CN108841989B - SSR molecular marker system of muskmelon and application thereof - Google Patents

SSR molecular marker system of muskmelon and application thereof Download PDF

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CN108841989B
CN108841989B CN201810763805.XA CN201810763805A CN108841989B CN 108841989 B CN108841989 B CN 108841989B CN 201810763805 A CN201810763805 A CN 201810763805A CN 108841989 B CN108841989 B CN 108841989B
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west
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molecular marker
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CN108841989A (en
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李超
孙玉萍
廖新福
杨英
陈伟
郑贺云
杨军
张翠环
杨咪
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Xinjiang Uygur Autonomous Region Grape And Fruit Research Institute
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Abstract

The invention discloses a melon SSR molecular marker system and application thereof, and relates to the technical field of molecular markers. The names of the primer pairs for identifying the melon varieties are respectively C30, MU5499, MU5554-1, SSR12083 and SSR 04219. The SSR molecular marker primer pair researched by the invention has high polymorphism, clear bands and stable repeatability, and can be used for constructing a melon DNA fingerprint and identifying melon varieties. Particularly, the effect is more obvious when the western state code 1, the western state code 3, the western state code 25, the western state code 17, the western state code 21 or the western state code 29 are identified.

Description

SSR molecular marker system of muskmelon and application thereof
Technical Field
The invention relates to the technical field of molecular markers, in particular to an SSR molecular marker system of melons and application thereof.
Background
SSR (simple Sequence repeats) markers are a molecular marking technology based on specific primer PCR developed in recent years, also called microsatellite DNA (microspatellite DNA), and are series-connected repetitive sequences which are composed of several nucleotides (generally 1-6) as repetitive units and have the length of dozens of nucleotides. The sequences flanking each SSR are typically relatively conserved single copy sequences.
The genome of an organism, particularly a higher organism, contains a large number of repetitive sequences, and it has been found that there is a high degree of variation in the number of repetitive units in microsatellites, and such variation is expressed as an integral fold variation in the number of microsatellites or the sequences in the repetitive unit sequences may not be completely identical, and thus polymorphisms at a plurality of sites are caused. If these variations can be revealed, polymorphisms of different SSRs between different species or even different individuals can be found. Because the flanking sequence of a specific microsatellite in a genome is usually a single sequence with strong conservation, the DNA segments flanking the microsatellite can be cloned and sequenced, and then primers can be artificially synthesized according to the flanking sequence of the microsatellite for PCR amplification, so that a single microsatellite locus is amplified. Due to the quantitative variation of the repeat units of a single microsatellite locus, the length variation of the amplification product of an individual results in a length polymorphism called simple sequence repeat length polymorphism (SSLP), each amplification locus representing a pair of alleles of the locus. Compared with other molecular markers, SSR markers have the following advantages: (1) the quantity is rich, the whole genome is covered, and the disclosed polymorphism is high; (2) the gene has the characteristics of multiple alleles, and the provided information amount is high; (3) inherited in mendelian fashion, co-dominant; (4) each site is determined by the designed primer sequence, so that different laboratories can conveniently communicate with each other to develop the primers. Therefore, the technology is widely used for researches such as construction of genetic maps, calibration of target genes, drawing of fingerprint maps and the like.
The technology for identifying the melon by adopting the SSR molecular marker has been researched, but the SSR molecular marker with high polymorphism, clear strip and stable repeatability needs to be researched so as to be used for constructing the DNA fingerprint of the melon.
Disclosure of Invention
In view of the above, the embodiment of the invention provides a melon SSR molecular marker system and an application thereof, and mainly aims to solve the problem that the variety of the melon SSR molecular markers for identifying melon varieties is too various to quickly and accurately identify the melon varieties.
In order to achieve the purpose, the invention mainly provides the following technical scheme:
in one aspect, the embodiment of the invention provides an SSR molecular marker system of melon; the molecular marker system comprises 5 primer pairs;
the primer pair 1 is named as C30, and the nucleotide sequences are SEQ ID NO.1 and SEQ ID NO. 2;
the name of the primer pair 2 is MU5499, and the nucleotide sequences are SEQ ID NO.3 and SEQ ID NO. 4;
the name of the primer pair 3 is MU5554-1, and the nucleotide sequences are SEQ ID NO.5 and SEQ ID NO. 6;
the name of the primer pair 4 is SSR12083, and the nucleotide sequences are SEQ ID NO.7 and SEQ ID NO. 8;
