CN106047996A - Method for identifying varieties of loose leaf lettuce - Google Patents

Method for identifying varieties of loose leaf lettuce Download PDF

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Publication number
CN106047996A
CN106047996A CN201610350280.8A CN201610350280A CN106047996A CN 106047996 A CN106047996 A CN 106047996A CN 201610350280 A CN201610350280 A CN 201610350280A CN 106047996 A CN106047996 A CN 106047996A
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Prior art keywords
leaf
ssr
leaf lettuce
lettuce
fingerprint
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范双喜
罗江
韩莹琰
张鹏航
高琦
谷建田
刘超杰
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Beijing University of Agriculture
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Beijing University of Agriculture
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a method for identifying varieties of loose leaf lettuce. The loose leaf lettuce is subjected to variety identification by using an SSR (Simple Sequence Repeat) molecular marker method. In the polymorphism study of the loose leaf lettuce, special primers of the SSR molecular marker method include SML026, SML022, SML021, SML013, SML007, SML002, SML001, SH86, SH66, SH49, SH43, SH42, KSL271, KSL173, KSL123, KSL115, KSL97, KSL92, KSL87, KSL51, KSL32 and KSL26. The special primers are used for obtaining the SSR fingerprint maps of the loose leaf lettuce and fingerprints of the varieties to be identified. The fingerprints of the varieties to be identified are compared with the SSR fingerprint maps to obtain an identification result. The varieties of the loose leaf lettuce can be conveniently and accurately identified.

