CN107130029A - A kind of fig germ plasm resource relationship authentication method based on SSR marker - Google Patents

A kind of fig germ plasm resource relationship authentication method based on SSR marker Download PDF

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Publication number
CN107130029A
CN107130029A CN201710348530.9A CN201710348530A CN107130029A CN 107130029 A CN107130029 A CN 107130029A CN 201710348530 A CN201710348530 A CN 201710348530A CN 107130029 A CN107130029 A CN 107130029A
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China
Prior art keywords
ssr
germ plasm
plasm resource
resource relationship
authentication method
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CN201710348530.9A
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Chinese (zh)
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郭正兵
韩柏明
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Jiangsu Polytechnic College of Agriculture and Forestry
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Jiangsu Polytechnic College of Agriculture and Forestry
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Priority to CN201710348530.9A priority Critical patent/CN107130029A/en
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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
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • 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

Abstract

The invention discloses a kind of fig germ plasm resource relationship authentication method based on SSR marker.The invention also discloses the primer pair for the SSR marker identified for fig germ plasm resource relationship.The product detection collection of illustrative plates of the SSR reaction systems of the present invention is clear, illustrates that stable system is reliable.The present invention 15 SSR markers show polymorphism in 30 fig variety sources, repeat, explanation is reliable and stable mark, can for carry out fig variety source genetic diversity and Genetic relationship.

