CN105331615A - InDel molecular marker for identifying watermelon fusarium wilt and primer and application thereof - Google Patents
InDel molecular marker for identifying watermelon fusarium wilt and primer and application thereof Download PDFInfo
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- CN105331615A CN105331615A CN201510899021.6A CN201510899021A CN105331615A CN 105331615 A CN105331615 A CN 105331615A CN 201510899021 A CN201510899021 A CN 201510899021A CN 105331615 A CN105331615 A CN 105331615A
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Abstract
The invention particularly relates to an InDel molecular marker for identifying watermelon fusarium wilt, a primer of the InDel molecular marker and an application of the InDel molecular marker in watermelon fusarium wilt resistance breeding. The nucleotide sequence of the InDel molecular marker is TTTATTTTTTATTTTTTATTT. The primer sequence of the InDel molecular marker comprises InDel1_fon1F:TTCCAAAAGTGCAGATTTC and InDel1_fon1R:CACATGGGGATTGACTAAG. According to the InDel molecular marker for identifying watermelon fusarium wilt and the primer and application thereof, main-effect QTL for resisting fon1 is positioned in a first chromosome in the similar way through QTL initial positioning, the InDel molecular marker which is tightly linked with the watermelon fusarium wilt resistance and the primer of the InDel molecular marker are prepared by combining parent re-sequencing, and the application of the InDel molecular marker in watermelon fusarium wilt resistance breeding is realized; novel means can be provided for watermelon fusarium wilt resistance, the improvement process of the watermelon fusarium wilt resistance character is accelerated, and the breeding accuracy and selecting efficiency are improved.
Description
Technical field
The invention belongs to biology field, be specifically related to a kind of identify watermelon blight InDel molecule marker and primer and application.
Background technology
Blight (Fusariumwilt, FW), is caused by Deuteromycotina fusarium bacterium watermelon specialized form (fon) parasitism, is that in world wide, watermelon causes yield and quality to reduce the most serious a kind of fungi soil-borne disease in producing.At present, the fon physiological strain reported has 0,1,2 and 3 totally four kinds, and except fon3, different commercial watermelon varieties has incorporated the resistance of physiological strain fon0, fon1 and fon2 to a certain extent.But most of kind not anti-blight, germ can the diffusion very fast in growth of watermelon district and spreading.Therefore, exploitation and the closely linked easy handling of fusarium wilt disease resistance, molecule marker with low cost, not only in Rapid identification anti-blight kind/be in seedling stage, can shorten breeding cycle, also help the fusarium wilt disease resistance of improvement watermelon, for watermelon breeding for disease resistance provides technical support.
Recently, Lambel etc. (2014) utilize SNP marker to located the main effect QTL site of a watermelon fon1 resistance, are positioned at 2.3 ~ 8.4cM of No. 1 chromosomal inheritance collection of illustrative plates, corresponding to 56050 ~ 6541994bp of No. 1 physical chromosomal map spectrum.Xu Yong seminar also develops closely linked two CAPs/dCAPs with fon1 according to SNP site and marks, be respectively 7716_fon and 502124_fon, corresponding to 459624 and 502124bp of No. 1 physical chromosomal map spectrum, and the chain degree of 502124_fon and fon1 more closely.
The existing closely linked molecule marker of watermelon fon1 resistance developed is CAPS/dCAPs mark, and testing process needs enzyme to cut, and cost is higher, complex steps.Be labeled as codominant marker based on the resurvey insertion/deletion (InDel) of sequence of full-length genome, detect easily, with low cost, but remain in some parts that do not comply with one's wishes, as make a variation less, stability is inadequate.
Summary of the invention
The present invention passes through QTL(quantitativetraitlocus, quantitative trait locus) at the beginning of be positioned at No. 1 chromosomal localization and arrived the main effect QTL of anti-fon1, and to be prepared into watermelon blight resistance closely linked InDel molecule marker and primer thereof and the application in Watermelon Resistances To Fusarium Wilt breeding in conjunction with parent's sequence of resurveying, can be watermelon blight resistance and new tool is provided, accelerate the improvement process of watermelon blight resistance trait, improve accuracy and the efficiency of selection of breeding.
