately at its 5 ' end or 3 ' ends, the increased base type of institute can be according to muskmelon genomic DNA
The base type in the upper region that matches with SEQ ID NO:2 and SEQ ID NO:3 simultaneously determines according to basepairing rule, thus
The amplified production of obtained primer pair and SEQ ID NO:2 and SEQ ID NO:3 are essentially identical (between upstream and downstream primer
DNA sequence dna is identical).Therefore, 1~10 base is increased separately simultaneously at the 5 ' ends of SEQ ID NO:2 and SEQ ID NO:3 or 3 ' ends
It can expand and obtain the primer pair of essentially identical DNA fragmentation, be included in primer pair of the invention.
According to another aspect of the invention, the present invention also provides a kind of for detecting the examination of mentioned-above molecular labeling
Agent box.According to an embodiment of the invention, the kit includes: being described previously for detecting the primer of molecular labeling of the invention
It is right.Include the primer pair with nucleotide sequence shown in SEQ ID NO:2-3 in kit i.e. of the invention.According to the present invention
Embodiment can effectively realize the above-mentioned and fruit to muskmelon to be measured using primer pair included in kit of the invention
The detection of the relevant molecular labeling of skin color trait, determines whether muskmelon to be measured has the molecular labeling, and then can effectively really
The fruit colour character of fixed muskmelon to be measured.Specifically, such as using above-mentioned primer pair to muskmelon genomic DNA to be measured PCR is carried out
When amplification and detected through gel electrophoresis, when purpose band is one, and the purpose band is about 886bp, it can determine to be measured
Muskmelon pericarp yellow green；When purpose band is one, and the purpose band is about 441bp, muskmelon fruit to be measured can be determined
Beijing opera color；When purpose band be two, and the length of the purpose band be respectively about 441bp and 886bp when, can determine to
Survey muskmelon pericarp yellow green.The kit for being used to detect mentioned-above molecular labeling of the invention of the invention as a result, can
Effective for the molecular mark of muskmelon, and then early stage can be assisted to realize the short time, low cost, select to high accuracy
Educate muskmelon excellent variety.
In accordance with a further aspect of the present invention, the present invention also provides mentioned-above molecular labelings of the invention, primer pair
Or kit, the purposes in muskmelon breeding.As previously mentioned, of the invention with muskmelon fruit colour by can be used in detecting
The reagent of the relevant molecular labeling of shape, such as primer pair above-mentioned or the kit comprising the primer pair etc., can effectively really
Whether fixed muskmelon genomic DNA to be measured has above-mentioned molecular labeling.Such as when using the specific primer for being directed to the molecular labeling
When carrying out PCR amplification and detected through gel electrophoresis to muskmelon genomic DNA to be measured, according to the quantity and length of amplified production purpose band
Degree, can determine the fruit colour character of muskmelon to be measured, effectively so as to effectively assist muskmelon breeding.
In turn, according to another aspect of the present invention, the present invention also provides a kind of sides for detecting muskmelon fruit colour character
Method.According to an embodiment of the invention, this method carries out the detection of mentioned-above molecular labeling to muskmelon to be measured, so as to determine to
Survey the fruit colour character of muskmelon.It specifically, can be of the invention with muskmelon fruit colour character phase by can be used in detecting
The reagent of the molecular labeling of pass, such as primer pair above-mentioned or the kit comprising the primer pair etc., to the gene of muskmelon to be measured
Group DNA carries out PCR amplification, gel electrophoresis, thus according to the quantity and length of amplified production purpose band, can effectively determine to
Survey the fruit colour character of muskmelon.Wherein, as previously mentioned, using above-mentioned primer pair or kit, Muskmelon Plants to be measured are carried out
It, specifically, can when purpose band is one, and the purpose band is about 886bp when PCR amplification and detected through gel electrophoresis
With determination muskmelon pericarp yellow green to be measured；When purpose band is one, and the purpose band is about 441bp, can determine
Muskmelon fruit yellow to be measured；When purpose band is two, and the length of the purpose band is respectively about 441bp and 886bp,
It can determine muskmelon pericarp yellow green to be measured.It, can be fast as a result, using the method for detection muskmelon fruit colour character of the invention
Speed efficiently and accurately detects muskmelon fruit colour character, so can effective for the molecular mark of muskmelon, from
And early stage can be assisted to realize short time, low cost, high accuracy ground breeding muskmelon excellent variety.