the name of the primer pair 5 is SSR04219, and the nucleotide sequences are SEQ ID NO.9 and SEQ ID NO. 10.
Preferably, the new variety of melon is West City Milo No.1, West Milo No.3, West Milo No. 25, West Milo No. 17, West Milo No. 21 or West Milo No. 29.
On the other hand, the embodiment of the invention provides the application of the melon SSR molecular marker system in identifying melon varieties.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, the SSR molecular marker system with high polymorphism, clear strips and stable repeatability is screened out by screening the SSR primers of the melon genome and recording the base sequence information and the annealing temperature of the primers, and is used for constructing the DNA fingerprint of the melon to quickly and accurately identify the melon variety.
Drawings
FIG. 1 is a diagram of gel electrophoresis after amplification of primer 73 provided in example 2 of the present invention;
FIG. 2 is a diagram of gel electrophoresis after amplification of primer 47 provided in example 2 of the present invention;
FIG. 3 is a diagram of gel electrophoresis after amplification of the primer 14 provided in example 2 of the present invention;
FIG. 4 is a diagram of gel electrophoresis after amplification of the primer 53 provided in example 2 of the present invention;
FIG. 5 is a diagram of gel electrophoresis after amplification of primer 5 provided in example 2 of the present invention;
FIG. 6 is SSR fingerprints of 6 melon varieties provided in example 3 of the present invention;
FIGS. 7A-7F are finger print QR codes for 6 melon varieties as provided in example 3 of the present invention;
fig. 8 is a two-dimensional code scan of west mil No.1 provided in embodiment 3 of the present invention.
Detailed Description
To further illustrate the technical means and effects of the present invention adopted to achieve the predetermined objects, the following detailed description of the embodiments, technical solutions, features and effects according to the present invention will be given with preferred embodiments. The particular features, structures, or characteristics may be combined in any suitable manner in the embodiments or embodiments described below.
Example 1 extraction of DNA of melon by CTAB method
a. Taking 0.2-0.5 g of young leaves, putting the young leaves into a mortar, adding sufficient liquid nitrogen, and grinding until the young leaves are powdery;
b. rapidly transferring the powder into a 2ml centrifuge tube; 760. mu.l of CTAB extraction buffer 100mmol/LTris (pH8.0), 1.4mol/LNaCl, 50mmol/L EDTA, 2% CTAB, 2% PVP, 20. mu.l of beta-mercaptoethanol were added to the tube and mixed well;
c, carrying out water bath at 65 ℃ for 30min, opening and closing the tube cover in the water bath process, and shaking for 2-3 times;
d. cooled to room temperature and an equal volume of 24: 1(V/V) chloroform: isoamyl alcohol, about 900 μ l, mixed well;
e. centrifuging at normal temperature at 12000rpm for 15 min;
f. taking 2ml centrifuge tube, transferring supernatant (about 750 μ l) into the centrifuge tube;
g. chloroform was added in equal volume to the supernatant: isoamyl alcohol (V/V is 24:1), inverting the centrifuge tube for several times, and mixing uniformly;
h. centrifuging at normal temperature at 12000rpm for 15 min;
i. repeating the step f-h for 1-2 times until white impurities disappear at the interface of the organic phase and the water phase;
j. taking 2ml or 5ml centrifuge tube, transferring supernatant (about 650 μ l) into the centrifuge tube; adding precooled absolute ethyl alcohol with the volume of 2 times that of the supernatant or isopropanol with the same volume, slightly rotating the test tube, then standing and precipitating DNA, and enabling flocculent DNA to appear in the tube;
k. adding 1ml of 70% ethanol into a 1.5ml centrifuge tube, picking DNA into the centrifuge tube, slightly shaking and washing, pouring out 70% ethanol, adding 1ml of absolute ethanol for washing once, pouring out the absolute ethanol, and sucking the absolute ethanol with a gun head. Placing the ethanol solution on a superclean workbench, and opening a fan for 10-15 minutes until the ethanol is completely volatilized;
mu.l of TE [10mmol/L of LTris. HCl (pH8.0), 1mmol/L of EDTA (pH8.0) ] was added to dissolve the DNA;
m. after the DNA was completely dissolved, 1. mu.l of 10mg/mL RNase (final concentration approximately 20. mu.g/mL) was added. Incubating for 30-60 min at 37 ℃;
n. an equal volume of chloroform may be added again: isoamyl alcohol (V/V is 24:1), inverting the centrifuge tube for several times, mixing uniformly, centrifuging at normal temperature, 12000rpm for 10min, taking 1.5ml of centrifuge tube, and transferring the supernatant into the centrifuge tube;
o, adding 2 Xvolume of precooled absolute ethyl alcohol, 1/10 volume of 3mol/L NaAc (pH5.2), standing at 20 ℃ for 1-2 hr or overnight, and precipitating DNA;
p. take 0.5ml centrifuge tube, wash DNA once each with 0.5ml 70% ethanol and absolute ethanol, suck dry ethanol-free with the rifle head. Fully drying on a superclean workbench;
q. adding 50-100 μ L TE [10mmol/L Tris-HCl (pH8.0), 1mmol/L EDTA (pH8.0) ] to dissolve DNA;
and r, packaging the DNA sample, wherein one part is used for the near term, and the other part is stored for a long time and is stored at the temperature of minus 20 ℃.
