CN105506149A - Linkage SNP locus and CAPS marker of watermelon fruit sugar accumulation gene STP1 - Google Patents
Linkage SNP locus and CAPS marker of watermelon fruit sugar accumulation gene STP1 Download PDFInfo
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Abstract
The invention discloses a linkage SNP locus and a CAPS marker of a watermelon fruit sugar accumulation gene STP1 and provides application of substances for detecting polymorphism or the genotype of the SNP locus in a watermelon genome to identification or auxiliary identification whether a watermelon is a high-sugar-content watermelon or a low-sugar-content watermelon. It is verified through tests that the SNP locus related to the sugar content of the watermelon is found; fruit flavor identification is conducted on the segregation population and the natural population of RILs, and the condition of linkage between the SNP lotus and a target character is detected through STP-CAPS labeled molecules; preliminary screening can be conducted o the variety through SNP polymorphism, so that the purpose of assistant breeding conducted through the molecular marker is achieved, the breeding cycle is greatly shortened, and great theoretical significance and practical significance are achieved.
Description
Technical field
The present invention relates to biological technical field, particularly relate to the chain SNP site of Watermelon Fruit sucrose accumulation gene STP1 and CAPS mark.
Background technology
Watermelon (Citrulluslanatus) belongs to Curcurbitaceae important crops, and account for world's vegetable crop area 7%, global annual production is about 90,000,000 ton (http://faostat.fao.org).China's watermelon volume of production and marketing is positioned at the first in the world, is the Horticultural crop that China has international competitiveness and larger economic growth space.The issue of recent watermelon whole genome sequence, greatly facilitates watermelon molecular biology research, for molecular designing assistant breeding provides solid basis more.Along with the arrival of watermelon genome era, further clear and definite gene function, finding the key gene controlling watermelon important character, is the primary content of current watermelon molecular biology research.
Watermelon originates from Africa, and the wild watermelon originating in Africa comprises two biochemical types, a kind of high sugared content fruit (pol 10-13 degree, another kind of low sugar content fruit (pol 1-3 degree); High sugared content watermelon is the major traits of mankind's seed selection.At present at Watermelon Germplasm storehouse (http://www.ars-grin.gov/) although have disease-resistant good character of Denging in the Watermelon Germplasm deposited, but the many parts of material sugar contents of fruit are all only less than 5 degree, but simultaneously due to the quantitative trait locus that the sugar content of fruit is a kind of complexity, by multiple gene co-controlling.When not having molecular marking supplementary breeding, breeding seed selection person hesitates to use the disease-resistant excellent resources of low sugar content to improve parent's disease resistance etc.Be tested and appraised sugar of watermelon additive gene, develop its closely linked molecule marker and carry out kind initial screening, reach the object of marker assisted selection, the cycle that greatly can shorten breeding improves breeding efficiency, realizes the target wild for low sugar excellent resources being used for main breed molecular improvement.Therefore, the chain SNP site of a kind of Watermelon Fruit sucrose accumulation gene STP1 and CAPS mark and acquisition methods thereof is needed to propose in the scientific research in watermelon breeding field and practice.
Summary of the invention
An object of the present invention is to provide and detect the polymorphism of SNP site or the purposes of genotypic material in watermelon genome.
The invention provides the polymorphism that detects SNP site in watermelon genome or genotypic material identify or assistant identification watermelon be high sugared watermelon or low sugar watermelon in application;
Or the invention provides the application in characterization or assistant identification watermelon are high sugared watermelon or low sugar watermelon products of the polymorphism that detects SNP site in watermelon genome or genotypic material;
Described SNP site is the 4037102nd Nucleotide or sequence 1 the 44th Nucleotide on watermelon genome No. 2 karyomit(e)s, and described SNP site genotype is that TT isozygotys, GG isozygotys or T/G heterozygosis.
Of the present invention also providing detects the polymorphism of SNP site or another purposes of genotypic material in watermelon genome.