In addition, the method for detection muskmelon fruit colour character according to the above embodiment of the present invention can also have it is following attached
The technical characteristic added:
According to an embodiment of the invention, the method for detection muskmelon fruit colour character of the invention further comprises: utilizing
Mentioned-above primer pair or kit carry out PCR amplification to the muskmelon genomic DNA to be measured；Detect amplified fragments
Length；And the length based on the amplified fragments, determine the fruit colour character of the muskmelon to be measured, wherein when the expansion
When increasing piece segment length 886bp, the muskmelon to be measured is pericarp yellow green；As the long 441bp of the amplified fragments, the muskmelon to be measured
For fruit yellow；When the amplified fragments are 441bp and two kinds of 886bp, the molecular labeling site of the muskmelon to be measured
Heterozygosis, pericarp yellow green.Thereby, it is possible to the detection muskmelons to be measured of efficiently and accurately whether to have molecular labeling of the invention, in turn
The fruit colour character of muskmelon to be measured can be effectively determined based on testing result.
It is not particularly limited, can adopt according to an embodiment of the invention, extracting the method for obtaining muskmelon genomic DNA to be measured
It is carried out with any of genome DNA extracting method or kit.Some specific examples according to the present invention, using hexadecane
Base trimethylammonium bromide method (CTAB method) extracts the genomic DNA of muskmelon to be measured.Thereby, it is possible to effectively obtain high-quality, purity
High genomic DNA carries out convenient for subsequent step.
According to an embodiment of the invention, detecting the amplified fragments using the preferred agarose gel electrophoresis of gel electrophoresis
Length.It is convenient and efficient as a result,.
Inventors have found that the method for detection muskmelon fruit colour of the invention can effective for muskmelon molecular labeling it is auxiliary
Breeding is helped, so as to assist early stage to realize short time, low cost, high accuracy ground breeding muskmelon excellent variety.
According to another aspect of the invention, the present invention also provides a kind of muskmelon auxiliary breeding means.It is according to the present invention
Embodiment, this method include detecting the molecular labeling by the method for mentioned-above detection muskmelon fruit colour character, with
Just the fruit colour character of the muskmelon to be measured is determined.Muskmelon auxiliary breeding means of the invention are utilized as a result, it can effectively really
Determine the fruit colour character of muskmelon, and then can be realized short time, low cost, high accuracy ground breeding muskmelon excellent variety.
It should be noted that molecular labeling relevant to muskmelon fruit colour of the invention and its application are with following excellent
Point:
(1) the relevant molecular labeling of muskmelon fruit colour character provided by the invention is not by the limit of the growth phase of muskmelon
System, can be used for the early stage breeding of muskmelon, can significantly promote the breeding process of muskmelon；
(2) method for detecting muskmelon relevant molecular labeling of muskmelon fruit colour character as shown in SEQ ID NO:1, it is quasi-
It is really reliable, it is easy to operate；
(3) detection of muskmelon relevant molecular labeling of muskmelon fruit colour character as shown in SEQ ID NO:1 is muskmelon
The marker assisted selection of growth traits provides scientific basis.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description
Obviously, or practice through the invention is recognized.
Detailed description of the invention
Above-mentioned and/or additional aspect of the invention and advantage will become from the description of the embodiment in conjunction with the following figures
Obviously and it is readily appreciated that, in which:
Fig. 1 is shown according to an embodiment of the present invention, utilizes drawing with nucleotide sequence shown in SEQ ID NO:2-3
Object carries out the electrophoresis detection result figure of the amplified production of PCR amplification to the DNA of parent and first familiar generation；
Fig. 2 is shown according to an embodiment of the present invention, utilizes drawing with nucleotide sequence shown in SEQ ID NO:2-3
Object carries out the electrophoresis detection result figure of the amplified production of PCR amplification to the DNA of part F2 generation individual.