For achieving the above object, the technical solution used in the present invention is as follows:
Design a kind of InDel molecule marker identifying watermelon blight, its nucleotides sequence is classified as:
TTTATTTTTTATTTTTTATTT。
The primer of above-mentioned InDel molecule marker, comprises following nucleotide sequence:
InDel1_fon1F：TTCCAAAAGTGCAGATTTC；
InDel1_fon1R：CACATGGGGATTGACTAAG。
The screening method of the InDel molecule marker of qualification watermelon blight, comprises the following steps:
(1) F that maternal " ZXG01478 " (high resistance to wilt good) and male parent " 14CB11 " (high sense blight) are hybridized is utilized
2the highdensity genetic linkage maps of informative population, and to Parent, F
1and F
293 individual plants of colony carry out fusarium wilt disease resistance qualification;
(2) in conjunction with genetic linkage maps and fusarium wilt disease resistance qualification, WinQTLcartographer2.5software (http://statgen.ncsu.edu/qtlcart/WQTLCart.htm) can be utilized to carry out QTL just locate, the relevant QTL site fon1 of a fusarium wilt disease resistance is identified at LG1, its LOD peak value is 26.05, explain the phenotypic variation of 80.18%, physical location corresponding to fiducial interval is No. 1 chromosomal 193331 ~ 2775577bp;
(3) in conjunction with the sequence of resurveying of two parents, find that 19 insertion and deletion fragments are greater than the InDels of 20bp in QTL interval, in order to reduce the scope of candidate gene, it is 5 sections QTL Region dividing further, according to the insertion and deletion design InDel molecule marker being positioned at endpoint location, the InDel primer that design is corresponding;
(4) utilize corresponding InDel primer at Parent, F
1and F
2genotype identification is carried out in colony;
(5) add that other SNP marker on the genotype identification result of InDel molecule marker and LG1 utilize JoinMap4.0 to carry out map construction, and utilize WinQTLcartographer2.5software to carry out rescaning of QTL; Result shows one of them molecule marker, and InDel1_fon1 appears at the peak dot of this QTL, and LOD value is 31.65, explains the phenotypic variation of 91.46%, and physical location corresponding to QTL fiducial interval is similarly No. 1 chromosomal 193331 ~ 2775577bp.
Utilize abovementioned technology, applicant finally obtains and marks InDel1_fon1 with the closely linked insertion and deletion of watermelon blight resistance, the position of the watermelon reference physical map of genome of its correspondence is No. 1 chromosomal 464377bp, sequence is TTTATTTTTTATTTTTTATTT, primer sequence is
InDel1_fon1F:TTCCAAAAGTGCAGATTTC;
InDel1_fon1R:CACATGGGGATTGACTAAG。
The LOD value that marker site InDel1_fon1 is corresponding is 31.65, explains the phenotypic variation of 91.46%.
The application method of InDel molecule marker in watermelon blight resistance breeding of above-mentioned qualification watermelon blight, comprises the steps:
(1) to F
293 individual plants of colony utilize described molecule marker InDel1_fon1 to carry out genotype identification;
(2) utilize above-mentioned (1) analytical results to classify to different individual plant according to the genotype of molecule marker InDel1_fon1, and utilize fusarium wilt disease resistance appraising datum to carry out identifying and verifying.
the present invention has following actively useful technique effect:
InDel molecule marker variation of the present invention is stable, detection is easy, the step that not need enzyme to cut etc. loaded down with trivial details, and (the CAPS/dCAPs mark relative to having developed) with low cost, improves efficiency of selection and the accuracy rate of watermelon blight resistance breeding, thus accelerates breeding process to greatest extent; In addition, this mark is positioned at the peak value of QTL, explains very large phenotype contribution rate (91.46%), accelerates clone and the functional verification of watermelon blight resistant gene.
Accompanying drawing explanation
Fig. 1 F
2the fusarium wilt disease resistance qualification result distribution plan of colony.
Fig. 2 screens QTL scanning result before and after InDel1_fon1 molecule marker and compares.Font-weight and underscore be the peak molecule marker of QTLfon1; The mark of the upper overstriking of Chromosome1 is the InDel molecule marker of new screening.
Fig. 3 utilizes the primer of molecule marker InDel1_fon1 at part F
2amplification in Meta-genomic DNA.Band for the purpose of band in Parent square frame.Title be all variety name latter two/tri-of code name.