Description

Identify the method dissipating leaf Leaf lettuce kind
Technical field
The present invention relates to a kind of qualification and dissipate leaf Leaf lettuce kind technology, particularly relate to a kind of qualification and dissipate leaf Leaf lettuce product The method planted.
Background technology
At present, country of China Germplasm Resources of Vegetables storehouse is collected and scattered leaf Leaf lettuce germ plasm resource more than 200 part of preservation, but mesh The cultivar used in front production is most quoted from abroad.Dissipating leaf Leaf lettuce is that a kind of low input, high yield, nutritive value are rich Rich Fast-growing vegetables, cultivated area expands rapidly in recent years, is increasingly becoming one of first-selected green vegetable that people eat.But, China dissipates the cultivar of leaf Leaf lettuce and has kind mainly by Introduced From Abroad, has kind and the type of independent intellectual property right The most limited, and almost blank in variety source collection, qualification.Accordingly, it would be desirable to multi-angle, multi-method carry out germ plasm resource Innovation work, develop colory scattered leaf Leaf lettuce new varieties, meet the needs of current production.Germ plasm resource is Vegetable Genetics Breeding and the stock of breed improvement, the qualification of germ plasm resource and screening are cultivating and improveing excellent kind Item element task.Owing to frequently introducing a fine variety between different regions so that variety name is the most chaotic, synonym and homonym Phenomenon is the most universal.So, scattered leaf Leaf lettuce kind is necessary to utilize SSR fingerprint pattern technology to identify.
Present stage, for the germplasm identification of vegetable, the DNA marker technology of the most multiplex PCR-based technology, can be comprehensive For three major types: one is the labelling technique based on molecule hybridizes, mainly RFLP (Restriction Fragment Length Polymorphism) and VNTR (Variable number tandem repeat)；Two is the mark based on PCR Note technology, such as RAPD (Random Amplified Polymorphic DNA), SCAR (Sequence Characterized Amplified Regions)、STS(Sequence Tagged Site)、SSR(Simple Sequence Repeat)、ISSR (Inter-Simple Sequence Repeat), DAF (DNA Amplified Fingerprinting) and ERPAR (Ex- Tended Random Primer Amplified Regions) etc.；Three are enzyme action and labelling technique that PCR combines, mainly There is AFLP (Amplified ragment Length Polymorphism) and CAPS (Cleaved Amplified Polymorphic Sequence).Additionally, DNA based on the Single nuclear polymorphism mark that also a class newly-developed gets up Note, i.e. SNP (Simple Nucleotide Polymorphism).Along with retrotransponsons is studied, one based on retrotransposition The plant molecular marker technology RTN technology of son has obtained the biggest development, mainly has SSAP (Sequence-l Variation Polymorphism)、RIAP(Inverse Retrotransposon Amplified Polymorphism)、RBIP (Retrotransposon based Insertion Polymorphism) etc. 5 kinds.
Summary of the invention
It is an object of the invention to provide a kind of method that easy to use, result identifies scattered leaf Leaf lettuce kind accurately.
It is an object of the invention to be achieved through the following technical solutions:
The present invention identifies the method dissipating leaf Leaf lettuce kind, uses SSR molecular marker method to enter scattered leaf Leaf lettuce Row cultivar identification, described SSR molecular marker method is in scattered leaf Leaf lettuce polymorphism research, and its primer special is as follows: (1) SML026
(2)SML022
(3)SML021
(4)SML013
(5)SML007
(6)SML002
(7)SML001
(8)SH86
(9)SH66
(10)SH49
(11)SH43
(12)SH42
(13)KSL271
(14)KSL173
(15)KSL123
(16)KSL115
(17)KSL97
(18)KSL92
(19)KSL87
(20)KSL51
(21)KSL32
(22)KSL26
Obtain with described primer special and dissipate leaf Leaf lettuce SSR finger printing, and obtain to be identified with described primer special The fingerprint of kind, contrasts the fingerprint of kind to be measured with described SSR finger printing, it is thus achieved that qualification result.
As seen from the above technical solution provided by the invention, the qualification that the embodiment of the present invention provides dissipates leaf Leaf lettuce The method of kind, can identify convenient, accurately and dissipate leaf Leaf lettuce kind.
Detailed description of the invention
The embodiment of the present invention will be described in further detail below.
The present invention identifies the method dissipating leaf Leaf lettuce kind, and its preferably detailed description of the invention is:
Using SSR molecular marker method that scattered leaf Leaf lettuce is carried out cultivar identification, described SSR molecular marker method is at scattered leaf In Leaf lettuce polymorphism research, its primer special is as follows: (1) SML026
(2)SML022
(3)SML021
(4)SML013
(5)SML007
(6)SML002
(7)SML001
(8)SH86
(9)SH66
(10)SH49
(11)SH43
(12)SH42
(13)KSL271
(14)KSL173
(15)KSL123
(16)KSL115
(17)KSL97
(18)KSL92
(19)KSL87
(20)KSL51
(21)KSL32
(22)KSL26
Obtain with described primer special and dissipate leaf Leaf lettuce SSR finger printing, and obtain to be identified with described primer special The fingerprint of kind, contrasts the fingerprint of kind to be measured with described SSR finger printing, it is thus achieved that qualification result.
Obtain by the following method and dissipate leaf Leaf lettuce SSR finger printing, comprise the following steps:
A, with the genomic DNA of Leaf lettuce as template, carry out PCR amplification with described primer special；
B, the amplified production obtained in step A is carried out capillary electrophoresis, respectively the length of SSR fragment in record kind；
C, peak value figure reading according to electrophoresis, have peak to be designated as 1, be designated as 0 without peak, according to (0,1) data matrix, builds SSR Finger printing.