Specific embodiment
The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and is only used for explaining this hair
It is bright, and be not considered as limiting the invention.Particular technique or condition are not specified in embodiment, according to text in the art
Offer described technology or conditions (such as with reference to J. Pehanorm Brooker etc. write, " Molecular Cloning:A Laboratory guide " that Huang Peitang etc. is translated,
The third edition, Science Press) or carry out according to product description.Reagents or instruments used without specified manufacturer is
Can be with conventional products that are commercially available, such as can purchase from Illumina company.
Embodiment 1F2 is constructed for segregating population and character is investigated
(1) material
The male parent " K3-92-1 " and female parent " K1-7 " of the muskmelon " gold honey six " provided with Xinjiang Baofeng Zhong Ye Co., Ltd
For material, F2 is constructed for segregating population.
Male parent " K3-92-1 " is self-mating system, the average long 21.6cm of leaf, mean blade width 29.3cm, and pachydermia netted melon is pale yellow
Skin, fine and closely woven reticulate pattern, without line is covered, pulp salmon pink, the long base of a fruit, averagely list melon quality 2.4kg, fruit shape index 1.55, edge are soluble
Solid content 7.4%, center soluble solid content 11.9%.
Maternal " K1-7 " is excellent single melon system, average leaf made of one farm variety of Kazakhstan " sheepshead " selfed breeding
Long 24.1cm, mean blade width 34.5cm, thick-skinned melon, melon is larger, and carpopodium end is yellow green skin, and areola end is golden yellow skin, carefully
Dilute reticulate pattern, without line, pulp white is covered, the short base of a fruit, averagely list melon quality 3.0kg, fruit shape index 1.52, edge soluble solid contain
Amount 5.1%, center soluble solid content 7.5%.
(2) informative population
Be female parent with K1-7 (P1), using K3-92-1 (P2) be male parent hybridized (male parent pericarp background color as yellow, female parent
Pericarp background color is yellow green), it obtains first-filial generation (F1), F2 group is obtained after stringent self-pollination, totally 500 single plants.
(3) character is investigated
In fructescence, according to retouching for " Germplasm Resources of Cucumis Melo L Description standard and data standard " (Ma Shuanwu etc., 2006)
It states, investigates the fruit colour (i.e. pericarp background color) of each single plant of P1, P2 and F2 group, according to phenotypic evaluation as a result, calculating segregation ratio,
Data statistic analysis is carried out using 2003 software of Microsoft Excel, Chi-square statistic is carried out to result using SPSS17.0.
The survey showed that for character: having 332 plants of pericarp background colors in F2 group is yellow green, and 114 plants of pericarp background colors are yellow
(being shown in Table 1).Chi-square statistic shows that the segregation ratio of pericarp background color is about 3:1, meets the law of segregation of Mendelian one pair of genes.
Therefore, muskmelon pericarp background color gene is the qualitative character of Dominant gene, and wherein pericarp background color yellow green shows as dominant character.
1 muskmelon F of table2Group's pericarp background color trait segregation ratio
χ2 0.05,1=3.84
Embodiment 2:SNP labeled analysis and genetic map are drawn
1, SNP marker analysis and genetic map building
1.1SNP labeled analysis
The tender leaf of parent " K1-7 " (P1), each single plant of " K3-92-1 " (P2) and F2 group in Example 1, with improvement
CTAB (cetyl trimethylammonium bromide) method extract genomic DNA, it is specific as follows:
Extract the genomic DNA of Parent, F1 generation and F2 generation individual respectively with CTAB method, the specific method is as follows:
(1) the fresh blade of 1.0g is weighed, shreds and is put into mortar, with 1.5 × CTAB of 3mL is added after liquid nitrogen grinding, is ground into
Homogenate is transferred in the centrifuge tube of 15mL, and 1.5 × CTAB of 1mL flushing is then added into mortar and is transferred in centrifuge tube again.After mixing
In 65 DEG C of water-bath 30min, during which slowly shake up frequently.
