CN107805672A - One kind identification giant pumpkin × Cucurbita moschata hybrid stock variety authenticity method - Google Patents
One kind identification giant pumpkin × Cucurbita moschata hybrid stock variety authenticity method Download PDFInfo
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Abstract
The present invention is entitled《One kind identification giant pumpkin × Cucurbita moschata hybrid stock variety authenticity method》, belong to biological field.Differential variety authenticity is to safeguarding that the interests of the producer and breeder become more and more important.India and China's hybridization pumpkin rootstock outward appearance various in style is similar, and the true and false, poor reliability that time-consuming are screened using morphological index, therefore, inventing a kind of method using molecular markers for identification variety authentication turns into the task of top priority.Technical scheme is that three stock varieties and control material are entered with performing PCR amplification using 5 pairs of SSR primers (SEQ ID NO.1 SEQ ID NO.10), " 1 " and " 0 " assignment is passed through in caused 9 difference sites, and structure Variety fingerprinting is to authenticity identification.Invention is hybridized pumpkin rootstock kind ' river in Zhejiang Province anvil 7 ', ' river in Zhejiang Province anvil 8 ' and ' river in Zhejiang Province anvil 10 ' to India and China and is carried out more accurate more effectively identification and protection with more sensitive identification of means, lower time cost of labor.
Description
First, technical field
The invention belongs to biological field, more particularly to river in Zhejiang Province anvil series giant pumpkin × Cucurbita moschata hybrid stock variety
A kind of authentication method of ' river in Zhejiang Province anvil 7 ', ' river in Zhejiang Province anvil 8 ' and ' river in Zhejiang Province anvil 10 ' authenticity.
2nd, background technology
Watermelon blight triggers continuous cropping obstacle, generally occurs in melon planting area, is the great difficult problem of melon production.According to system
Meter, the incidence of disease of annual watermelon blight even have no harvest up to 10-30%, serious plot, cause huge economic losses.At present,
Due to land resource limitation and the intractable of Pathogen of Fusarium Wilt, crop rotation and chemopreventive effects are bad, and environmental pollution be present
And the problems such as food security, therefore, grafting is overcoming the effective means of continuous cropping obstacle and droop in watermelon production.
Pumpkin is one of ancient crop that the mankind cultivate earliest, and its species is various, and variation is abundant, is not only on mankind's dining table
Health care colorful vegetable, and melon produce overcome the important rootstock resource of continuous cropping obstacle.Common pumpkin kind is main in production
Including musky gourd (Cucurbita moschata), giant pumpkin (Cucurbita maxima) and american pumpkin
Species such as (Cucurbita pepo).These pumpkin kinds origin is different, and annidation is also different.Giant pumpkin originates from America
South of Peru, the northern dry ground band of Bolivia and Argentina, under hot conditions, virosis and powdery mildew morbidity are serious.In
State's pumpkin originates from Mexico and Central and South America, with a long history in Chinese cultivated, and common manifestation is heat-resisting, impoverishment tolerant, disease-resistant, adapts to
Property is wide.By interspecific hybridization, the advantages of different parents can be polymerize, new phenotypic variation is formed, is provided for stock breeding
Abundant germ plasm resource.' river in Zhejiang Province anvil 7 ', ' river in Zhejiang Province anvil 8 ' and ' river in Zhejiang Province anvil 10 ' is that Ningbo City Agriculture Science Academy's emphasis is directed to
Grafting watermelon is cultivated, a series of giant pumpkin × Cucurbita moschata hybrid stock variety being independently bred as.The series has China concurrently
The resistance of pumpkin and the graft compatibility of giant pumpkin, have no adverse effects to quality of watermelon, deep to be welcome by melon grower.
With the popularization of river in Zhejiang Province anvil series India and China hybridization pumpkin rootstock kind, differential variety authenticity is to safeguarding the producer and educating
The interests of kind person become more and more important.However, the identification of stock variety at present is more with conventional methods such as plant forms identifications.These
Time-consuming for method, easily affected by environment, poor reliability, the India and China similar in face of various in style, exophenotype hybridize pumpkin rootstock
Material, the identification true and false are hard to work.
