CN108456684A - Watermelon seed size gene and its SNP marker and application - Google Patents

Watermelon seed size gene and its SNP marker and application Download PDF

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CN108456684A
CN108456684A CN201810315311.5A CN201810315311A CN108456684A CN 108456684 A CN108456684 A CN 108456684A CN 201810315311 A CN201810315311 A CN 201810315311A CN 108456684 A CN108456684 A CN 108456684A
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gene
watermelon
seed size
granule
watermelon seed
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CN108456684B (en
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李娜
马双武
尚建立
王吉明
周丹
李楠楠
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Zhengzhou Fruit Research Institute CAAS
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Abstract

The invention discloses a kind of watermelon seed size gene and its SNP marker and applications.The gene nucleotide series such as SEQ ID NO.1, the protein sequence such as SEQ ID NO.2 of coding；The homology for having 90% or more with the gene has also been devised, and encodes the gene of identical function protein；It specifies a kind of SNP site that the watermelon seed size gene isolates, is located at No. 6 chromosome 5418365bp of watermelon genome, base sports A by G；Design the dCAPs molecular labelings of SNP site；A kind of identification method of watermelon seed size gene type is designed, watermelon seed size gene or dCAPs molecular labelings are applied in watermelon seed sized molecules mark assisting sifting and breeding.New tool can be provided for identification watermelon seed size, accelerate the improvement process of watermelon seed size character.

Description

Watermelon seed size gene and its SNP marker and application
Technical field
The present invention relates to technical field of molecular biology, and in particular to a kind of watermelon seed size gene and its SNP molecules Label and application.
Background technology
First purposes of watermelon is fresh food, and pulp is edible part, seed is bigger than normal influence it is edible, consumer like no seed or It is the smaller watermelon of seed, therefore granule kind is one of breeding objective of cultivated watermelons.Another purposes of watermelon is that seed is used, kind Son is the main product of seed water melon.Studies have shown that seed water melon seed is good protein and vegetable oil resource, and contain Abundant vitamin D, therefore, big grain kind are the main breeding objectives of seed water melon.
Collected in vast resources and during data acquisition, the inventors discovered that big grain be typically wild watermelon, it is viscous Seed watermelon and local varieties, quality are often poor；Granule is mostly common cultivation watermelon, and quality is preferable, is planted between different cultivars Sub- seed size difference is apparent, therefore seed size is also a classification character of watermelon.
The research of domestic and international watermelon seed size is concentrated mainly on carries out tradition to the gene of control seed size, mass of 1000 kernel Genetic analysis.Weetman (1937) Buddhist monk foundation etc.（2015）Research shows that grain tends to the quality of Dominant gene again Character.Zhang Yang（2013）And Zhou Yanfeng（2014）Research show grain weight be all by a pair of of key-gene control.Pool etc. （1941）It is found that the allele of this pair of control seed length of l and s, Kensler etc.（1958）With Shimotsuma etc. （1963）Research also demonstrate this result.However, Tanaka etc.（1995）The gene Ti of control granule is found that, with l and s For non-allelic genes.Zhang etc.（1996）Research shows that tomato watermelon seeds are controlled by single recessive gene ts.Although using Test material it is different from method, conclusion is also not quite identical, but these researchs have some common ground, all think that seed is big Small to belong to qualitative character, small-sized seed is dominant in large seed, and by Dominant gene.On the other hand, Zhai Wen is strong etc.（1995） With grain again two watermelon height with significant difference for the grain weight that self-mating system is parent research filial generation, watermelon seed mass of 1000 kernel The characteristics of showing as Inheritance of Quantitative Characters.Prothro etc.（2012）Utilize recombinant inbred lines and F2Population research seed size, table Bright grain is typical quantitative character again.
The method that traditional science of heredity uses mathematical statistics can only will control one or more bases of quality/quantitative character Because being studied as a whole, single quality/Quantitative Trait Genes position and effect (Xu Yun on chromosome not can determine that It is green, 1992).
It is perfect with molecular marking technique development and molecular quantitative, in recent years, the work that a batch QTL is positioned It has carried out（Model is quick etc., and 2000；Hawkins etc., 2001；Yi Ke, 2002；Prothro etc., 2012；Meru and McGregor, 2013）, to disclose the hereditary basis of watermelon seed size, but the section of these research institutes positioning is larger, waits It selects gene more and is difficult to screen, neither one watermelon seed size gene is determined so far.