Wherein 1.5 × CTAB is formulated following (1L):
| CTAB | 15g |
| The Tris.Cl (pH 8.0) of 1mol/L | 75mL |
| The EDTA of 0.5mol/L | 30mL |
| NaCl | 61.4g |
Add deionized water to be settled to 1L, uses the preceding mercaptoethanol that final concentration of 0.2% (2ml) is added.
(2) it is cooled to room temperature, isometric chloroform/isoamyl alcohol (24:1) is added, mixes gently, until subnatant becomes dark green
Color.
(3) 4200rpm is centrifuged 10min, and upper strata aqueous phase is moved on to new 15mL centrifuge tube, adds the anhydrous of 2 times of volume pre-coolings
Ethyl alcohol mixes static 5min.DNA is precipitated in -20 DEG C of placement 30min.
(4) 4200rpm is centrifuged 10min, discards supernatant, and 75% ethanol washing of 1mL is added and precipitates 1 time, it is dry to be inverted centrifuge tube
200 μ L TE dissolving DNAs are added in dry DNA.
(5) genomic DNA is detected with 0.8% Ago-Gel.
(6) by the genomic DNA of obtained Parent and F1 generation, F2 generation individual be stored in -20 DEG C it is spare.
Then, 1 μ g genomic DNA is taken, is interrupted it at random with restriction enzyme, electrophoresis recycles 400-600bpDNA piece
Section, be then based on Hiseq2000 high-flux sequence platform builds library strategy, constructs the genomic library of each sample, then will
After various kinds Ben Wenku mixing (each sample carries the label that do not allow mutually) by 2100 biological analyser of Agilent and
Hiseq2000 high-flux sequence platform carries out sequencing analysis.
According to identification sequence label obtain the sequencing reads of each individual, with SOAP2.20 software (reference can be made to: Li
R.et al., 2009a, SOAP2:an improved ultrafast tool for short read
Alignment.Bioinformatics, by referring to be incorporated by herein), with muskmelon DHL92 be (can with reference to genome
Referring to: Garcia-Mas et al., 2012, The genome of melon (Cucumis melo L.), by referring to general
It is incorporated by herein), alignment is carried out, as a result with SAMtools 0.1.8, the conversion of 0.983 realSFS software, analysis
(reference can be made to: Li et al., 2009b, SNP detection for massively parallel whole-genome
Resequencing., by referring to be incorporated by herein), identify each site, then result be filtered, filter
Standard is 50≤depth≤2500, and site mutation probability >=95% obtains the SNP collection of high confidence level.It is reference, mistake with SNP collection
Filter obtains the genotype of parent and group.
The building of 1.2 genetic maps
A large amount of SNP causes to be difficult to construct Genetic linkage map, the Bin being composed according to continuous SNP to each label
Gene constructed Bin map can be simpler effective.
It according to the genotype of parent and group, selects with every 15 SNP as a window, slides a SNP every time, determine
The exchange site of the genotype of each window and each individual obtains the Bin genotype of each individual.Ready Bin base
Because of type data, using MSTMap software, graphing method selects Regression mapping algorithm, and mapping function selects Kosambi '
S function building genetic map (reference can be made to: Wu et al., 2008, Efficient and accurate construction
Of genetic linkage maps from the minimum spanning tree of a graph., by referring to general
It is incorporated by herein).Chromosome serial number is added to name chromosome with abbreviation chr, as chr01 indicates No. 1 chromosome.
Wherein, analysing and comparing to sequencing result, the results are shown in Table 2, removes non-anchor sequence, in parents' sheet and
44722 SNP markers are detected in F2 group altogether, averagely each a length of 8.21Kb of SNP, the SNP quantity on every chromosome is not
Together, minimum with the SNP on o.11 chromosome, it is 1894, the SNP on No. 6 chromosomes is most, up to 5865；SNP is in each dye
Distribution on colour solid is from 1SNP/4.7Kb (No. 3 chromosome) to 1SNP/4.714.5Kb (No. 11 chromosome).After combined,
Common property gives birth to 2277 Bin segments in F2 group, and fragment length is differed from 20Kb to 4.83Mb, average length 138.95kb, structure
The Bin genetic map that total genetic distance is 1296.174cM is built, No. 10 chromosome maps are away from most short (77.997cM), No. 1 dyeing
Body map distance longest (143.278cM).In the genotype of entire F2 group whole, have 25.36% from male parent, 24.67%
From female parent, 49.96% is heterozygous, illustrate ratio of 3 kinds of genotype in total genotype close to 1:2:1 segregation ratio.