When being applied to cultivar identification, the DNA of kind to be identified is expanded through PCR primer with described primer special, by product Corresponding band is filled in into the fingerprint drawing kind to be measured in corresponding (0,1) labelling table, then by the fingerprint of kind to be measured with Above-mentioned SSR finger printing contrasts, and directly obtains qualification result.
Specific embodiment:
Example 1, the SSR molecular marker method foundation of using dissipate leaf Leaf lettuce finger printing.
Operating process is: prepare template DNA → DNA cloning → capillary electrophoresis → statistic analysis result.Concrete grammar and Process is as follows:
1, the Leaf lettuce genomic DNA that the CTAB method of use improvement is extracted:
The genomic DNA extracted measures OD through Eppendorf Biophotometer260/OD280Value is in 1.7-1.9 scope In.DNA extraction liquid is diluted to 100ng/ μ l, takes 3 μ l DNA sample and add 1 μ l loading buffer diluent, 1% Agarose gel on detect, voltage stabilizing 4V/cm, electrophoresis 45min.Electrophoresis result shows, DNA master tape is clear, without signs of degradation, RNA removes clean, can be as template.
2, the design of SSR primer and screening:
In SSR molecular marker is tested, different primers combination is different to the amplification efficiency of a certain specific gene group.Draw The polymorphic bands number that the height of thing amplification efficiency is produced by it determines.109 pairs of primer combinations are carried out by this example respectively Amplification, has therefrom filtered out 22 pairs of bands of a spectrum and has been evenly distributed, and band enriches, and polymorphism is higher and the preferable primer sets of bands of a spectrum quality Close, can be used for providing the research of sample Genetic Diversity of Germplasm.This ten pair of primers is as follows:
Primer is to 1:SML026；GGGTTCTCATTGGCTGACAT/TGTCTTCCAACCAAAACATACA(GAA)11
Primer is to 2:SML022；GGGCCTCAAATCCTCTCTG/TGTTCTTCCCCTCTTTGGAA(ATC)13
Primer is to 3:SML021；TTGGGAGAATTTTCATTTCCA/AGTCATCTTTTTCACCCCACA(TA)8
Primer is to 4:SML013；TCCCATGATGGAGAGACTCA/CCCAAAAGGGAATAGCAACC(GAA)14(CTG)5
Primer is to 5:SML007；ACACTTGCCGATTCCTTCAC/ACCCGTGTTGAAAATGGAGA(TCACCA)19
Primer is to 6:SML002；GTGATTGCATGCCAAATGAA/TTAGTAGCCCGCATGCTTTT(TTC)17
Primer is to 7:SML001；CCATGGATCCTGTGTGAAGA/CACCATGTTCCACTTCCACTT(CATGAT)6
Primer is to 8:SH86；GTCTGTGTGGTTTTGGT/TGTGGTGGAGTGTGATTT(CT)20
Primer is to 9:SH66；GGTAGGGCAGTCAAGCAAGA/AATGATGATTTTGCCCTTGG(TC)28
Primer is to 10:SH49；GGAGATTGAGAGGAAAA/GAGTGGAAGGGAAATAG(AG)25
Primer is to 11:SH43；CTCTCGTTCCTTTTGTTGGTTT/TGGTAGTGGCTTCCTTGCTATT(AC)10
Primer is to 12:SH42；GCAAGCTAAAGGGCTTTTTGT/CAGCCTGGGAATATTTACTCTGA(ATT)27
Primer is to 13:KSL271；ACAAAGGCAAGATTGGGTCA/GCGGATATGCAGCCATAACA(ATG)12
Primer is to 14:KSL173；ATAGTCACGACTCACGCCCA/CCATTTTCCTCTTTCTGCGA(CT)14
Primer is to 15:KSL123；ATTGTAACTTCTGCGGGCCT/GCCTCACATGTTCTTCCCCT(ATC)13
Primer is to 16:KSL115；CATTGCACTCCGTCATCTCC/GGGTTGATTCCGAAAGTTCC(CT)11
Primer is to 17:KSL97；CGCAGAAAAGGGATCAGACA/TCAGAGACACTGCAAAAGGGA(CT)11
Primer is to 18:KSL92；GGTCTCTTTCCTCTGCCCTG/TCGCGTTCTGAAGTAGCCAT(CT)20
Primer is to 19:KSL87；GCGGGATCGATACTTACCCT/ATCATCGACGGGCTTTTCTT(CT)17
Primer is to 20:KSL51；CCCCTACCACCACCAAAGTC/TACCAAATGACATGCACCCC(ATG)10
Primer is to 21:KSL32；CGGGGAGCATTTAGTGTGTG/AATTTGGGGTCCGATTTGAG(CT)14
Primer is to 22:KSL26；GGGCTTTCTCTCCTTTCCTTT/AATTTGGATCCTGTCGAGGG(TC)16
3, the amplification of DNA, such as table 1 below:
94 DEG C of denaturations 5min；94 DEG C of degeneration 30sec, 58 DEG C of annealing 30sec, 72 DEG C extend 45sec, circulate 38 times；? Latter 72 DEG C extend 10min.4 DEG C of heat preservation for standby use.
Table 2.SSR expands statistical result:
4. the finger printing of the scattered leaf Leaf lettuce of acquisition:
According to the power of amplified band, and definition determines the band of hereditary variation, eliminates illusion band.Electrophoresis pattern enters Row compares, and determines singlet band and polymorphic band.Choose electrophoretic band clear and legible on electrophoresis flat board, with " 1 " and " 0 " record respectively The presence or absence of band, has band assignment " 1 " in same migration distance, without band assignment " 0 ".
Example 2, the SSR fingerprint map analyzing dissipating leaf Leaf lettuce and application:
According to the amplification of 22 pairs of primers, draw the finger printing drawing scattered leaf Leaf lettuce by (0,1) method, such as table 3:
Table 3: dissipate the finger printing (part) of leaf Leaf lettuce
When being applied to cultivar identification, by the DNA of kind to be identified, with 22, primer is expanded through PCR primer, product is corresponding Band fill in into the fingerprint drawing kind to be measured in corresponding (0,1) labelling table, then by the fingerprint of kind to be measured with above-mentioned Finger printing (table 3) contrast, qualification result can be directly obtained.
As through above-mentioned flow process, a certain kind show that fingerprint is:
000101000100010000100000010010010000000000001000000010000000001001100 1000000100000100010001000000000100001000010000000000101000001000000000
The fingerprint contrast this fingerprint and table 3 provided, then understanding this kind is GS-61.
The above, the only present invention preferably detailed description of the invention, but protection scope of the present invention is not limited thereto, Any those familiar with the art in the technical scope of present disclosure, the change that can readily occur in or replacement, All should contain within protection scope of the present invention.Therefore, protection scope of the present invention should be with the protection model of claims Enclose and be as the criterion.