Invention content
The technical problem to be solved in the present invention is to provide a kind of watermelon seed size gene and its SNP marker and answer With to disclose the hereditary basis of watermelon seed size, determining the gene of watermelon seed size.
In order to solve the above technical problems, the present invention adopts the following technical scheme that：
A kind of watermelon seed size gene is filtered out, is named asClamdtK, nucleotide sequence as shown in SEQ ID NO.1, The protein sequence of coding is as shown in SEQ ID NO.2.
Designing a kind of and watermelon seed size gene has 90% or more homology, and encodes identical function albumen The gene of matter.
A kind of SNP site that the watermelon seed size gene isolates is specified, No. 6 chromosomes of watermelon genome are located at At 5418365bp, base sports A by G.
A kind of dCAPs molecular labelings of the SNP site are designed, sense primer is named as dcaps9_S6F, nucleosides Acid sequence is as shown in SEQ ID NO.3；Primer is named as dcaps9_S6R, nucleotide sequence such as SEQ ID NO.4 downstream Shown, restriction enzyme is used in the primer endonuclease reactionTagI。
A kind of identification method of watermelon seed size gene type is designed, is included the following steps：
A. DNA is extracted：Watermelon plant total DNA is extracted using CTAB methods；
B. PCR amplification：
Reaction system is：100 ng/ μ L watermelon plant total DNA, 1 μ L, 1 dcaps9_S6F μ L, 1 dcaps9_S6R μ L, 2 × Power Taq PCR MasterMix 12.5 μL、ddH2O 9.5 μL；
Response procedures are：94 DEG C of 5 min, 35 cycle 94 DEG C of 20 s, 55 DEG C of 1 min, 72 DEG C of 30 s, 72 DEG C 5 min；
C. endonuclease reaction system and program：
Reaction system is：5 μ l of PCR product,TagI0.5 1.5 8 μ l of μ l, ddH2O of μ l, 10Xbuffer of restriction enzyme.
Response procedures are：65 DEG C of constant temperature are handled 10 hours.
D. Electrophoretic：Polyacrylamide gel electrophoresis, development, dyeing and banding pattern are carried out to the digestion products Target stripe is found in interpretation, and affiliated genotype is determined according to the stripe size of the amplified production and position relationship, 142bp's Band represents big seed genotype, and the band of 113bp represents fine grain genotype.
By the application of the watermelon seed size gene or the gene in watermelon seed size gene is cloned or is expressed.
The gene, the SNP site or the dCAPs molecular labelings are marked in watermelon seed sized molecules and assisted Application in screening and breeding.
Above application includes the following steps：
（1）To F2The each strain of group carries out genotype identification using the molecular labeling；
（2）Using above-mentioned（1）Middle F2The genotype identification of group is as a result, carry out different single plants according to the genotype of molecular labeling Classification, and identified and verified using seed phenotypic data.
Compared with prior art, the beneficial technical effect of the present invention lies in：
1. the present invention makes public for the first time watermelon multi-drug resistance protein gene using the method for forward geneticsClamdtKRegulating and controlling Application in the function of seed size.
2. the present invention devises the molecular labeling that a watermelon seed size gene isolates, divided using the label Sub- marker assisted selection breeding, can be more rapidly, accurately to carry out the orientation genetic improvement of watermelon seed size.
3. the present invention is applied in watermelon seed size breeding, new hand can be provided for identification watermelon seed size Section, and then accelerate the improvement process of watermelon seed size character, improve the accuracy and efficiency of selection of breeding.
Description of the drawings
Fig. 1 is that 6 difference expression gene RT-PCR verify situation；
Fig. 2 isClamdtKThe gene expression amount of different development stage in RIL_L and RIL_S；
Fig. 3 is to mark amplification figure of the primer of dcaps9_S6 in the F2 Meta-genomic DNAs of part using seed size；
Wherein, band is the purpose band of Parent in box, and title is all latter two/tri- of variety name/code name.
Specific implementation mode
Illustrate the specific implementation mode of the present invention with reference to the accompanying drawings and examples, but following embodiment is used only in detail It describes the bright present invention in detail, does not limit the scope of the invention in any way.