The distribution of SNP, Bin on 2 genetic map of table
| Chromosome | SNP | Bin | Genetic distance (cM) |
| chr01 | 3319 | 244 | 143.278 |
| chr02 | 3743 | 169 | 109.473 |
| chr03 | 5675 | 275 | 114.580 |
| chr04 | 3056 | 191 | 134.128 |
| chr05 | 4150 | 204 | 111.275 |
| chr06 | 5865 | 253 | 125.153 |
| chr07 | 4623 | 179 | 100.867 |
| chr08 | 2984 | 160 | 101.795 |
| chr09 | 3554 | 186 | 101.119 |
| chr10 | 2764 | 108 | 77.997 |
| chr11 | 1894 | 127 | 86.461 |
| chr12 | 3095 | 181 | 90.048 |
| Total | 44722 | 2277 | 1296.174 |
3, the assignment of genes gene mapping
Using winQTLCart2.5 software, the assignment of genes gene mapping is carried out using CIM (composite interval mapping method).Specifically: according to
The individual phenotype of F2 group, similar with male parent type character to be denoted as a, similar with maternal type character to be denoted as b, character occupy male parent
H is denoted as between female parent.The phenotypic datas of all individuals are obtained, by the phenotypic data of individual and the genotype number that before obtains
According to being compared, the assignment of genes gene mapping is carried out to fruit colour (i.e. pericarp background color) character.The results show that the pericarp background color assignment of genes gene mapping exists
In the section 409828bp~835625bp of No. 4 ends of chromosome, length is about 425kb, additive effect 0.3003, phenotype
Variation accounts rate is 0.4918.
4, molecular markers development
Parent is carried out to full-length genome and resurveys sequence (10X), according to genome sequencing as a result, utilizing SOAP software
(reference can be made to application link: http://soap.genomics.org.cn/, by referring to be incorporated by herein) compare survey
Then sequence reads finds the biggish molecular labeling of Fragment Differential with SOAPsv, identify convenient for being distinguished with gel electrophoresis.
As a result, selecting the label M040048 (nucleic acid shown in SEQ ID NO:1 close to section where fruit colour gene
Sequence) as candidate.
The nucleotide sequence of molecular labeling M040048 (445bp) is as follows:
AGAAATCTGTATATGAGCGGGAGCTCATGGCTATTGTCTTATCGATGGAAAAGTGGCGACATTACATA
TTAGGACAGTGTATACTGACCAGAAAGCTCTTCACCACATTCTTGAGCAAAGAGAAGTCCCTTCTGACGTGCAAAA
ATGGGTGACAATATTAATAGAGTTTGACTTTGAAATTTGTTATAGATCAAGAGCAGAAAATAAGCCAGCGGATGCT
CTCTCGCATGCCTGAGGAGGGATATCAGTGCTGGTGATCATGGATTGCCTTAGCAAATACGCTCATTTTTTGATCT
TGGGACACCCTTTTTTGAGGACAGTTGACATGATTTTCATTCAAGAAGTGGTAAGAGTCCATGGATACCCACAATC
CATTGTCTTAGATCGTGGTAGGGTTTCTCTGAGTCACTTTTGGATGGATTTATTACGATTGGAAGAAACTCAA(SEQ
ID NO:1).
Embodiment 3: the fruit colour relevance verification of molecular labeling
To the muskmelon fruit colour related molecular marker M040048 (core shown in SEQ ID NO:1 determined in embodiment 2
Acid sequence) it is verified, specific as follows:
For above-mentioned molecular labeling design primer, primer sequence is as follows:
Forward primer: 5 '-GCTATGGTAACATTGCTGGT-3 ' (SEQ ID NO:2)；
Reverse primer: 5 '-ATGGTATCAGAGCAAGTGGT-3 ' (SEQ ID NO:3).