Fig. 4 utilizes the primer of molecule marker fon_7716 at part F
2amplification in Meta-genomic DNA.Band for the purpose of band in Parent square frame.Title be all variety name latter two/tri-of code name.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with specific embodiment, the present invention is described in more detail.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.Raw material involved in following examples, be commercially available if no special instructions, involved detection method if no special instructions, is then ordinary method.
embodiment 1
Watermelon blight Resistance Identification, concrete steps are as follows:
(1) preparation of bacterium soil: by bacterial classification from separation and Culture the watermelon blight plant that incubator is cultivated, mix in 1:50 ratio with sterilizing sandy soil after using front wheat to expand numerous cultivation, for subsequent use;
(2) seed preparation: every kind is got and planted 100, and room temperature is soaked seed 24 hours, 33 DEG C of vernalization sowing in 36 hours;
(3) inoculated identification: adopt the husky bacterium local method of wheat, installed by ready bacterium soil nutrition pot, seeding in nursery bed of heating in greenhouse, every nutrition pot 5, twice repetition, if susceptible and disease-resistant contrast and protection row;
(4) Disease investigation: sow after 10 days and start morbidity, record and emerge and incidence, within about 4 weeks, reach after the sick level of expection respectively until susceptible and disease-resistant check variety, the number of live vaccine of each kind of last statistics, calculates the state of an illness;
(5) state of an illness method of calculation:
Death rate (%)=(number-number of live vaccine of emerging)/number × 100% of emerging;
(6) disease resistance classification:
High resistance (HR): 0≤death rate≤20%;
Disease-resistant (R): 20% < death rate≤40%;
In anti-(MR): 40% < death rate≤60%;
Susceptible (S): 60% < death rate≤80%;
High sense (HS): death rate > 80%.
embodiment 2
The development approach of qualification watermelon blight InDel molecule marker, concrete steps are as follows:
(1) fusarium wilt disease resistance full-length genome QTL just locates
Utilize the F that maternal " ZXG01478 " (high resistance to wilt good) and male parent " 14CB11 " (high sense blight) hybridization obtain
2colony has constructed highdensity genetic linkage maps (carrying out chain hiving off, in table 1 by mLOD value between molecular label), and to Parent, F
1and F
293 individual plants of colony carry out fusarium wilt disease resistance qualification; In conjunction with genetic linkage maps and F
2colony's fusarium wilt disease resistance qualification result (Fig. 1), utilize WinQTLcartographer2.5software (http://statgen.ncsu.edu/qtlcart/WQTLCart.htm) to carry out QTL just to locate, the relevant QTL site fon1 of a fusarium wilt disease resistance is identified in LG1 linkage group, its LOD peak value is 26.05, explain the phenotypic variation of 80.18%, physical location corresponding to fiducial interval is No. 1 chromosomal 193331 ~ 2775577bp(table 2).
Table 1 genetic linkage maps
table 2 high-density collection of illustrative plates and QTL scanning informations after adding two InDel mark
。
(2) sequence of resurveying of Parent
The high-throughput utilizing maternal " ZXG01478 " (high resistance to wilt good) and male parent " 14CB11 " (high feel blight) to carry out the 22x degree of depth is resurveyed sequence.Two parents detect 45134 little InDel altogether.
(3) exploitation of QTL region InDel mark
Find that 19 insertion and deletion fragments are greater than the InDels of 20bp in the interval of QTL location.In order to reduce the region of candidate gene, being 5 sections QTL Region dividing, designing InDel primer according to being positioned at endpoint location/neighbouring insertion and deletion, designing 6 pairs of InDel primers altogether.Utilize the sequence of each 500bp before and after Program extraction insertion and deletion corresponding position, the InDel molecule marker that design is corresponding.