Mixing 10 different individual plants (such as 10 cultivation individual plants of western Mimi No.1, western Mimi No.3, western Mimi No. 25, western Mimi No. 17, western Mimi No. 21 or western Mimi No. 29) of the same melon, establishing gene pool, and extracting respective genome DNA by the CTAB method; checking the integrity and concentration of the DNA fragment by agarose electrophoresis; OD measurement using UV spectrophotometer230、OD260、OD280Calculating the OD260/OD230And OD260/OD280Detecting the purity of the DNA; if OD260/OD280The ratio is between 1.60 and 1.90, OD260/OD230Greater than 2.0 for PCR amplification, otherwise further purification is required.
Example 2(PCR amplification)
Using the melon DNA extracted in example 1 (e.g., west mi No.1, west mi No.3, west mi No. 25, west mi No. 17, west mi No. 21, or west mi 29) as an amplification template, amplifying the DNA of the above 6 melon varieties using primer 73(C30), primer 47(MU5499), primer 14(MU5554-1), primer 53(SSR04219), or primer 5(SSR12083), respectively; the primer pair information is shown in table 1, and the PCR reaction system is shown in table 2;
TABLE 1 muskmelon SSR primer system
Figure BDA0001728533460000041
TABLE 2 PCR reaction System
Reaction system Volume (20 μ L)
ddH2O 7.9μL
DNA template 1.5μL
Primers P1, P2 0.3. mu.L each
10×Buffer 10.0μL
PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 15s, annealing at a suitable temperature for 15s, extension at 72 ℃ for 30s, and circulation for 35 times; extending for 4min at 72 ℃;
after PCR amplification is finished, a pipette is used to spot on 8% of non-deformable polyacrylamide gel for vertical electrophoresis, the gel is immediately taken down after electrophoresis is finished for silver staining, and the electrophoresis gel result is photographed and recorded by a digital camera, as shown in figures 1-5.
Example 3 (construction of SSR fingerprint of melon)
The 5 pairs of SSR primers designed in example 2 are used to amplify the genomic DNA (total DNA) of 18 melon materials shown in Table 3, and each primer has a different banding pattern (different numbers represent different banding patterns, and the banding patterns sequentially appearing in primer 73 in FIG. 6 are respectively represented by 1, 2, 3, 4 and 5, i.e. 1, 2, 3, 4 and 5 represent 5 different banding patterns), so that the fingerprint patterns of 18 materials can be obtained by using primer pairs 73, 47, 14, 53 and 5, wherein the fingerprint patterns of new species are Xizhou Mi No.1, Xizhou Mi No.3, Xizhou Mi No. 25, Xizhou Mi No. 17, Xizhou Mi No. 21 and Xizhou Mi No. 29 are shown in FIG. 6, and 1-18 in FIG. 6 represent the Xizhou Mi No.1 female parent, the Xizhou Mi No.1 male parent and the Xizhou Mi No.1 male parent respectively; a western honey 3 female parent, a western honey 3 female parent and a western honey 3 female parent; a female parent of western honey 25, a male parent of western honey 25 and a male parent of western honey 25; the female parent of western honey No. 17, the male parent of western honey No. 17 and the male parent of western honey No. 17; a female parent of western honey 21, a male parent of western honey 21 and a male parent of western honey 21; the female parent of western honey 29, the male parent of western honey 29 and the male parent of western honey 29.
TABLE 3 melon materials
Figure BDA0001728533460000051
As can be seen from fig. 6, the west secret 1 fingerprint code is 33131, the west secret 3 fingerprint code is 33253, the west secret 25 fingerprint code is 33311, the west secret 17 fingerprint code is 53215, the west secret 21 fingerprint code is 15511, and the west secret 29 fingerprint code is 36611.
The fingerprint QR code is coded by using a two-dimensional code editor, and the material name, type, plant taxonomy and SSR fingerprint codes of each new variety (6 varieties) are recorded into the software together to form the fingerprint QR code, as shown in figures 7A-7F, and the two-dimensional code scanning image is as shown in figure 8.
The 5 pairs of primers of the invention have more obvious identification effects among the West City Mil No.1, the West Mil No.3, the West Mil No. 25, the West Mil No. 17, the West Mil No. 21 and the West Mil No. 29.
The embodiments of the present invention are not exhaustive, and those skilled in the art can select them from the prior art.
The above disclosure is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and shall be covered by the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the above claims.
Sequence listing
<110> research institute for grapevines and fruits in Xinjiang Uygur autonomous region
<120> SSR molecular marker system of melon and application thereof
<160> 10
<170> SIPOSequenceListing 1.0
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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cgactgtgtc ttgtggagga 20
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<213> Artificial Sequence (Artificial Sequence)
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ccttcatgat cctctactaa accc 24
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tcttccatgc ttttctcgct 20
<210> 7
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gaattggccc atccttcatt 20
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<213> Artificial Sequence (Artificial Sequence)
<400> 8
gccattccaa aaacttttca ac 22
<210> 9
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gagacattgt gggcatttga 20
<210> 10
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ctcattttca tccaaagggc 20