Using above-mentioned primer, the polymorphism and expansion of the label are verified by PCR amplification and agarose gel electrophoresis detection
Increase stability.
Specifically, using male parent, female parent, F1 generation or the genomic DNA in F2 generation extracted in embodiment 2 as template, in utilization
It states amplimer and carries out PCR amplification, wherein
PCR reaction system is as follows:
| Sterile water | 20.2μl |
| 10*Buffer (contains Mg2+) | 2.5μl |
| dNTPs(25mM) | 0.15μl |
| Taq enzyme (5U/ μ l) | 0.15μl |
| Forward primer | 0.5μl |
| Reverse primer | 0.5μl |
| Template | 1.0μl |
| Total volume | 25μl |
PCR response procedures are as follows:
94 DEG C initial denaturation 5 minutes；94 DEG C are denaturalized 30 seconds, and 60 DEG C are annealed 30 seconds, and 72 DEG C extend 40 seconds, run 35 circulations；
Last 72 DEG C extend 3 minutes.Pcr amplification product can be saved at 4 DEG C.
Then, part is taken to carry out agarose gel electrophoresis detection, the result is shown in Figure 1 and Fig. 2 each pcr amplification product.Such as Fig. 1
Shown, male parent amplified production is the amplified fragments of 441bp size, and maternal amplified production is the amplified fragments of 886bp size, hybridization
F1 generation then contains the two amplified fragments simultaneously.Shown in Fig. 2 part F2 generation individual amplified production electrophoresis detection as a result,
The single plant of fruit yellow amplifies the band of 441bp, and the single plant of pericarp yellow green amplifies the band of 886bp, middle part
The single plant for dividing pericarp yellow green is heterozygosis, amplifies above-mentioned two band.Thus, it was demonstrated that molecular labeling M040048 (SEQ ID
Nucleic acid sequence shown in NO:1) there is polymorphism, the molecular labeling and the close phase of muskmelon fruit colour character between Parent
It closes.
Then, each amplified production is sequenced using 3730 sequenators, as a result, male parent and fruit yellow single plant in F2 generation
Amplified band, lacked a Duan Xulie compared with maternal and F2 is for the amplified band of pericarp yellow green single plant, which is
Molecular labeling M040048 (nucleic acid sequence shown in SEQ ID NO:1) above-mentioned.
Wherein, male parent amplified fragments sequence (441bp) is as follows:
GGGGCTCGGATCACTTACCCTTCGAATCGATGCTTCAGGATTTGGGTTAGGGGCTGTTCTTTCGCAAA
ATAAGAAGCCAATTGCATATTTTAGTCAGAAACTGTCTGTGGAACTTTATTGAAGCGTAGCACTGCGTACCACCCT
TAAACTAACAGCCAATCGGAGATGGTTAACAAATTTCTGGAACTTTATTTAAGCTGTTTTTGCAGTGAGAAGCCTA
AAACTTAGAGTGAAGTCTCGAATCACCAATTGACCCAAAAACTTAAGTTGATGGGTAAAGGAAAATTTAATTATAT
ATCACTAACACTCTCCTTCACTTGTGGGCTTGAAATATATGAAAGCCCCAACAGATGGTAATCAATTTTAATTGGG
GATGAAACAACAATGCAGAGACTTGAATACGACTTTCCTGAACCACTTGTTTGAATACCCATTAAAGCA(SEQ ID
NO:4),
Maternal amplified fragments sequence (886bp) is as follows:
GGGGTCGGATCACTTACCCTTCGAATCGATGCTTCAGGATTTGGGTTAGGGGCTGTTCTTTCACAAAA
TAAGAAGCCAATTGCATATTTTAGTCAGAAACTGTCTGTGGAACGGCCGGGAGAAATCTGTATATGAGCGGGAGCT
CATGGCTATTGTCTTATCGATGGAAAAGTGGCGACATTACATATTAGGACAGTGTATACTGACCAGAAAGCTCTTC
ACCACATTCTTGAGCAAAGAGAAGTCCCTTCTGACGTGCAAAAATGGGTGACAATATTAATAGAGTTTGACTTTGA
AATTTGTTATAGATCAAGAGCAGAAAATAAGCCAGCGGATGCTCTCTCGCATGCCTGAGGAGGGATATCAGTGCTG