Involved instrument and equipment is routine instrument device unless otherwise instructed in the examples below；Involved Reagent is commercially available conventional reagent unless otherwise instructed；Involved test method is unless otherwise instructed conventional method.
Embodiment one：The determination of watermelon seed size gene and the exploitation of molecular labeling
1. the finely positioning of watermelon seed size main effect QTL
F is obtained using the big grain of watermelon maternal " ZXG01478 " and granule male parent " 14CB11 " hybridization2Informative population it is highdensity Genetic linkage maps, to Parent, F1And F2The single melon of 93 of group carries out the intuitive identification of seed size；In conjunction with genetic linkage Collection of illustrative plates and F2Group's phenotypic evaluation just positions as a result, carrying out QTL using Rqtl-IM-binary methods, identifies seed size Main effect QTL, LOD peak values are 46.94, explain 26.16% phenotypic variation, confidence interval（2-LOD）It is 3.68~31.67 cM。
2. the relevant relatively transcription group analysis of seed size
（1）Recombinant inbred lines is built using the big grain of watermelon maternal " ZXG01478 " and granule male parent " 14CB11 "（RILs）, screening The big grain of a copy of it（RIL_L）With a granule（RIL_S）7,8,9,10,11,12 and 13 days after spending（DAF）Seed turned Record group is sequenced.The essential information of 14 sample transcript profiles sequencing is as shown in table 1.Q30 is both greater than 90%, and G/C content is joined with watermelon It is almost the same to examine genome.
The essential information of 1 transcript profile of table sequencing
。
（2）The RIL_L and RIL_S for being utilized respectively different development stage carry out Differential expression analysis, and select 6 at random Difference expression gene is verified using RT-PCR.
The results are shown in Figure 1, that RT-PCR demonstrates transcriptome analysis well as a result, illustrating transcript profile sequencing and analysis The result is that reliable.
（3）In the regions QTL having determined, only a gene,ClamdtK, multi-drug resistance albumen is encoded, in 7 differences The differential expression of all significant differential expression of developmental stage, other genes is not notable.AndClamdtKIn the difference hair of RIL_L Period is educated not express substantially, and with the extension of pollination time, its gene expression amount is the trend continuously decreased in RIL_S（Such as Shown in Fig. 2）.
Thereby determine that the seed size gene of control main effect QTL isClamdtK, nucleotide sequence such as SEQ ID NO.1 Shown, the protein sequence of coding is as shown in SEQ ID NO.2.
3. the SNP site resurveyed sequence and excavate full-length genome level of " ZXG01478 " and " 14CB11 " is utilized, in geneClamdtKNeighbouring one SNP site of discovery, it is 5418365 which, which is located at No. 6 chromosome base positions of watermelon genome, Base variation is that G sports A.
4. developing a dCAPs molecular labeling according to above-mentioned SNP site, sense primer is named as dcaps9_S6F, under Trip primer is named as dcaps9_S6R, and the nucleotides sequence of the primer pair is classified as：
dcaps9_S6F：CTAAAAATACAGGATTAAAATTGTACATTC；
dcaps9_S6R：TGTAAAACACATATATAACG.
The restriction enzyme used forTagI。
Embodiment two：Watermelon F2Group's molecular marker analysis
1. utilizing CTAB methods extraction watermelon F2Colony leaves total DNA, is as follows：
（1）It takes the fresh blades of 1 g to be put into mortar, liquid nitrogen grind into fine powder is added, is transferred to immediately added with 1 ml CTAB extracts Centrifuge tube in, both make to mix well, be subsequently placed in 65 DEG C of 60 min of water bath with thermostatic control, overturn mixing 2~3 times therebetween；
（2）After water-bath taking-up, 8000 rpm centrifuge 1 min；
（3）It takes supernatant to be placed in another centrifuge tube, adds isometric chloroform：Isoamyl alcohol, gently overturning makes it mix well；Its Middle chloroform and isoamyl alcohol volume ratio are 24：1；
（4）10000 rpm centrifuge 5 min, take supernatant；
（5）The pre- cold isopropanol mixing of 0.7 times of volume of addition is placed on -20 DEG C of freezings allows DNA to be precipitated no more than 30 min；It takes out 10000 rpm centrifuge 5 min afterwards, and precipitation is stayed to abandon supernatant；
（6）For several times with washes of absolute alcohol precipitation, soak is outwelled, opens centrifuge tube lid and dries；
（7）The distilled water dissolving DNA of 200 μ l is added；The concentration that DNA is measured with ultraviolet specrophotometer, in -20 DEG C of refrigerators It saves backup.