The invention provides the application in the pol state of qualification or assistant identification watermelon of the polymorphism that detects SNP site in watermelon genome or genotypic material;
Or the invention provides the application in the pol phase product of characterization or assistant identification watermelon of the polymorphism that detects SNP site in watermelon genome or genotypic material;
Described SNP site is the 4037102nd Nucleotide on watermelon genome No. 2 karyomit(e)s, and described SNP site genotype is that TT isozygotys, GG isozygotys or T/G heterozygosis.
In above-mentioned application, in described detection watermelon genome, the polymorphism of SNP site or genotypic material comprise the primer pair of the described SNP site that increases;
The primer sets that described primer pair is specifically made up of the single strand dna shown in the single strand dna shown in sequence 2 and sequence 3.
In above-mentioned application, in described detection watermelon genome, the polymorphism of SNP site or genotypic material also comprise MesI and limit restriction endonuclease.
The present invention's the 3rd object is to provide a kind of to identify or assistant identification watermelon to be measured is the method for high sugared watermelon or low sugar watermelon.
Method provided by the invention, comprise the steps: that detecting watermelon genome SNP site genotype to be measured is that TT isozygotys, GG isozygotys or T/G heterozygosis, if described watermelon genome SNP site genotype to be measured is that TT isozygotys, then described watermelon to be measured is or candidate is high sugared watermelon; If described watermelon genome SNP site genotype to be measured is that GG isozygotys or T/G heterozygosis, then described watermelon to be measured is or candidate is low sugar watermelon;
Described SNP site is the 4037102nd Nucleotide on watermelon genome No. 2 karyomit(e)s.
In aforesaid method, the genotypic method of described detection watermelon to be measured genome SNP site is as follows:
1) direct Sequencing;
2) pcr amplification is carried out with the primer pair Chinese cabbage to be measured can increased containing SNP site fragment, obtain amplified production, again described amplified production is cut through enzyme, detect digestion products size, determine watermelon genome SNP site genotype to be measured according to described digestion products size.
In aforesaid method, the described primer pair containing SNP site fragment that can increase is following 1) or 2):
1) described primer pair is for being made up of the single strand dna shown in the single strand dna shown in sequence 1 and sequence 2;
2) described primer pair is for being made up of the single strand dna shown in the single strand dna shown in sequence A and sequence B;
Sequence A is the Nucleotide that sequence 1 was deleted or increased one or several Nucleotide and obtains;
Sequence B is the Nucleotide that sequence 2 was deleted or increased one or several Nucleotide and obtains;
Described enzyme is that MesI limits restriction endonuclease.
In aforesaid method, describedly determine that watermelon genome SNP site Genotypic methods to be measured is according to digestion products size:
If digestion products only has 144bp size fragment, then watermelon genome SNP site genotype to be measured is that GG isozygotys,
If digestion products has 144bp size fragment and 72bp size fragment, then watermelon genome SNP site genotype to be measured is T/G heterozygosis,
If digestion products only has 72bp size fragment, then watermelon genome SNP site genotype to be measured is that TT isozygotys.
The present invention's the 3rd object is to provide the test kit of qualification or assistant identification watermelon pol to be measured.
Test kit provided by the invention, comprises the polymorphism or genotypic material that detect SNP site in watermelon genome.
In mentioned reagent box, in described detection watermelon genome, the polymorphism of SNP site or genotypic material comprise the primer pair for increasing containing SNP site fragment, and described SNP site is the 4037102nd Nucleotide on watermelon genome No. 2 karyomit(e)s;
Describedly be specially following 1 for the primer pair increased containing SNP site fragment) or 2):
1) described primer pair is for being made up of the single strand dna shown in the single strand dna shown in sequence 1 and sequence 2;
2) described primer pair is for being made up of the single strand dna shown in the single strand dna shown in sequence A and sequence B;
Sequence A is the Nucleotide that sequence 1 was deleted or increased one or several Nucleotide and obtains;
Sequence B is the Nucleotide that sequence 2 was deleted or increased one or several Nucleotide and obtains.