GTGATCATGGATTGCCTTAGCAAATACGCTCATTTTTTGATCTTGGGACACCCTTTTTTGAGGACAGTTGACATGA
TTTTCATTCAAGAAGTGGTAAGAGTCCATGGATACCCACAATCCATTGTCTTAGATCGTGGTAGGGTTTCTCTGAG
TCACTTTTGGATGGATTTATTACGATTGGAAGAAACTCAATTGAAGCATAGCACTGCGTACCACCCTTAAACTAAC
AGCCAATCGGAGATGGTTAACAAATTTCTGGAACTTTATTTAAGCTGTTTTTTGCAGTGAGAAGCCTAAAACTTAG
AGTGAAGTCTCGAATCACCAATTGACCCAAAAACTTAAGTTGATGGGTAAAGGAAAATTTAATTATATATCACTAA
CACTCTCCTTCACTTGTGTGCTTGAAATATATGAAAGCCCCAACAGATGGTAATCGATTTTATTTGGGGATGAAAC
AACAATGCAGAGACTTGAATACGACTTTCCTGAACCACTCTCGTATATCACTCTAT TA (SEQ ID NO:5).
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
Centainly refer to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be any
One or more embodiment or examples in can be combined in any suitable manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that: not
A variety of change, modification, replacement and modification can be carried out to these embodiments in the case where being detached from the principle of the present invention and objective, this
The range of invention is defined by the claims and their equivalents.
Claims (3)
1. a kind of relevant molecular labeling of muskmelon fruit colour character, which is characterized in that the nucleotide sequence of the molecular labeling
As shown in SEQ ID NO:1, with nucleotide sequence shown in SEQ ID NO:1 and in of the molecular labeling site homozygosis
Body shows as pericarp yellow green；Lack nucleotide sequence shown in SEQ ID NO:1 and homozygous in the molecular labeling site
Body shows as fruit yellow；With nucleotide sequence shown in SEQ ID NO:1 and in of molecular labeling site heterozygosis
Body shows as pericarp yellow green.
2. a kind of method for detecting muskmelon fruit colour character, which is characterized in that
The detection of molecular labeling described in claim 1 is carried out, to muskmelon to be measured to determine the muskmelon fruit colour to be measured
Character.
3. a kind of muskmelon auxiliary breeding means, which is characterized in that the described method includes:
By method as claimed in claim 2, molecular labeling described in claim 1 is detected, to determine muskmelon pericarp to be measured
Color trait.
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| CN108676905B (en) * | 2018-05-29 | 2021-08-24 | 中国农业科学院郑州果树研究所 | Watermelon flesh color major gene and InDel molecular marker and application thereof |
| CN109487002B (en) * | 2019-01-04 | 2021-10-19 | 西北农林科技大学 | SNP (single nucleotide polymorphism) marker of melon pericarp and fruit surface groove, detection method and application |
| CN110106270B (en) * | 2019-04-24 | 2022-03-22 | 北京市农林科学院 | Molecular marker coseparated from melon yellow seed coat and application thereof |
| CN111996285B (en) * | 2020-09-27 | 2021-09-14 | 广东省农业科学院蔬菜研究所 | Molecular marker related to towel gourd powdery mildew and application thereof |
| CN114763579B (en) * | 2021-01-13 | 2023-08-08 | 北京市农林科学院 | Molecular marker related to melon fruit shape, and method and application thereof |
| CN113005221B (en) * | 2021-04-16 | 2023-01-06 | 黑龙江八一农垦大学 | Molecular marker SNP-392 closely linked with muskmelon pedicle-resistance gene CmAL3 and application thereof |
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