2. PCR reaction systems and program
Reaction system is：
Water melon leaf total DNA (100ng/ μ l) 1 μ l, Forward primer (10 μm of ol/L) 1 μ l, Reverse primer (10μmol/L) 1 μl、2×Power Taq PCR MasterMix 12.5 μl、ddH2O 9.5 μl。
PCR response procedures are：
94 DEG C 5 minutes, 35 cycle 94 DEG C 20 seconds, 55 DEG C 1 minute, 72 DEG C 30 seconds, 72 DEG C 5 minutes.
3. endonuclease reaction system and program
Reaction system is：5 μ l of PCR product,TagIRestriction enzyme 0.5 μ l, 10 × buffer 1.5 μ l, ddH2O 8 μ l。
65 DEG C of constant temperature are handled 10 hours.
4. pair PCR product carries out 8% polyacrylamide gel electrophoresis, development, dyeing and banding pattern interpretation.
（1）Preparation of reagents：
A.5 × TBE：
53.9 grams of Tris-base, EDTA3.72 grams, 27.5 grams of boric acid are taken, 1 liter is settled to distilled water.
B. 40% polyacrylamide solution：
193.34 grams of polyacrylamide, 6.66 grams of methylene diacrylamide are taken, 500 milliliters are settled to distilled water.
C. 8% polyacrylamide gel：
40% 10 milliliters of polyacrylamide solution, 5 milliliters of 5 × TBE, 10% ammonium persulfate（APS）200 microlitres, tetramethyl second two Amine（TEMED）80 microlitres, 22 milliliters of distilled water.
D. silver staining liquid：
1 gram of silver nitrate, 5 milliliters of glacial acetic acid, 50 milliliters of absolute ethyl alcohol are taken, 500 milliliters are settled to deionized water.
E. developer solution：
Take 15 grams of sodium hydroxide, formaldehyde（37%）2.5 milliliters, 500 milliliters are settled to deionized water.
（2）Gel slab prepares：
Glass plate cleaned with distilled water, dry after, wiped, dried with the rayon balls for impregnating absolute ethyl alcohol.By notch board peace It is put into the clip that its both sides is compressed and buckled well in gel maker after plate overlapping is close.It is molten that 8% polyacrylamide gel is configured in wash bottle Liquid is rapidly injected in the gap among two plates after mixing, comb with teeth is inserted into after filling；It, can if liquid level is declined at this time Not solidified solution is drawn with pipettor to supplement.Solution is waited for fully to solidify.
（3）Electrophoresis：
Gel maker holder is removed from pedestal, is directly placed into mating electrophoresis tank, two pieces on electrophoresis trench bottom and holder Suitable 1 × tbe buffer liquid is poured among glass plate.6 × DNA Loading of 0.2 times of volume are added in PCR product Buffer takes after mixing 0.8 microlitre to be added in loading wells, 260 volts of electrophoresis 35 minutes.
（4）Dyeing and development：
After the completion of electrophoresis, glass plate is taken out from electrophoresis tank, sled goes notch board, gel that can be attached on tablet, and gel upwardly will Tablet is put into silver staining liquid, is placed in jog 15 minutes on decolorization swinging table, and gel meeting Automatic-falling gets off；Silver staining finishes, by gel Taking-up is put into deionized water and cleans 10 seconds；Gel is transferred in developer solution after cleaning, jog shaking table, after band is clear Gel is taken out, is placed on the position difference for visually observing band on diagosis device, and preservation of taking pictures.
（5）Banding pattern interpretation：
The glass plate spontaneously dried after development is placed on diagosis platform, the position difference of two parent's bands is visually observed.
Using dcaps9_S6 molecular labelings in F2The results are shown in Figure 3 for genotype identification in group, and title is all product Latter two or three of kind of title/code name, band is purpose band in Parent box, and maternal target fragment size is 142bp, father This target fragment size is 113bp.