Mentioned reagent box also comprises MesI and limits restriction endonuclease.
The polymorphism or the application of genotypic material in the sugared watermelon breeding of height that detect SNP site in watermelon genome are also the scope of protection of the invention.
In the 103 strain RILs colonies shown in selfing acquisition of many generations table 1 that above-mentioned watermelon to be measured is PI296341FR and 97103 any one; Or in portion watermelon of 132 shown in table 2 any one.
High sugared watermelon: Watermelon Fruit pol is more than or equal to 6 degree;
Middle sugared watermelon: Watermelon Fruit pol is 3-6 degree, and does not comprise 3 and 6;
Low sugar watermelon: Watermelon Fruit pol is less than or equal to 3 degree;
Experiment of the present invention proves, the present invention finds one and sugar of watermelon content SNP site; Taste of fruit qualification is carried out to RILs segregating population and natural population and sharp STP-CAPS tagged molecule detects this SNP site and object linkage of characters situation; Discovery can carry out initial screening by SNP polymorphism to kind, reach and detect sugar content seedling stage, set up that the molecule marker of sugar of watermelon screening is auxiliary educates system, greatly shorten breeding cycle, there is important theory and practice significance, after reality is implemented, find that this invention effectively can detect sugar distribution (specifically seeing the following form 2) of 132 parts of materials.
Accompanying drawing explanation
Fig. 1 is Watermelon Fruit sucrose accumulation gene STP1 positioning result figure in embodiment 1.
Fig. 2 is the result electrophorogram after primer pair STP-F and STP-R utilizes MesI enzyme to cut to the pcr amplification reaction band of male parent (low sugar), female parent (high sugar), F1 generation (low sugar) and RIL population segment material and this pcr amplification product in embodiment 1.
Fig. 3 is the candidate SNP locus figure obtained after 132 the natural population's material sequence comparisons of Primary Location gene interval watermelon in embodiment 1.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The discovery of embodiment 1, the chain SNP site of Watermelon Fruit sucrose accumulation gene STP1 and CAPS mark
1, for examination material selection
Male parent, female parent, F1 generation, RILs colony and natural population's material is comprised for examination material;
Male parent PI296341FR (can from obtaining as follows: https: //npgsweb.ars-grin.gov/gringlobal/search.aspx; Or obtain from Vegetable Research Centre, Beijing Academy of Agriculture and Forest Sciences's germplasm resource bank): be typical agriotype watermelon material, the sugar content of fruit is extremely low;
Maternal 97103 (can from obtaining as follows: https: //npgsweb.ars-grin.gov/gringlobal/search.aspx; Or obtain from Vegetable Research Centre, Beijing Academy of Agriculture and Forest Sciences's germplasm resource bank): be typical East Asia type watermelon cultivars, the sugar content of fruit is high;
F1 generation: obtain F1 generation with male parent PI296341 and maternal 97103 hybridization;
High sugared watermelon: Watermelon Fruit pol is more than or equal to 6 degree;
Middle sugared watermelon: Watermelon Fruit pol is 3-6 degree, and does not comprise 3 and 6;
Low sugar watermelon: Watermelon Fruit pol is less than or equal to 3 degree;
RILs colony: the RILs colony that this F1 generation selfing many generations obtain, totally 103 strains (table 1);
Natural population's material: 132 parts of watermelon individual plants (table 2);
Each in the present embodiment all can obtain from Vegetable Research Centre, Beijing Academy of Agriculture and Forest Sciences's germplasm resource bank for examination material.
2, for determination and the watermelon pol gene genetic law-analysing of examination material fruit pol
Adopt hand-held saccharimeter to identify the fruit supplying examination material, obtain fruit pol, every strain plants 3 years continuously, utilizes the data of 3 years different fields phenotypes, goes mean value to be the final sugar degree of every strain material.
Carry out the qualification of fruit pol to 103 RILs segregating population individual plants, result is as shown in table 1, and in 103 individual plants, pol distribution presents continuous regime, sugared content strain and 70 strain low sugar strains in the high sugared strain of 10 strain fruits, 23 strains.