Stock new varieties ' river in Zhejiang Province anvil 7 ', ' river in Zhejiang Province anvil 8 ' and ' river in Zhejiang Province anvil 10 ' have only made crop field morphology purity mirror so far
It is fixed, still lack convenient effective Varieties identification method, protected for kind also far from enough.
3rd, the content of the invention
In view of the above-mentioned problems, the invention provides one kind to utilize DNA fingerprinting identification river in Zhejiang Province anvil series India and China hybridization pumpkin
The method of stock variety ' river in Zhejiang Province anvil 7 ', ' river in Zhejiang Province anvil 8 ' and ' river in Zhejiang Province anvil 10 ' variety authentication.
To realize the purpose of the present invention, inventor provides following technical scheme：Using CTAB methods extraction ' river in Zhejiang Province anvil 7 ', ' river in Zhejiang Province
Anvil 8 ' and the spire DNA of ' river in Zhejiang Province anvil 10 ' and its check variety；Using 5 pairs of SSR primers enter performing PCR amplification (Primer with
The corresponding relation of sequence number is as shown in table 1 in sequence table, and primer particular sequence is shown in this specification finally appended sequence table)；Amplification
Product is separated, after ethidium bromide staining through high-resolution agarose electrophoresis, and polymorphism is detected on ultraviolet scenograph.Amplified production
Polymorphism reflect the polymorphism of genome.Discrepant loci, amplifies being designated as " 1 " for band, same position without
Band is designated as " 0 "；These differential amplification fragments are converted to the character string being made up of 1 and 0, that is, DNA fingerprinting are formed, to area
Divide 3 parts of India and China's hybridization pumpkin rootstock kinds and its control.
Advantages of the present invention：Compared with conventional Morphological Identification, qualification result of the invention accurately and reliably, it is time-consuming it is short, not by
Environment influences, is simple to operate, can identify multiple kinds simultaneously, convenient and swift.
4th, illustrate
Fig. 1 is SSR primer N11/12, N41/42, N51/52, N121/122 and N155/156 ' rivers in Zhejiang Province in the embodiment of the present invention
Anvil 7 ', ' river in Zhejiang Province anvil 8 ', ' river in Zhejiang Province anvil 10 ' and control material 5558,5559, the amplification in ' bright show ' and ' tortoise beetle field '.
Three width pictures of left side are SSR primers N11/12, N41/42 and N51/52 respectively from top to bottom in ' river in Zhejiang Province anvil 7 in Fig. 1
Number ', ' river in Zhejiang Province anvil 8 ', ' river in Zhejiang Province anvil 10 ' and control material 5558,5559, the amplification in ' bright show ' and ' tortoise beetle field '；The width of right side two
Picture be SSR primer N121/122 and N155/156 respectively from top to bottom ' river in Zhejiang Province anvil 7 ', ' river in Zhejiang Province anvil 8 ', ' river in Zhejiang Province anvil 10 ' and
Amplification in control material 5558,5559, ' bright show ' and ' tortoise beetle field '.Two groups of picture samples put in order to be followed successively by from left to right
DNA Marker, ' river in Zhejiang Province anvil 7 ', ' river in Zhejiang Province anvil 8 ', ' river in Zhejiang Province anvil 10 ', 5558,5559, ' bright show ' and ' tortoise beetle field '.On the left of picture
Mark is DNAmarker standard molecular weights, and right side mark is clip size where Primer and difference site.
5th, embodiment
1st, sample：Sow ' river in Zhejiang Province anvil 7 ', ' river in Zhejiang Province anvil 8 ' and ' river in Zhejiang Province anvil 10 ' and its compare, plant to be planted length is true to 5-6 pieces
Ye Shi, collection tender leaf 1g, -80 DEG C of preservations.
2nd, DNA is extracted：With reference to CTAB methods (see：Murray HG, Thompson WF.Rapid isolation of
Higher weight DNA.NucleicAcids Res, 1980,8：4321) fresh blade total genomic dna, is extracted.