Description

A kind of fig germ plasm resource relationship authentication method based on SSR marker
Technical field
The invention belongs to molecular biology DNA marker technology and application field, more particularly to a kind of nothing based on SSR marker Flowers and fruits germ plasm resource relationship authentication method.
Background technology
Fig (scientific name：Ficus carica L.), also known as reflect day fruit, diverseleaf fig fruit, mamoncillo, lacerateleaf fig-tree root and fruit, Fructus Fici Beecheyanae, bright The holy fruit of mesh fruit, bodhi, is a kind of flowering plant, is under the jurisdiction of Moraceae Ficus, is mainly grown on the place in some torrid zones and temperate zone, category Subtropical zone defoliation small arbor.Fig has stronger salt tolerance, barren-resistant, and well developed root system, management are simple, and its fruit is nutritious, It is often edible to strengthen resistance against diseases with the effect for improving immunologic function.Fig is rapid in China's cultivated area in recent years Expand.
China fig cultivar is except the early Huang in Xinjiang that early stage introduces, the late Huang in Xinjiang, rascal, Blanc Rake and purple fruit Outside etc. kind, it has been introduced sequentially into from the U.S., Deng Guodeng states of Japan that some fruits are big, quality is excellent, yield is high since the generation in 20th century 80 Fig new varieties, be greatly enriched the variety source of China's fig.And carried out breeding research, 10 have been cultivated successively Several new varieties.But current China also extremely lacks to the research in terms of fig Genetic Diversity of Germplasm and genealogical relationship It is weary.This Sustainable use to fig resource is all very unfavorable.In order to which the current fig resource of more preferable many China is entered Row, which is evaluated, to be utilized, and it is necessary element task that genetic diversity and Genetic relationship are carried out to it.
Molecular labeling be study Genetic Diversity powerful, with accuracy it is high, reproducible, not by environment The advantages of condition is disturbed, SSR be it is a kind of apply quite varied molecular labeling, be widely used in plant Genetic relationship, In terms of analysis of genetic diversity, linkage map foundation, Molecular Identification, with simple to operate, the features such as accuracy is high.
The content of the invention
Goal of the invention：The technical problems to be solved by the invention there is provided a kind of fig germplasm based on SSR marker Resource relationship authentication method.The invention provides the stabilising system of the fig resource analysis based on SSR marker, polymorphism is high Primer.
Technical scheme：The invention provides a kind of fig germ plasm resource relationship authentication method based on SSR marker, it is wrapped Include following steps：
1) genomic DNA of the fig of different cultivars is extracted；
2) screening of SSR primers；
3) performing PCR is entered using the genomic DNA of the fig of SSR primer pair different cultivars；
4) Capillary Electrophoresis is carried out to PCR primer；
5) polymorphism analysis is carried out to electrophoresis result and draws genetic diversity, show that fig germplasm is provided by clustering Source affiliation.
Wherein, above-mentioned steps 2) in screening obtain 15 pairs of SSR primer pairs, the primer pair sequence such as SEQ ID NO：1~ 30。
Wherein, above-mentioned steps 3) PCR reaction systems be：1 × PCR cushioning liquid, 2mmol/LdNTPs, 0.4mmol/L Primer, DNA profiling 40ng, 1U Taq.
Wherein, above-mentioned steps 3) PCR reaction conditions be 94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 54 DEG C of annealing renaturation 35s, 72 DEG C of extension 40s, totally 35 circulations；Final 72 DEG C of extensions 3min.
Wherein, above-mentioned steps 4) Capillary Electrophoresis in formamide and molecular weight internal standard press 100:After 1 volume ratio is mixed, Take in 9 μ L additions in model, add the PCR primer that 1 μ L dilute 10 times.
Present invention also includes being used for the primer pair for the SSR marker that fig germ plasm resource relationship is identified, the primer To sequence such as SEQ ID NO：1~30.
Beneficial effect：Compared with prior art, the present invention possesses advantages below：The product of the SSR reaction systems of the present invention Detect that collection of illustrative plates is clear (visible Fig. 2 and Fig. 3), this collection of illustrative plates reflects the reliability and stability of this detection technique very accurately.This 15 SSR markers of invention show polymorphism in 30 fig variety sources, repeat (table 4), explanation is reliable and stable Mark, can for carry out fig variety source genetic diversity and Genetic relationship.SSR marker be using PCR as The molecular marking technique on basis, the sizes of amplified fragments often difference very little, using agarose and polyacrylamide as the inspection of representative Survey technology fails effectively to separate, and directly determines clip size using Capillary Electrophoresis graphical spectrum technology, is very accurate and has The detection technique of effect.
Brief description of the drawings
Fig. 1 is the dendrogram of fig kind；
Show that amplification obtains two equipotential positions in the Capillary Electrophoresis collection of illustrative plates of Fig. 2 primers Fs CUP069-6 amplified production, figure Point；
Show that amplification obtains two etc. in the Capillary Electrophoresis collection of illustrative plates of Fig. 3 primers Fs CUP044-6-22 amplified production, figure Position site.
Because the electrophoretogram of the present invention has 15*30=450, because electrophoretogram is more or less the same, therefore it have selected with generation The electrophoretogram of table.