Embodiment three：F2Group's genotyping
Dcaps9_S6 molecular labelings are carried out through embodiment two, and F is being built by " ZXG01478 " and " 14CB11 "2Gene in group After type identification, check two kinds of genotype of dcaps9_S6 molecular labelings in 93 F2Distribution situation in group is found The genotype of dcaps9_S6 isolates completely with seed size phenotype.The results are shown in Table 2, and wherein A represents maternal banding pattern, B generations Table male parent banding pattern, H represent heterozygous genotypes.
The genotype of molecular labeling dcaps9_S6 is in 69 parts of granules（Mass of 1000 kernel<37g）24 parts are B in strain（113bp）, 44 parts are H.And all A in 24 parts big grain strain（142bp）.
The genotype of molecular labeling InDel14_S6 is entirely big grain strain for 24 strains of A, and is B's in genotype 24 strains are all granule strains.The accuracy rate of its genotype identification is 100%.
2 dcaps9_S6 of table is in F2Identification in group and verification
Variety name/code name dcaps9_S6 Mass of 1000 kernel（g） Classification
It is maternal A 93.5 Big grain
Male parent B 20.8 Granule
F1 H 30.1 Granule
13QB135-001 H 30.3 Granule
13QB135-002 H 26.3 Granule
13QB135-003 H 29 Granule
13QB135-004 H 29.2 Granule
13QB135-005 A 110.6 Big grain
13QB135-006 A 141.7 Big grain
13QB135-007 A 86.8 Big grain
13QB135-008 B 16.8 Granule
13QB135-009 H 32 Granule
13QB135-010 H 32.9 Granule
13QB135-011 H 28.8 Granule
13QB135-013 A 90.4 Big grain
13QB135-014 A 101.2 Big grain
13QB135-015 H 29 Granule
13QB135-016 B 19.5 Granule
13QB135-017 H 24.8 Granule
13QB135-018 H 25.4 Granule
13QB135-019 B 16.6 Granule
13QB135-020 H 27.4 Granule
13QB135-021 H 24.5 Granule
13QB135-022 H 27.8 Granule
13QB135-023 B 18.2 Granule
13QB135-024 A 116 Big grain
13QB135-025 B 18.8 Granule
13QB135-026 A 95.6 Big grain
13QB135-027 B 23.5 Granule
13QB135-028 H 31.6 Granule
13QB135-029 H 20 Granule
13QB135-030 A 113.2 Big grain
13QB135-031 B 20.3 Granule
13QB135-032 H 28.9 Granule
13QB135-033 H 33.3 Granule
13QB135-034 B 23 Granule
13QB135-035 B 19.7 Granule
13QB135-036 H 28.8 Granule
13QB135-037 H 32.2 Granule
13QB135-038 H 25.4 Granule
13QB135-040 H 21.5 Granule
13QB135-041 A 97.9 Big grain
13QB135-042 H 26.8 Granule
13QB135-043 A 118.9 Big grain
13QB135-044 H 29.3 Granule
13QB135-045 B 20.8 Granule
13QB135-046 B 20.2 Granule
13QB135-047 A 90.3 Big grain
13QB135-048 H 27 Granule
13QB135-049 A 99.3 Big grain
13QB135-050 H 25.5 Granule
13QB135-051 B 23.1 Granule
13QB135-052 B 22.4 Granule
13QB135-053 B 17.8 Granule
13QB135-054 H 31.8 Granule
13QB135-055 H 29.9 Granule
13QB135-056 B 18.8 Granule
13QB135-057 B 20.9 Granule
13QB135-058 H 27.1 Granule
13QB135-059 H 30.3 Granule
13QB135-060 H 30.4 Granule
13QB135-061 H 31.2 Granule
13QB135-062 H 27.6 Granule
13QB135-064 A 93.7 Big grain
13QB135-066 H 25.8 Granule
13QB135-067 B 20.4 Granule
13QB135-068 B 18.6 Granule
13QB135-069 A 111.6 Big grain
13QB135-071 A 129.4 Big grain
13QB135-075 B 23.5 Granule
13QB135-076 B 17 Granule
13QB135-078 H 27.4 Granule
13QB135-079 H 27.5 Granule
13QB135-080 H 28.9 Granule
13QB135-081 A 123.6 Big grain
13QB135-083 H 25 Granule
13QB135-086 H 31.2 Granule
13QB135-087 B 21.8 Granule
13QB135-090 H 28.6 Granule
13QB135-094 H 33.6 Granule
13QB135-095 A 98.7 Big grain
13QB135-097 A 100.6 Big grain
13QB135-099 H 24 Granule
13QB135-100 A 122.8 Big grain
13QB135-101 H 26.9 Granule
13QB135-103 A 92.6 Big grain
13QB135-104 A 108.8 Big grain
13QB135-105 B 18.6 Granule
13QB135-106 H 31.1 Granule
13QB135-107 B 23.7 Granule
13QB135-109 B 18.3 Granule
13QB135-111 H 35.5 Granule
13QB135-113 A 100 Big grain
13QB135-115 B 20.4 Granule
13QB135-118 A 105.4 Big grain
13QB135-122 A 97.8 Big grain
The present invention is described in detail above in conjunction with drawings and examples, still, those of skill in the art Member is it is understood that without departing from the purpose of the present invention, can also carry out each design parameter in above-described embodiment Change, forms multiple specific embodiments, is the common variation range of the present invention, is no longer described in detail one by one herein.