3rd, PCR is expanded：Sample mixing system is 15 μ l (Fazio G, Staub JE, Stevens MR.Genetic mapping
and QTL analysis of horticultural traits in cucumber(Cucumis sativus L.)using
Recombinant inbred lines.Theor Appl Genet, 2003,107：864-874), including 1.5 μ l dNTP
(1.25mM), 1.5ul 10 × buffer, 1.5ul MgCl2(25mM), 2.0ul primers (5mM；Two primers of SSR add respectively
1.0ul), 5ul DNA (10ng/ul), 0.05ul Taq (1U), 3.45ul ultra-pure waters.PCR amplification programs are：94 DEG C of pre-degeneration
1min；93 DEG C of 1min are denatured, anneal 40 DEG C of 1min, extends 72 DEG C of 2min (40 circulations)；Extend 72 DEG C of 1min.
4th, PCR primer detects：Use 2.5% high-resolution standard gel agarose (DNA fragmentation separating ranges 40-
1Kbp).Add 20 μ l EB (ethidium bromide, ethidium bromide) colour developings.2 μ l Loading are added in PCR primer
Dye, electrophoresis 2-3 hours, voltage 200-250v.After electrophoresis terminates, result is observed using gel imaging system.
5th, Data Collection and analysis：To 5 couples of SSR primers N11/12, N41/42, N51/52, N121/122 and N155/156
In ' river in Zhejiang Province anvil 7 ', ' river in Zhejiang Province anvil 8 ', ' river in Zhejiang Province anvil 10 ' and control material 5558,5559, the amplification knot in ' bright show ' and ' tortoise beetle field '
Fruit carries out assignment, and certain amplified allele shaping band is designated as " 1 ", and same site is designated as " 0 " without amplified band.These are poor
Different amplified fragments are converted to the character string being made up of 1 and 0, that is, form Variety fingerprinting (table 1).Produced using 5 molecular labelings
9 raw pleomorphism sites, the finger-print of structure ' river in Zhejiang Province anvil 7 ' is 1-1-0-1-1-0-1-0-1, the fingerprint of ' river in Zhejiang Province anvil 8 '
Collection of illustrative plates is 0-0-1-0-1-1-0-1-0, the finger-print of ' river in Zhejiang Province anvil 10 ' is 0-0-0-0-0-0-0-0-0,5558 fingerprint image
To compose as 1-0-1-0-1-1-0-1-0,5559 finger-print be 1-1-1-0-1-1-0-1-0, the finger-print of ' bright show ' is 1-
1-0-1-1-0-1-1-0 and the finger-print in ' tortoise beetle field ' are 0-1-0-1-1-0-1-0-1.These finger-prints can not only have
Effect distinguishes above three kind, can also screen out to come from the confusing similar varieties of form by them, plays cultivar identification
With the effect of protection.
Table 1 is for examination India and China hybridization pumpkin rootstock kind and the title and SSR finger-prints of control material
Claims (1)
1. the method for one kind identification giant pumpkin × Cucurbita moschata hybrid stock variety authenticity, comprises the following steps：
(1) sample：Sow ' river in Zhejiang Province anvil 7 ', ' river in Zhejiang Province anvil 8 ' and ' river in Zhejiang Province anvil 10 ' and its control material, plant to be planted length are true to 5-6 pieces
Ye Shi, collection tender leaf 1g, -80 DEG C of preservations；
(2) DNA is extracted：Fresh blade total genomic dna is extracted with reference to CTAB methods；
(3) PCR is expanded：Sample mixing system is 15 μ l, including 1.5 μ l dNTP (1.25mM), 1.5ul 10 × buffer, 1.5ul
MgCl2(25mM), 2.0ul primers (5mM；Two primers of SSR add 1.0ul respectively), 5ul DNA (10ng/ul), 0.05ul
Taq (1U), 3.45ul ultra-pure waters.PCR amplification programs are：94 DEG C of 1min of pre-degeneration；93 DEG C of 1min are denatured, anneal 40 DEG C of 1min,
72 DEG C of 2min of extension (40 circulations)；Extend 72 DEG C of 1min；
(4) PCR primer detects：Use 2.5% high-resolution standard gel agarose (DNA fragmentation separating ranges 40-1Kbp).Add
20 μ l EB (ethidium bromide, ethidium bromide) are added to develop the color.2 μ l Loading Dye, electrophoresis are added in PCR primer