Comprehensive above-mentioned parents, a F1 and 103 RILs segregating population individual plant fruit pol qualification result, show that Watermelon Fruit pol gene is the recessive character of controlled by multiple genes.
Shown in his-and-hers watches 2 132 part individual plant carries out the qualification of fruit pol, result as table 2, the high sugared material of 61 parts of fruits, sugared material and 42 parts of low sugar materials in 30 parts.
Table 1 is 103 RILs segregating population individual plant fruit pol multiple years qualification results
Table 2 effectively can detect 132 natural population's material individual plant fruit pols for SNP genotype
3, genetic linkage analysis
Utilize Joinmap4.0 software to mark genetic map to the RILs colony flavor High Density Molecular identified constructed by the individual plant of fruit pol and this RILs colony and carry out genetic linkage analysis, Primary Location target gene is interval.
The construction process of High Density Molecular mark genetic map is recorded in applicant delivered AHighResolutionGeneticMapAnchoringScaffoldsoftheSequence dWatermelonGenome.PLoSONE7 (1): e29453.doi:10.1371/journal.pone.0029453RenY, ZhaoH, KouQ, JiangJ, GuoS, etal. (2012).
Fruit sucrose accumulation gene STP1, as Fig. 1, is positioned on watermelon No. 2 karyomit(e)s, is specifically positioned at the scope of 2bin3-2bin4 by result.
4, the acquisition of candidate SNP
Utilize watermelon full genome natural population result, in Primary Location gene interval, genome sequence comparison is carried out to natural population's material, obtain the candidate SNP locus met with watermelon material phenotypic character height;
Find SNP site closely chain with its pol in natural population material, find just to have in positioning area the change of place's SNP site (T → G) and pol chain.
Utilize 132 parts of watermelon representative species matter full-length genome natural population information, to all SNP site linkage analysises in No. 2 karyomit(e) 2bin3 ~ 2bin4 interval range of Primary Location, obtain 1 SNP site conformed to 132 parts of material fruit pol phenotype height, analytical results display be positioned at the change of the T → G of the generation of 4037102bp place on watermelon genome No. 2 karyomit(e)s and fruit pol chain, as shown in figure 3 and table 2.
Therefore relevant to fruit pol SNP site to be positioned on watermelon genome No. 2 karyomit(e)s the 4037102nd, is sequence 1 the 44th Nucleotide.The Nucleotide shown in sequence 1 at this SNP site place marks as CAPS.In low pol kind, this SNP site is that GG isozygotys or T/G heterozygosis, and in high sugar products kind, this SNP site is that TT isozygotys, and presents low sugar phenotype in heterozygosis kind.
Therefore, can determine that watermelon to be measured belongs to high sugar products kind or low sugar kind according to SNP site, method is as follows: the genotype detecting SNP site in watermelon genome to be measured, isozygoty if the genotype of the SNP site of watermelon to be measured is TT, then this watermelon to be measured is or candidate is high sugar products kind, isozygoty or T/G heterozygosis if the genotype of the SNP site of watermelon to be measured is GG, then this watermelon to be measured is or candidate is low sugar kind.
Whether embodiment 2, SNP site are application in high sugar products kind detecting watermelon
One, the design of CAPS labeled primer
According to the SNP site design CAPS labeled primer obtained in embodiment 1, amplified production size is that 144bp, CAPS labeled primer is made up of primer STP-F (sequence 2) and primer STP-R (sequence 3).
STP-F (upstream primer sequence): 5 '-AAAACAGCATCCACCACCGC-3 ',
STP-R (downstream primer sequence): 5 '-CATTGAAGTTAGAAATGGG-3 ';
According to difference place base sequence design CAPS labeled primer STP-F and STP-R, 144bpPCR product after this primer amplification is after DNA restriction restriction endonuclease MesI enzyme is cut, occur following situation: being cut into two sections of sizes is 72bp fragment, the SNP site in corresponding PCR primer is TT; Be only 144bp fragment, the SNP site in corresponding PCR primer is GG.