Embodiment
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real Apply specific material proportion, process conditions and its result described by example and be merely to illustrate the present invention, without that will not also should limit The present invention described in detail in claims processed.
Embodiment 1
1st, test material：
This experiment collects 30 parts of fig variety sources referring to table 1 altogether
Fig kind of the table 1 for examination
2nd, DNA is extracted
Spring in 2015 takes (30 experimental cultivar fig young leaflet tablets are as material), using Tiangeng DNA extracts reagents Box, which extracts STb gene, is used for SSR Fluorometric assay of fluorescence-labeled.
3rd, SSR primer screenings
49 pairs are collected from existing SSR primers related to fig, according to primer polymorphism, 15 pairs of screening fig primer For this research, forward primer is marked with fluorescent marker FAM.15 pairs of primers are referring to table 2；
Explanation：49 pairs of primers are selected from the related SSR primers of the fig delivered.49 couple of selection is drawn Thing is according to the polymorphism of its pcr amplification product, and further screening polymorphism is high, and 15 pairs high of primer of repetitive rate is used as this experiment Primer.
The primer of table 2 and its sequence
4th, performing PCR is entered using the genomic DNA of the fig of 30 kinds of SSR primer pairs
The foundation and optimization of 4.1 reaction systems
SSR fluorescent primer PCR reaction systems (totally 25 μ l)：1 × PCR cushioning liquid, 2mmolL-1DNTPs, 0.4mmol·L-1Primer, DNA profiling 40ng, 1U Taq.94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30S, 54 DEG C (annealing temperature exists About 54 DEG C fluctuations, are specifically shown in Table 3) renaturation 35s, 72 DEG C of extension 40s, totally 35 circulations；Final 72 DEG C of extensions 3min.
Explanation：The PCR reaction volumes of each sample are 25 μ l, are used as the μ l of DNA sample volume 1 of amplification template.
The primer annealing temperature of table 3
Primer Annealing temperature
FCUP038-6 59℃
FCUP044-6 55℃
FCUP045-6 57℃
FCUP066-7 56℃
FCUP069-6 55℃
LMFC17 54℃
LMFC19 54℃
LMFC27 56℃
LMFC28 56℃
LMFC30 56℃
LMFC37 48℃
LMFC38 48℃
MFC2 58℃
MFC7 48℃
MFC8 57℃
5th, capillary electrophoresis method
Formamide and molecular weight internal standard are pressed 100:After 1 volume ratio is mixed, take in 9 μ L additions in model, add 1 μ L The PCR primer of 10 times of dilution.Then carry out Capillary Electrophoresis using 3730XL sequenators (electrophoretogram result is referring to Fig. 2~3).
6th, data processing and data analysis
6.1 original numbers obtained using Fragment (Plant) fragment analysis softwares in Genemarker to sequenator According to being analyzed, the position of target position in molecular weight in each swimming lane and each sample peak value is compared into analysis, fragment is obtained big It is small.Form according to the software requirements of Convert 1.31 is entered into EXCEL, is then changed into the softwares of Convert 1.31 Form required by POPGENE softwares.Statistical analysis is carried out using POPGENE1.32 softwares.Calculate number of alleles (A), observation Heterozygosity (Ho), expectation heterozygosity (He) and Shannon information indexes (I).Clustering is carried out using UPGMA methods.
The polymorphism analysis of 6.2SSR marks
15 pairs of bands of a spectrum are clear, polymorphism is high primers from 49 pairs of fig SSR primers.Utilize the 15 pairs of figs selected 30 fig kind materials of primer pair are expanded, and amplified production passes through capillary electrophoresis detection.79 equipotentials are detected altogether Variation, each pair primer detection goes out 2-10 allelic variation, and average 5.27, what each primer was expanded is polymorphism equipotential base Cause.The fragment length of 15 site amplifications is between 133~326bp；Each site PIC value 0.3328-0.8757, average out to 0.5303；Observe heterozygosity (Ho) 0.1667~0.8667, average out to 0.4768；Expect heterozygosity (He) 0.3328~ 0.8757, average out to 0.5929；The Shannon information indexes (I) in each site, 0.6049~2.1273, average out to 1.1360.Knot Fruit shows that the fig resource that this experiment is collected has more rich genetic diversity.
4 15 pairs of SSR primer extension products analyses of table
6.3 clustering
By 30 parts of fig variety sources of clustering at genetic similarity 0.67, two monoids can be divided into, Monoid I and monoid II, in monoid I, only 3 fig kinds are that magnificent, Israel and Xinjiang are early yellow respectively；Other 27 Individual fig kind is gathered for monoid II.In monoid II, tri- subclass of A, B, C can be divided into again at genetic similarity 0.76, Wherein A subclass includes 19 fig kinds such as Blanc Rake, white Genoa, banana, purple Bordeaux, wherein Blanc Rake and Got together in white Genoa；The kind quilt such as banana, purple Bordeaux, black California, Ma Siyitaofen, Adam, Luo Yier, Du Lu Get together, wherein banana and purple Bordeaux, California is black and unknown, Roy you and Du Lu are got together, shown respectively More close affiliation；It is B1011, ripple Ji Hong, Baima match, D110, beautiful Asia, Jin Aofen, A1213, Huang of getting bumper crops, king, beautiful The kinds such as Sha are got together, wherein B1011 and ripple Ji Hong, D110 and beauty Asia, king and Li Sha, are got together respectively, Show more close affiliation；There was only one kind of Hardy in B subclass；In C subclass, including Chinese purple fruit, rascal, The kinds such as purple treasured, Grice, Japanese purple fruit, whitish honey, purple great waves sweet smell, wherein rascal and purple is precious, Grice and Japan's purple fruit, respectively by Get together, show more close affiliation (referring to Fig. 1).