SEQUENCE LISTING
<110>Zhengzhou Fruit-tree Inst., Chinese Agriculture Science Academy
<120>Watermelon seed size gene and its SNP marker and application
<130> 2018
<160> 4
<170> PatentIn version 3.2
<210> 1
<211> 1533
<212> DNA
<213> watermelon
<400> 1
atgcttgcag aagagaagtc ccaaaagtac ccgacaatgc cagaggtcct tgaagagctg 60
aagcaaatgg ctgacattgg tttccctgtt ttggcaatgg gcttagtggg ttatctcaaa 120
aatatgatct ctgttatttg catgggcaga cttggaactc ttcatctcgc tgctggttct 180
ttggccattg gtttcactaa tatcactggc tattcagttc tttcaggctt ggctatgggc 240
atggagccac tctgtagtca agcttttggt tctcataatt cttccattgc ctttctcact 300
ttgcaaagaa cggttcttat tttgcttttt gctactattc ccattgggtt tctttggcta 360
aatttggagc ctcttatgtt ggttctacat cagaacccag aaatcactag aattgcaact 420
gtttattgcc gttttgcagt tcctgatttg gtattgaata gccttttaca tcctttgcgt 480
atttacctta gaaacaaagg caccacgtgg attgtcatgt ggtgcaattt gttggctatt 540
ctcctacatg ttcccatcgc tattttcttg acttttcctc ttgatcttgg aatccgtggg 600
attgctatct ccaattttat agctaatttc aatacccttt tcttcctttt actctatttg 660
atattctgta ctcgtactac tttttcctcc tcttcttcta aggaggctaa tctgtttgtg 720
ccactgaaaa gcagcaccgt ggttagcgcc gctacggttg gggaggaatg gggaatgctg 780
atcaagttgg ccattcctag ctgtcttgga gtttgcttgg aatggtggtg gtatgagttc 840
atgaccattc tcactggcta cctttataac ccgcggattg cactcgccac ttcaggcatt 900
gtaatccaaa caacttcact aatgtacaca ttaccaatgg ctctcagtgc cgctgtctcg 960
actagagttg gtcacgagct cggcgctggt cggcccaaaa aggctcgact agcggcggtg 1020
gtggcgatag gattggcctt ggtgggctca ttgatgggac tctcactaac caccattggc 1080
agaaggacat ggggaagagt tttcacaaaa gatgaggaaa ttctagagct gacaatggcg 1140
gttctgccca taatcgggct gtgcgagcta gcaaattgcc cgcaaacaac aagctgcggg 1200
attctgaggg gaagtgcaag gccggggatc ggagcaggaa taaacttctg ttcattttac 1260
atggtggggg cgccgatggc cgtcttgtcg gcgtttgttt ggaaatctgg gttcgtgggt 1320
ctttgctacg ggcttttggc agcccagatg gcatgtgtgg tctcaatctt aatagtggtc 1380
ttcaacacag attgggaaat ggagtcaatc aaagccgaag acttagtagg caaaaacacc 1440
aataacgtct ttgcacatgc aatccacaca gccatacgtg aggaaggtcc tgaattcctc 1500
aaagaatcac ctgttgaaag acaagacaca taa 1533
<210> 2
<211> 510
<212> PRT
<213> watermelon
<400> 2
Met Leu Ala Glu Glu Lys Ser Gln Lys Tyr Pro Thr Met Pro Glu Val
1 5 10 15
Leu Glu Glu Leu Lys Gln Met Ala Asp Ile Gly Phe Pro Val Leu Ala
20 25 30
Met Gly Leu Val Gly Tyr Leu Lys Asn Met Ile Ser Val Ile Cys Met
35 40 45
Gly Arg Leu Gly Thr Leu His Leu Ala Ala Gly Ser Leu Ala Ile Gly
50 55 60
Phe Thr Asn Ile Thr Gly Tyr Ser Val Leu Ser Gly Leu Ala Met Gly