embodiment 3
Watermelon F
2colony's molecular marker analysis, comprises the following steps:
(1) utilize CTAB method to extract blade STb gene, concrete steps are as follows:
get the fresh blade of 1g and put into mortar, add liquid nitrogen grind into fine powder, proceed to immediately in the centrifuge tube being added with 1mlCTAB extraction liquid, make the two fully mixing, be then placed in 65 DEG C of water bath with thermostatic control 60min, put upside down mixing 2-3 time therebetween;
after taking out from water-bath, the centrifugal 1min of 8000rpm;
get supernatant liquor and be placed in another centrifuge tube, add isopyknic chloroform: primary isoamyl alcohol (24:1, V/V), put upside down gently and make it fully mix;
the centrifugal 5min of 10000rpm, gets supernatant liquor (as far as possible not getting middle level precipitation);
add the Virahol (need shift to an earlier date precooling 30min) of 0.7 times of volume, mixing is placed on-20 ° of freezing 30min of being no more than of C allows DNA separate out; The centrifugal 5min of 10000rpm after taking out, stays precipitation to abandon supernatant liquor;
with washes of absolute alcohol precipitation several, outwell soak solution, open centrifuge tube lid and dry;
add the distilled water dissolving DNA of 200 μ l; Measure the concentration of DNA with ultraviolet spectrophotometer, save backup in-20 ° of C refrigerators.
(2) PCR reaction system and program
Reaction system is as follows:
PCR response procedures is: 94 DEG C 5 minutes, 35 circulation 94 DEG C of 20 second, 55 DEG C 1 minute, 72 DEG C 30 seconds, 72 DEG C 5 minutes.
(3) 8% polyacrylamide gel electrophoresis, development, dyeing and banding pattern interpretation are carried out to PCR primer.
preparation of reagents:
A．5×TBE：
Tris-base53.9 gram
EDTA 3.72 grams
Boric acid 27.5 grams
1 liter is settled to distilled water.
B.40% polyacrylamide solution:
Polyacrylamide 193.34 grams
Methylene diacrylamide 6.66 grams
500 milliliters are settled to distilled water.
C.8% polyacrylamide gel (now with the current):
40% polyacrylamide solution 10 milliliters
5 × TBE5 milliliter
10% ammonium persulphate (APS) 200 microlitre
Tetramethyl Ethylene Diamine (TEMED) 80 microlitre
Distilled water 22 milliliters.
D. silver-colored dye liquor:
Silver Nitrate 1 gram
5 milliliters, Glacial acetic acid
Dehydrated alcohol 50 milliliters
500 milliliters are settled to deionized water.
E. developing solution:
15 grams, sodium hydroxide
(37%) 2.5 milliliter, formaldehyde
500 milliliters are settled to deionized water.
gel slab prepares:
Sheet glass distilled water is cleaned, dry after, with the rayon balls wiping of soaking dehydrated alcohol, dry.By notch board and flat board superimposed closely after put into gel maker and compress and the clip (gel maker can make two plate gels) buckling well its both sides.In wash bottle, configure 8% polyacrylamide gel solution (two parts), inject fast after mixing in the gap in the middle of two plates, after filling, be inserted with the comb (not allowing the below of comb tooth produce bubble) of tooth; If now liquid level declines to some extent, not solidified solution can be drawn supplement with pipettor.Wait for that solution fully solidifies.
electrophoresis:
Gel maker support is taken off from base, directly puts into supporting electrophoresis chamber, bottom electrophoresis chamber and on support, in the middle of two pieces of sheet glass, pour appropriate 1 × tbe buffer liquid into.In PCR primer, add 6 × DNALoadingBuffer of 0.2 times of volume, get 0.8 microlitre after mixing and add in loading wells, 260 volts of electrophoresis 35 minutes.
dyeing and development:
After electrophoresis completes, from electrophoresis chamber, take out sheet glass, sled removes notch board, and gel can be attached on flat board, gel towards on flat board is put into silver-colored dye liquor, to be placed on decolorization swinging table jog 15 minutes, gel can get off by Automatic-falling; Silver dye is complete, is taken out by gel and puts into deionized water cleaning 10 seconds; Proceed in developing solution by gel after cleaning, jog shaking table, takes out gel after band is clear, is placed on the position difference of visual inspection band on diagosis device, and preservation of taking pictures.
banding pattern interpretation:
Sheet glass good for seasoning after development is placed on diagosis platform, the position difference of visual inspection two parent band.
embodiment 4
F
2population genetic linkage map rebuild and QTL reorientation, its step is as follows:
(1) add that InDel molecule marker is at F
2the JoinMap4.0 that on the genotype identification of colony and LG1, other SNP marker utilizes commercial sources to obtain carries out map construction.After adding InDel mark, having occurred in sequence end to end of LG1 is put upside down, but the SNP marker of this mark and surrounding is all positioned on same scaffold, and consistent with the collinearity of physical map of genome (Fig. 2).