Two, whether SNP site is high sugar products kind at detection watermelon
1, extracting genome DNA:
Extract the genomic dna of the male parent in embodiment 1, female parent, F1 generation, RILs colony, natural population respectively;
DNA extraction method is at the method (MurrayM with reference to (1980) such as Murry, ThompsonWF.RapidisolationofhighmolecularweightplantDNA [J] .NuclAcidRes, 1980,8:668-673.) basis on improvement form; Concrete steps are as follows:
I. above-mentioned each 1.5 grams, blade for examination material, grind into powder in liquid nitrogen, add 9ml2%CTAB extracting solution (2%CTAB, 1.4mMNaCl, 100mMTris-HClpH8.0 again, 20mMEDTApH8.0,1%PVP-40,0.2% beta-mercaptoethanol), mixing, in 65 DEG C of water-baths 1 hour, obtain mixture A;
II. by mixture A stop water-bath, add the liquor kalii acetici of the 5M of 1/3 volume, ice bath 20 minutes after mixing; Add isopyknic chloroform/primary isoamyl alcohol (24:1) extracting twice again, obtain supernatant A;
III. to supernatant A in add 2/3 volume isopropanol be used for precipitate DNA; Use lavation buffer solution (75% ethanol, 10mM ammonium acetate) to wash once again, add TE damping fluid (10mMTris-HCl, 1mMEDTA, pH7.4) after drying up and dissolve, obtain solution A;
IV. to solution A in add RNaseA, make its final concentration reach 100 μ g/ml, then mix 37 DEG C of water-baths 1 hour; Use equal-volume chloroform/primary isoamyl alcohol (24:1) extracting more once, obtain supernatant liquor B;
V. to get in supernatant liquor B and add 1/2 volume 7.5M ammonium acetate, 2 times of volume dehydrated alcohols, obtain DNA precipitation;
VI. precipitate with the DNA of 70% washing with alcohol, after drying up, add appropriate ddH
2o dissolving DNA, obtains respective genomic dna; Use ultraviolet spectrophotometer (ShimadzuUV-1201, Japan) with the concentration of the respective genomic dna of OD260 pH-value determination pH again, then detect the extraction quality of respective genomic dna with 1.2% agarose gel electrophoresis.
2, pcr amplification and enzyme are cut
1) with above-mentioned 1 genomic dna of each strain extracted for template, carry out pcr amplification with primer STP-F and primer STP-R, obtain the PCR primer of about 144bp;
2) above-mentioned each PCR primer is limited endonuclease digestion with MesI respectively, obtain digestion products.
The reaction system of above-mentioned pcr amplification reaction is: 2.5 μ L10 × Buffer; 2.5 μ L concentration are the dNTPs of 2.5mM; 1UTaqDNA polysaccharase; 2 μ L concentration are 10mMPCR upstream and downstream mix primer (each 1 μ L of upstream and downstream primer); 50ng template DNA; ddH
2o supplies 25 μ L; Taq DNA polymerase and reaction buffer are purchased from TaKaRa company.DNTPs is purchased from Beijing Quanshijin Biotechnology Co., Ltd;
The response procedures of above-mentioned pcr amplification reaction is: stage 1:94 DEG C denaturation 5min; Stage 2:94 DEG C 20s, 56 DEG C of 20s, 72 DEG C of 30s, circulate 34 times altogether; Stage 3:72 DEG C extends 5min; Stage 4:4 DEG C of maintenance.Wherein PCR instrument is the Veriti96wellThermalCycler purchased from AppliedBiosystems company;
The reaction system of above-mentioned endonuclease reaction is: 1.0 μ L10 × NEBBuffer, 0.1 μ L100 × BSA, and 0.2 μ LMesI limits restriction endonuclease, 4 μ LPCR products, distilled water polishing to 10 μ L;
The response procedures of above-mentioned endonuclease reaction is: 37 DEG C of enzymes cut 4 DEG C of maintenances after 2h;
Electrophoresis detection digestion products,
If digestion products only has 72bp band individual plant, its SNP site is TT, then this individual plant is high sugar;
If digestion products only has 144bp band individual plant, its SNP site is GG, then this individual plant is low sugar;
If digestion products has 144bp and 72bp band individual plant, its SNP is T/G heterozygosis, then this individual plant is low sugar.