65 70 75 80
Met Glu Pro Leu Cys Ser Gln Ala Phe Gly Ser His Asn Ser Ser Ile
85 90 95
Ala Phe Leu Thr Leu Gln Arg Thr Val Leu Ile Leu Leu Phe Ala Thr
100 105 110
Ile Pro Ile Gly Phe Leu Trp Leu Asn Leu Glu Pro Leu Met Leu Val
115 120 125
Leu His Gln Asn Pro Glu Ile Thr Arg Ile Ala Thr Val Tyr Cys Arg
130 135 140
Phe Ala Val Pro Asp Leu Val Leu Asn Ser Leu Leu His Pro Leu Arg
145 150 155 160
Ile Tyr Leu Arg Asn Lys Gly Thr Thr Trp Ile Val Met Trp Cys Asn
165 170 175
Leu Leu Ala Ile Leu Leu His Val Pro Ile Ala Ile Phe Leu Thr Phe
180 185 190
Pro Leu Asp Leu Gly Ile Arg Gly Ile Ala Ile Ser Asn Phe Ile Ala
195 200 205
Asn Phe Asn Thr Leu Phe Phe Leu Leu Leu Tyr Leu Ile Phe Cys Thr
210 215 220
Arg Thr Thr Phe Ser Ser Ser Ser Ser Lys Glu Ala Asn Leu Phe Val
225 230 235 240
Pro Leu Lys Ser Ser Thr Val Val Ser Ala Ala Thr Val Gly Glu Glu
245 250 255
Trp Gly Met Leu Ile Lys Leu Ala Ile Pro Ser Cys Leu Gly Val Cys
260 265 270
Leu Glu Trp Trp Trp Tyr Glu Phe Met Thr Ile Leu Thr Gly Tyr Leu
275 280 285
Tyr Asn Pro Arg Ile Ala Leu Ala Thr Ser Gly Ile Val Ile Gln Thr
290 295 300
Thr Ser Leu Met Tyr Thr Leu Pro Met Ala Leu Ser Ala Ala Val Ser
305 310 315 320
Thr Arg Val Gly His Glu Leu Gly Ala Gly Arg Pro Lys Lys Ala Arg
325 330 335
Leu Ala Ala Val Val Ala Ile Gly Leu Ala Leu Val Gly Ser Leu Met
340 345 350
Gly Leu Ser Leu Thr Thr Ile Gly Arg Arg Thr Trp Gly Arg Val Phe
355 360 365
Thr Lys Asp Glu Glu Ile Leu Glu Leu Thr Met Ala Val Leu Pro Ile
370 375 380
Ile Gly Leu Cys Glu Leu Ala Asn Cys Pro Gln Thr Thr Ser Cys Gly
385 390 395 400
Ile Leu Arg Gly Ser Ala Arg Pro Gly Ile Gly Ala Gly Ile Asn Phe
405 410 415
Cys Ser Phe Tyr Met Val Gly Ala Pro Met Ala Val Leu Ser Ala Phe
420 425 430
Val Trp Lys Ser Gly Phe Val Gly Leu Cys Tyr Gly Leu Leu Ala Ala
435 440 445
Gln Met Ala Cys Val Val Ser Ile Leu Ile Val Val Phe Asn Thr Asp
450 455 460
Trp Glu Met Glu Ser Ile Lys Ala Glu Asp Leu Val Gly Lys Asn Thr
465 470 475 480
Asn Asn Val Phe Ala His Ala Ile His Thr Ala Ile Arg Glu Glu Gly
485 490 495
Pro Glu Phe Leu Lys Glu Ser Pro Val Glu Arg Gln Asp Thr
500 505 510
<210> 3
<211> 30
<212> DNA
<213>It is artificial synthesized
<400> 3
ctaaaaatac aggattaaaa ttgtacattc 30
<210> 4
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 4
tgtaaaacac atatataacg 20

Claims (9)

1. a kind of watermelon seed size geneClamdtK, which is characterized in that its nucleotide sequence as shown in SEQ ID NO.1, The protein sequence of coding is as shown in SEQ ID NO.2.