(2) utilize the new collection of illustrative plates built to reorientate QTL to carry out according to (1) in embodiment 2.Result is as shown in table 2, a molecule marker InDel1_fon1 wherein newly developed appears at the peak value of this QTL, and LOD value is 31.65, explain the phenotypic variation of 91.46%, physical location corresponding to QTL fiducial interval is similarly No. 1 chromosomal 193331 ~ 2775577bp.
The molecule marker InDel1_fon1 being positioned at summit corresponds to a 21bp insertion and deletion of No. 1 karyomit(e) 464377bp, and its sequence is TTTATTTTTTATTTTTTATTT, and its primer sequence is InDel1_fon1F:TTCCAAAAGTGCAGATTTC; InDel1_fon1R:CACATGGGGATTGACTAAG.The amplified production of this primer in maternal (high resistance to wilt good kind) is 335bp, and the amplified production in male parent (susceptible blight kind) is 356bp.After adding that this InDel marks, the LOD peak value of fusarium wilt disease resistance QTLfon1 is higher, and the phenotype contribution rate of explanation is larger, illustrates that the linkage degree of this mark and QTL is very tight.
embodiment 5
Embodiment 4 screen the application of InDel molecule marker in breeding, its applying step is as follows:
(1) as embodiment 3 carries out InDel1_fon1 molecule marker at F
2genotype identification (Fig. 3) in colony.
(2) as embodiment 3 carries out F
2the fusarium wilt disease resistance qualification of colony.
(3) check that two kinds of genotype (A represents maternal banding pattern, and B represents male parent banding pattern, and AB represents heterozygous genotypes) of InDel1_fon1 molecule marker are at 93 F
2distribution situation (table 3) in colony.
Result shows, the genotype of molecule marker InDel1_fon1 in 71 parts of disease-resistant (death rate <60%) strains 22 parts be A(335bp), 1 part is B(356bp), 49 parts is AB.And in 20 parts of susceptible strains, have 18 parts for B(356bp), 2 parts be AB, and all 1 B gene type individual plants are all highly feel (dead seedling >80%) strain.
The genotype of molecule marker InDel1_fon1 is 22 strains of A is all Resistant variants, and is high sense strain in 19 strains 18 that genotype is B, and 1 is Resistant variants (death rate=32.6%).The rate of accuracy reached of its genotype identification is to 97.56%.
(4) also utilize the CAPs that delivered mark fon_7716 as in embodiment 3 to F
2each individual plant of colony carries out pcr amplification, then uses restriction enzyme Tag1 to carry out enzyme and cuts, last as used 8% polyacrylamide gel electrophoresis, development, dyeing and banding pattern interpretation (Fig. 4) in embodiment 3.Result display (table 3 ~ 6), fon_7716 and InDel1_fon1 is at F
2the genotype of colony's 93 individual plants is completely the same, demonstrates qualification result of the present invention further.
Above result is enough to illustrate predicts watermelon blight resistance by molecule marker InDel1_fon1, can improve the efficiency of selection of watermelon blight resistance breeding, thus accelerate breeding process.
Table 3InDel1_Fon1 and fon_7716 is at F
2qualification in colony and checking
Table 4InDel1_Fon1 and fon_7716 is at F
2qualification in colony and checking (Continued)
Table 5InDel1_Fon1 and fon_7716 is at F
2qualification in colony and checking (Continued)
Table 6InDel1_Fon1 and fon_7716 is at F
2qualification in colony and checking (Continued)
。
SEQUENCELISTING
<110> Zhengzhou Fruit-tree Inst., Chinese Agriculture Science Academy
<120> identifies the InDel molecule marker of watermelon blight and primer thereof and application
<130>/
<160>3
<170>PatentInversion3.5
<210>1
<211>21
<212>DNA
<213> artificial sequence
<400>1
tttattttttattttttattt21
<210>2
<211>19
<212>DNA
<213> artificial sequence
<400>2
ttccaaaagtgcagatttc19
<210>3
<211>19
<212>DNA
<213> artificial sequence
<400>3
cacatggggattgactaag19
Claims (6)
1. identify an InDel molecule marker for watermelon blight, its nucleotides sequence is classified as: TTTATTTTTTATTTTTTATTT.