103 strain RILs population segment strain detected results as shown in Figure 2, from left to right number consecutively, from top to bottom number consecutively, swimming lane 1 is molecular weight Marker, is followed successively by 50bp-100bp-200bp from down to up; Swimming lane 2 is maternal 97103, swimming lane 3 is male parent PI296341-FR, swimming lane 4 is F1, other swimming lanes RILs each strain PCR primer enzyme is cut, result can be found out, the digestion products of maternal 97103 is 72bp band, and the digestion products of male parent PI296341-FR is 144bp band, be divided into 72bp band in the middle part of RILs colony, part is 144bp band.
The RILs colony individual plant that digestion products is only 72bp band has 16 strains, and its SNP site is TT, and method of the present invention is accredited as high sugared individual plant, detects through pol, and 13 strains are high sugar, the high sugared material tests goodness of fit 81.3% of RIL colony;
The RILs colony individual plant that digestion products is only 144bp band has 86 strains, and its SNP site is GG, and method of the present invention is accredited as low sugar individual plant, and detect through pol, 71 strains are low sugar, the high sugared material tests goodness of fit 82.5% of RIL colony.
Digestion products has the RILs colony individual plant of 144bp and 72bp band to have 1 strain, and its SNP is T/G heterozygosis, and method of the present invention is accredited as low sugar individual plant, and detect through pol, this strain is low sugar.
Above-mentioned experimental result display pol result and marker detection result present and are identical.
Three, whether SNP site is high sugar products kind at detection watermelon
Method according to above-mentioned two detects 132 parts of natural population's materials.
In 132 parts of natural population's materials, part is only 72bp fragment, and part be only 144bp fragment, partly has 72bp and 144bp fragment.
Add up as follows,
In 132 parts of natural population's materials:
Digestion products only has the fragment of 72bp to be 95 parts, and its SNP site is that TT isozygotys, and the inventive method thinks that it is high sugar products kind; Detect according to hand-held saccharimeter, in 95 parts, 85 parts is high sugar products kind 10 parts is middle sugar products kind, and therefore, method of the present invention detects the accuracy rate 89% of high sugared material.
Digestion products only has the fragment of 144bp to be 36 parts, and its SNP site is that GG isozygotys, and the inventive method thinks that it is low sugar kind; Detect according to hand-held saccharimeter, have 0 part in 36 parts for high sugar products kind, 36 parts is low sugar kind, and therefore, the accuracy rate that method of the present invention detects low sugar material in 132 parts of natural population's materials is 100%.
Digestion products has 144bp and 72bp band to be 1 part, and its SNP site is T/G heterozygosis, and the inventive method thinks that it is low sugar kind; Detect according to hand-held saccharimeter, this part of material is low sugar kind.
Therefore, in 132 parts of natural population's materials, the accuracy rate that method of the present invention detects low sugar material is 100%; Detect the accuracy rate 89% of high sugared material.
Claims (10)
1. detect the polymorphism of SNP site in watermelon genome or genotypic material identify or assistant identification watermelon be high sugared watermelon or low sugar watermelon in application;
Or detect the application in characterization or assistant identification watermelon are high sugared watermelon or low sugar watermelon products of the polymorphism of SNP site in watermelon genome or genotypic material;
Described SNP site is the 4037102nd Nucleotide on watermelon genome No. 2 karyomit(e)s, and described SNP site genotype is that TT isozygotys, GG isozygotys or T/G heterozygosis.