2. a kind of there is 90% or more homology with watermelon seed size gene described in claim 1, and encodes identical function egg The gene of white matter.
3. the SNP site that watermelon seed size gene isolates described in a kind of claim 1, which is characterized in that be located at watermelon base At No. 6 chromosome 5418365bp of group, base sports A by G.
4. a kind of dCAPs molecular labelings of SNP site described in claim 3, which is characterized in that its upstream primer nucleotide sequences As shown in SEQ ID NO.3；Primer nucleotide sequences are as shown in SEQ ID NO.4 downstream.
5. the dCAPs molecular labelings according to SNP site described in claim 4, which is characterized in that used in the primer endonuclease reaction Restriction enzyme isTagI。
6. a kind of identification method of watermelon seed size gene type, which is characterized in that include the following steps：
A. DNA is extracted：Watermelon plant total DNA is extracted using CTAB methods；
B. PCR amplification：
Reaction system is：1 μ L of sense primer, claim described in 100 ng/ μ L watermelon plant total DNA, 1 μ L, claim 4 4 downstream primer, 1 μ L, 2 × Power Taq PCR MasterMix, 12.5 μ L, ddH2O 9.5 μL；
Response procedures are：94 DEG C of 5 min, 35 cycle 94 DEG C of 20 s, 55 DEG C of 1 min, 72 DEG C of 30 s, 72 DEG C 5 min；
C. endonuclease reaction system and program：
Reaction system is：5 μ l of PCR product,TagIRestriction enzyme 0.5 μ l, 10 × buffer 1.5 μ l, ddH2O 8 μl；
Response procedures are：65 DEG C of constant temperature are handled 10 hours；
D. Electrophoretic：Polyacrylamide gel electrophoresis, development, dyeing and banding pattern interpretation are carried out to the digestion products, Target stripe is found, affiliated genotype, the band generation of 142bp are determined according to the stripe size of the amplified production and position relationship The big seed genotype of table, the band of 113bp represent fine grain genotype.
7. gene described in watermelon seed size gene or claim 2 described in claim 1 is cloned in watermelon seed size gene Or the application in expression.
8. the dCAPs described in SNP site or claim 4 described in gene, claim 3 described in claim 1, claim 2 Application of the molecular labeling in watermelon seed sized molecules mark assisting sifting and breeding.
9. according to being applied described in claim 8, which is characterized in that include the following steps：
（1）To F2The each strain of group carries out genotype identification using the molecular labeling；
（2）Using above-mentioned（1）Middle F2The genotype identification of group is as a result, carry out different single plants according to the genotype of molecular labeling Classification, and identified and verified using seed phenotypic data.
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CN110904264A (en) * 2019-12-19 2020-03-24 中国农业科学院郑州果树研究所 InDel molecular marker co-separated from watermelon minimal seed gene ts, primers and application thereof
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109679971A (en) * 2019-01-14 2019-04-26 浙江大学 The PCR molecular labeling and application thereof of the important adversity gene AOX of watermelon
CN110904264A (en) * 2019-12-19 2020-03-24 中国农业科学院郑州果树研究所 InDel molecular marker co-separated from watermelon minimal seed gene ts, primers and application thereof
CN110904264B (en) * 2019-12-19 2022-04-26 中国农业科学院郑州果树研究所 InDel molecular marker co-separated from watermelon minimal seed gene ts, primers and application thereof
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CN111549172B (en) * 2020-06-12 2023-02-28 中国农业科学院郑州果树研究所 Watermelon leaf posterior green gene linkage site and CAPS marker
CN116103428A (en) * 2022-10-27 2023-05-12 黑龙江省农业科学院园艺分院 dCAPS molecular marker related to watermelon seed size and application thereof
CN116103428B (en) * 2022-10-27 2023-08-18 黑龙江省农业科学院园艺分院 dCAPS molecular marker related to watermelon seed size and application thereof
CN118127227A (en) * 2024-04-30 2024-06-04 浙江大学海南研究院 InDel and KASP molecular marker for size and shape of watermelon seeds and application

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