Claims (3)

1. identify the method dissipating leaf Leaf lettuce kind for one kind, it is characterised in that use SSR molecular marker method to scattered leaf leaf lettuce Lettuce carries out cultivar identification, and described SSR molecular marker method is in scattered leaf Leaf lettuce polymorphism research, and its primer special is as follows:
(1)SML026
(2)SML022
(3)SML021
(4)SML013
(5)SML007
(6)SML002
(7)SML001
(8)SH86
(9)SH66
(10)SH49
(11)SH43
(12)SH42
(13)KSL271
(14)KSL173
(15)KSL123
(16)KSL115
(17)KSL97
(18)KSL92
(19)KSL87
(20)KSL51
(21)KSL32
(22)KSL26
Obtain with described primer special and dissipate leaf Leaf lettuce SSR finger printing, and obtain kind to be identified with described primer special Fingerprint, the fingerprint of kind to be measured is contrasted with described SSR finger printing, it is thus achieved that qualification result.
Qualification the most according to claim 1 dissipates the method for leaf Leaf lettuce kind, it is characterised in that obtain by the following method Take scattered leaf Leaf lettuce SSR finger printing, comprise the following steps:
A, with the genomic DNA of Leaf lettuce as template, carry out PCR amplification with described primer special；
B, the amplified production obtained in step A is carried out capillary electrophoresis, respectively the length of SSR fragment in record kind；
C, peak value figure reading according to electrophoresis, have peak to be designated as 1, be designated as 0 without peak, according to (0,1) data matrix, builds SSR fingerprint Collection of illustrative plates.
Qualification the most according to claim 2 dissipates the method for leaf Leaf lettuce kind, it is characterised in that be applied to cultivar identification Time, the DNA of kind to be identified is expanded through PCR primer with described primer special, band corresponding for product is filled in into corresponding Drawing the fingerprint of kind to be measured in (0,1) labelling table, it is right then the fingerprint of kind to be measured and above-mentioned SSR finger printing to be carried out Ratio, directly obtains qualification result.
CN201610350280.8A 2016-05-24 2016-05-24 Method for identifying varieties of loose leaf lettuce Pending CN106047996A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103194537A (en) * 2013-03-13 2013-07-10 山东省农业科学院作物研究所 Cabbage SSR fingerprint construction method
CN103243158A (en) * 2013-04-08 2013-08-14 山东省农业科学院作物研究所 Method for constructing wheat SSR (single sequence repeat) fingerprint

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103194537A (en) * 2013-03-13 2013-07-10 山东省农业科学院作物研究所 Cabbage SSR fingerprint construction method
CN103243158A (en) * 2013-04-08 2013-08-14 山东省农业科学院作物研究所 Method for constructing wheat SSR (single sequence repeat) fingerprint

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GILDA RAUSCHER 等: ""Development of genomic SSR markers for fingerprinting lettuce ( Lactuca sativa L.) cultivars and mapping genes"", 《BMC PLANT BIOLOGY》 *
JEE-HWA HONG 等: ""Construction of EST-SSR Databases for Effective Cultivar Identification and Their Applicability to Complement for Lettuce (Lactuca sativa L.) Distinctness Test"", 《AMERICAN JOURNAL OF PLANT SCIENCES》 *

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Application publication date: 20161026