2. detect the application in the pol state of qualification or assistant identification watermelon of the polymorphism of SNP site in watermelon genome or genotypic material;
Or detect the application in the pol phase product of characterization or assistant identification watermelon of the polymorphism of SNP site in watermelon genome or genotypic material;
Described SNP site is the 4037102nd Nucleotide on watermelon genome No. 2 karyomit(e)s, and described SNP site genotype is that TT isozygotys, GG isozygotys or T/G heterozygosis.
3. application according to claim 1 and 2, is characterized in that:
In described detection watermelon genome, the polymorphism of SNP site or genotypic material comprise the primer pair of the described SNP site that increases;
The primer sets that described primer pair is specifically made up of the single strand dna shown in the single strand dna shown in sequence 2 and sequence 3.
4. application according to claim 3, is characterized in that: in described detection watermelon genome, the polymorphism of SNP site or genotypic material also comprise MesI and limit restriction endonuclease.
5. to identify or assistant identification watermelon to be measured is the method for high sugared watermelon or low sugar watermelon for one kind, comprise the steps: that detecting watermelon genome SNP site genotype to be measured is that TT isozygotys, GG isozygotys or T/G heterozygosis, if described watermelon genome SNP site genotype to be measured is that TT isozygotys, then described watermelon to be measured is or candidate is high sugared watermelon; If described watermelon genome SNP site genotype to be measured is that GG isozygotys or T/G heterozygosis, then described watermelon to be measured is or candidate is low sugar watermelon;
Described SNP site is the 4037102nd Nucleotide on watermelon genome No. 2 karyomit(e)s.
6. method according to claim 5, is characterized in that: the genotypic method of described detection watermelon to be measured genome SNP site is as follows:
1) direct Sequencing;
2) pcr amplification is carried out with the primer pair Chinese cabbage to be measured can increased containing SNP site fragment, obtain amplified production, again described amplified production is cut through enzyme, detect digestion products size, determine watermelon genome SNP site genotype to be measured according to described digestion products size.
7. method according to claim 6, is characterized in that:.
The described primer pair containing SNP site fragment that can increase is following 1) or 2):
1) described primer pair is for being made up of the single strand dna shown in the single strand dna shown in sequence 1 and sequence 2;
2) described primer pair is for being made up of the single strand dna shown in the single strand dna shown in sequence A and sequence B;
Sequence A is the Nucleotide that sequence 1 was deleted or increased one or several Nucleotide and obtains;
Sequence B is the Nucleotide that sequence 2 was deleted or increased one or several Nucleotide and obtains;
Described enzyme is that MesI limits restriction endonuclease;
Describedly determine that watermelon genome SNP site Genotypic methods to be measured is according to digestion products size:
If digestion products only has 144bp size fragment, then watermelon genome SNP site genotype to be measured is that GG isozygotys,
If digestion products has 144bp size fragment and 72bp size fragment, then watermelon genome SNP site genotype to be measured is T/G heterozygosis,
If digestion products only has 72bp size fragment, then watermelon genome SNP site genotype to be measured is that TT isozygotys.
8. the test kit of qualification or assistant identification watermelon pol to be measured, comprises the polymorphism or genotypic material that detect SNP site in watermelon genome.
9. test kit according to claim 8, it is characterized in that: in described detection watermelon genome, the polymorphism of SNP site or genotypic material comprise the primer pair for increasing containing SNP site fragment, and described SNP site is the 4037102nd Nucleotide on watermelon genome No. 2 karyomit(e)s;
Describedly be specially following 1 for the primer pair increased containing SNP site fragment) or 2):
1) described primer pair is for being made up of the single strand dna shown in the single strand dna shown in sequence 1 and sequence 2;
2) described primer pair is for being made up of the single strand dna shown in the single strand dna shown in sequence A and sequence B;
Sequence A is the Nucleotide that sequence 1 was deleted or increased one or several Nucleotide and obtains;
Sequence B is the Nucleotide that sequence 2 was deleted or increased one or several Nucleotide and obtains;
Described test kit also specifically comprises MesI and limits restriction endonuclease.
10. detect polymorphism or the application of genotypic material in the sugared watermelon breeding of height of SNP site in watermelon genome.
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