2. a primer for InDel molecule marker described in claim 1, comprises following nucleotide sequence:
InDel1_fon1F：TTCCAAAAGTGCAGATTTC；
InDel1_fon1R：CACATGGGGATTGACTAAG。
3. a screening method for InDel molecule marker described in claim 1, comprises the following steps:
(1) F of high resistance to wilt good type female parent and high sense blight type paternal hybrid is utilized
2the highdensity genetic linkage maps of informative population, and to Parent, F
1and F
2the individual plant of colony carries out fusarium wilt disease resistance qualification;
(2) in conjunction with genetic linkage maps and fusarium wilt disease resistance qualification, carry out QTL and just locate, at the QTL site fon1 that LG1 linkage group qualification fusarium wilt disease resistance is relevant;
(3) in conjunction with the sequence of resurveying of two parents, being greater than the InDels of 20bp in QTL range lookup insertion and deletion fragment, in order to reduce the region of candidate gene, is 5 sections by QTL Region dividing, according to the insertion and deletion design InDel molecule marker being positioned at endpoint location, the InDel primer that design is corresponding;
(4) utilize corresponding InDel primer at Parent, F
1and F
2genotype identification is carried out in colony;
(5) add that other SNP marker on the genotype identification result of InDel molecule marker and LG1 utilize JoinMap4.0 to carry out map construction, and utilize WinQTLcartographer2.5software to carry out rescaning of QTL.
4. the application of primer in the molecular marker assisted selection of watermelon blight resistance described in InDel molecule marker described in claim 1 or claim 2.
5. the application method of InDel molecule marker described in claim 1 in the molecular marker assisted selection of watermelon blight resistance, comprises the steps:
(1) to F
2the individual plant of colony utilizes described molecule marker to carry out genotype identification;
(2) utilize above-mentioned (1) analytical results to classify to different individual plant according to the genotype of molecule marker, and utilize fusarium wilt disease resistance appraising datum to carry out identifying and verifying.
6. the application of primer in watermelon blight resistant gene map based cloning described in InDel molecule marker described in claim 1 or claim 2.
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| CN107254542A (en) * | 2017-08-09 | 2017-10-17 | 中国农业科学院郑州果树研究所 | Watermelon color traits major gene loci and its InDel molecular labelings and application |
| CN107881252A (en) * | 2017-11-29 | 2018-04-06 | 湖南省农业生物技术研究中心 | Identify dCAPS marks, primer and its acquisition methods and the application of watermelon blight |
| CN110622852A (en) * | 2019-09-10 | 2019-12-31 | 黑龙江省农业科学院园艺分院 | Method for cultivating watermelon variety with high fusarium wilt resistance |
| CN112195263A (en) * | 2020-10-21 | 2021-01-08 | 北京市农林科学院 | SNP (Single nucleotide polymorphism) locus and primer set for identifying purity of watermelon hybrid and application |
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| CN106191249A (en) * | 2016-07-13 | 2016-12-07 | 河南牧业经济学院 | A kind of identify that Citrullus vulgaris peel covers the InDel molecular marker of stricture of vagina feature and primer thereof and application |
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| CN107254542A (en) * | 2017-08-09 | 2017-10-17 | 中国农业科学院郑州果树研究所 | Watermelon color traits major gene loci and its InDel molecular labelings and application |
| CN107254542B (en) * | 2017-08-09 | 2020-05-19 | 中国农业科学院郑州果树研究所 | Watermelon flesh color character major gene locus and InDel molecular marker and application thereof |
| CN107881252A (en) * | 2017-11-29 | 2018-04-06 | 湖南省农业生物技术研究中心 | Identify dCAPS marks, primer and its acquisition methods and the application of watermelon blight |
| CN107881252B (en) * | 2017-11-29 | 2020-07-14 | 湖南省农业生物技术研究中心 | dCAPS marker and primer for identifying watermelon fusarium wilt, and acquisition method and application thereof |
| CN110622852A (en) * | 2019-09-10 | 2019-12-31 | 黑龙江省农业科学院园艺分院 | Method for cultivating watermelon variety with high fusarium wilt resistance |
| CN112195263A (en) * | 2020-10-21 | 2021-01-08 | 北京市农林科学院 | SNP (Single nucleotide polymorphism) locus and primer set for identifying purity of watermelon hybrid and application |
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