2-3 hours, voltage 200-250v.After electrophoresis terminates, result is observed using gel imaging system；
(5) Data Collection and analysis：5 couples of SSR primers N11/12, N41/42, N51/52, N121/122 and N155/156 are existed
' river in Zhejiang Province anvil 7 ', ' river in Zhejiang Province anvil 8 ', ' river in Zhejiang Province anvil 10 ' and control material 5558,5559, the amplification in ' bright show ' and ' tortoise beetle field '
Assignment is carried out, certain amplified allele shaping band is designated as " 1 ", and same site is designated as " 0 " without amplified band.By these differences
Amplified fragments are converted to the character string being made up of 1 and 0, that is, form Variety fingerprinting (table 1).Produced using 7 molecular labelings
15 pleomorphism sites, build ' river in Zhejiang Province anvil 7 ' finger-print；
Wherein, in 5 pairs of SSR primers N11/12 sequence as shown in SEQ ID NO.1 and SEQ ID NO.2, N41/42's
Sequence is as shown in SEQ ID NO.3 and SEQ ID NO.4, N51/52 sequence such as SEQ ID NO.5 and SEQ ID NO.6 institutes
Show, N121/122 sequence as shown in SEQ ID NO.7 and SEQ ID NO.8, N155/156 sequence such as SEQ ID NO.9 and
Shown in SEQ ID NO.10.
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| CN201610837480.6A CN107805672B (en) | 2016-09-09 | 2016-09-09 | Method for identifying authenticity of Indian pumpkin and Chinese pumpkin hybrid stock varieties |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110607391A (en) * | 2019-11-05 | 2019-12-24 | 东北农业大学 | Molecular marker closely linked with character control of Indian pumpkin red fruit peel, primer and application |
| CN114107535A (en) * | 2020-08-31 | 2022-03-01 | 北京市农林科学院 | Molecular marker for identifying character of removing wax powder of grafted cucumber fruit from Chinese pumpkin rootstock and application of molecular marker |
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| EP2949204A1 (en) * | 2013-06-14 | 2015-12-02 | Keygene N.V. | Directed strategies for improving phenotypic traits |
| CN105907869A (en) * | 2016-06-01 | 2016-08-31 | 青岛科技大学 | SSR primer and method for rootstock pumpkin "No. 2 Huang Chenggen" hybrid seed purity identification |
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2016
- 2016-09-09 CN CN201610837480.6A patent/CN107805672B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2949204A1 (en) * | 2013-06-14 | 2015-12-02 | Keygene N.V. | Directed strategies for improving phenotypic traits |
| CN105907869A (en) * | 2016-06-01 | 2016-08-31 | 青岛科技大学 | SSR primer and method for rootstock pumpkin "No. 2 Huang Chenggen" hybrid seed purity identification |
Non-Patent Citations (5)
| Title |
|---|
| GONG, L. ET AL.: "Microsatellites for the genus Cucurbita and an SSR-based genetic linkage map of Cucurbita pepo L", 《THEOR. APPL. GENET.》 * |
| L GONG ET AL.: "SSR-based genetic linkage map of Cucurbita moschata and its synteny with Cucurbita pepo", 《GENOME》 * |
| 宋慧 等: "甬砧系列葫芦类型砧木品种分子指纹图谱构建", 《江苏农业科学》 * |
| 王迎儿 等: "南瓜耐盐种质的筛选鉴定及耐盐基因的标记", 《浙江农业学报》 * |
| 陈蛟龙: "砧用南瓜品种多样性分析及新组合选育", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110607391A (en) * | 2019-11-05 | 2019-12-24 | 东北农业大学 | Molecular marker closely linked with character control of Indian pumpkin red fruit peel, primer and application |
| CN114107535A (en) * | 2020-08-31 | 2022-03-01 | 北京市农林科学院 | Molecular marker for identifying character of removing wax powder of grafted cucumber fruit from Chinese pumpkin rootstock and application of molecular marker |
| CN114107535B (en) * | 2020-08-31 | 2023-08-18 | 北京市农林科学院 | Molecular marker for identifying properties of removing wax powder of grafted cucumber fruits of pumpkin stock and application of molecular marker |
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