Claims (1)

1. An application of a melon SSR molecular marker system in identifying melon varieties; the molecular marker system comprises 5 primer pairs;
the primer pair 1 is named as C30, and has the nucleotide sequences of SEQ ID NO.1 and SEQ ID NO. 2;
the name of the primer pair 2 is MU5499, and the nucleotide sequences are SEQ ID NO.3 and SEQ ID NO. 4;
the name of the primer pair 3 is MU5554-1, and the nucleotide sequences are SEQ ID NO.5 and SEQ ID NO. 6;
the name of the primer pair 4 is SSR12083, and the nucleotide sequences are SEQ ID NO.7 and SEQ ID NO. 8;
the name of the primer pair 5 is SSR04219, and the nucleotide sequences are SEQ ID NO.9 and SEQ ID NO. 10;
the muskmelon is West-Zhou-Mi No.1, West-Zhou-Mi No.3, West-Zhou-Mi No. 25, West-Zhou-Mi No. 17, West-Zhou-Mi No. 21 or West-Zhou-Mi No. 29.
CN201810763805.XA 2018-07-12 2018-07-12 SSR molecular marker system of muskmelon and application thereof Expired - Fee Related CN108841989B (en)

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CN112961931B (en) * 2021-03-02 2023-01-10 宁波市农业科学研究院 Rapid identification method for purity of Yongtian No.5 melon seeds

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
甜瓜株型性状的遗传分析与分子标记;熊姜玲;《中国优秀硕士学位论文全文数据库》;20180515;附录 *
甜瓜育种亲本材料遗传多样性及群体结构的SSR标记分析;王美荣;《华北农学报》;20100828;第41-46页 *

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