Claims (6)

1. a kind of fig germ plasm resource relationship authentication method based on SSR marker, it is characterised in that comprise the following steps：
Extract the genomic DNA of the fig of different cultivars；
The screening of SSR primers；
Genomic DNA using the fig of SSR primer pair different cultivars enters performing PCR；
Capillary Electrophoresis is carried out to PCR primer；
Polymorphism analysis is carried out to electrophoresis result and draws genetic diversity, fig germ plasm resource relationship is drawn by clustering Relation.
2. a kind of fig germ plasm resource relationship authentication method based on SSR marker according to claim 1, its feature exists In the step 2）Middle screening obtains 15 pairs of SSR primer pairs, the primer pair sequence such as SEQ ID NO：1~30.
3. a kind of fig germ plasm resource relationship authentication method based on SSR marker according to claim 1, its feature exists In the step 3）PCR reaction systems be：1 × PCR cushioning liquid, 2mmol/LdNTPs, 0.4 mmol/L primers, DNA Template 40ng, 1U Taq.
4. a kind of fig germ plasm resource relationship authentication method based on SSR marker according to claim 1, its feature exists In the step 3）PCR reaction conditions be 94 DEG C of min of pre-degeneration 5；94 DEG C of denaturation 30s, 54 DEG C of annealing renaturation 35 s, 72 DEG C Extend 40s, totally 35 circulations；Final 72 DEG C of extensions 3min.
5. a kind of fig germ plasm resource relationship authentication method based on SSR marker according to claim 1, its feature exists In the step 4）Capillary Electrophoresis in formamide and molecular weight internal standard press 100:After 1 volume ratio is mixed, 9 μ L are taken to add In upper model, the PCR products that 1 μ L dilute 10 times are added.
6. the primer pair for the SSR marker identified for fig germ plasm resource relationship, it is characterised in that the primer pair sequence is such as SEQ ID NO：1~30.
CN201710348530.9A 2017-05-17 2017-05-17 A kind of fig germ plasm resource relationship authentication method based on SSR marker Pending CN107130029A (en)

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CN108220402A (en) * 2017-12-25 2018-06-29 山东省农业科学院蔬菜花卉研究所 A kind of identification method of Chinese cabbage germplasm and kind genealogical relationship
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CN112481406A (en) * 2020-12-04 2021-03-12 新疆农业科学院园艺作物研究所 SSR marker-based genetic identification method for germplasm resources of Munage grapes

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108220402A (en) * 2017-12-25 2018-06-29 山东省农业科学院蔬菜花卉研究所 A kind of identification method of Chinese cabbage germplasm and kind genealogical relationship
CN108220402B (en) * 2017-12-25 2020-07-07 山东省农业科学院蔬菜花卉研究所 Method for identifying pedigree relationship between Chinese cabbage germplasm and variety
CN110699480A (en) * 2019-11-18 2020-01-17 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） Primer group for hybridizing orchidaceae EST-SSR (expressed sequence tag-simple sequence repeat) markers and screening method
CN110699480B (en) * 2019-11-18 2023-10-24 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） Primer group for hybridization of EST-SSR (expressed sequence tag-simple sequence repeat) markers of cymbidium kanran and screening method
CN112481406A (en) * 2020-12-04 2021-03-12 新疆农业科学院园艺作物研究所 SSR marker-based genetic identification method for germplasm resources of Munage grapes
CN112481406B (en) * 2020-12-04 2024-03-26 新疆农业科学院园艺作物研究所 SSR (simple sequence repeat) marker-based method for genetic identification of germplasm resources of Vitis vinifera

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Application publication date: 20170905