CN110317894A - With the SNP marker and its application of the total root weight close linkage of Radix Notoginseng - Google Patents
With the SNP marker and its application of the total root weight close linkage of Radix Notoginseng Download PDFInfo
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Abstract
The invention discloses a kind of SNP markers and its application with the total root weight close linkage of Radix Notoginseng.Substance the present invention provides SNP site following in Radix Notoginseng genome or for detecting the single nucleotide polymorphism of following SNP site in Radix Notoginseng genome is being identified or is assisting to identify the application in total root principal characteristic shape of Radix Notoginseng to be measured；The SNP site are as follows: using Radix Notoginseng genomic DNA as template, the 199th nucleotide from 5 ' ends of amplified production obtained by PCR amplification is carried out using the primer pair being made of SEQ ID No.1 and SEQ ID No.2；Nucleotide at the SNP site is C or T.The present invention is by sequencing technologies, on the basis of studying different weight Radix Notoginseng germ plasm resource, is accurately positioned total root weight gene loci, and then develop the SNP marker closely coupled with it.And then the screening of high yield strain is realized in notoginseng planting early stage, improve notoginseng planting benefit.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of SNP marker with the total root weight close linkage of Radix Notoginseng
And its application.
Background technique
Radix Notoginseng Panax notoginseng (Burk.) F.H.Chen also known as pseudo-ginseng, Yunnan Radix Notoginseng, radix notoginseng, blood ginseng, field three
Seven etc., it is under the jurisdiction of the perennial Constantly allogamous herbaceous plant (2n=24) of Araliaceae (Araliaceae) Panax (Panax).
Radix Notoginseng distribution is only limitted to 23 ° of 30 ' neighbouring middle-high altitude area of north latitude.Due to excessively excavating, wild Radix Notoginseng scarcity of resources is existing
Radix Notoginseng is mostly artificial cultivation, and the ground such as domestic Radix Notoginseng main producing region Yunnan mountain of papers, Yanshan County, Maguan, cultivated area and yield are more than complete
The 98% of state.
Existing 600 years or more medicinal histories of Radix Notoginseng, it is compound danshen dripping pills, Yunnan that root, stem, leaf, Hua Junke, which are used as medicine,
One of the main component of the common Chinese medicines preparation such as baiyao, Pien Tze Huang, FUFANG SANQI KOUFUYE is at present that formula enters with Radix Notoginseng
The preparation of " country substantially medicinal catalogue " and " national Chinese medicine protection kind catalogue " is up to more than 20.The rare medicine traditional as China
Material, the research in terms of Radix Notoginseng biological characteristics are also very full and accurate.The pharmacological action of Radix Notoginseng is mainly reflected in hematological system, the heart
The influence of vascular system, cerebrovascular system, nervous system, metabolism, immunological regulation system etc..There are many correlations of Radix Notoginseng in recent years
Document report, in terms of content is concentrated mainly on separation and Extraction and the pharmacological effect of Radix Notoginseng chemical component.
Root is one of main medicinal part of Radix Notoginseng, containing there are many saponin components.The pharmacological action of Radix Notoginseng is with itself
Chemical component be it is inseparable, saponins compound be Radix Notoginseng main chemical compositions and Radix Notoginseng in generally acknowledge it is main
One of effective component is broadly divided into ginsenoside, notoginsenoside and seven leaf gallbladder saponin(es etc..Saponin(e amount is in Radix Notoginseng with ginsenoside
Rg1And Rb1Highest is also according to ginsenoside Rg in quality standard1、Rb1And Panax Notoginseng saponin R1Amount summation be no less than 5.0% work
For the standard for measuring Radix Notoginseng quality.Saponin(e type contained by Radix Notoginseng different parts is different with amount.It is then led in sanchi leaf and sanchi flower
Containing PDS, and PDS and PTS is mainly contained in the root of Radix Notoginseng, such as ginsenoside Rb1、Rb2、Rd、Re、Rg1、Rg2、Rh1With three
Seven saponin(e R1、R2、R3、R4、R6And seven leaf gallbladder saponins X VII etc..
The amount of saponin constituent is influenced by many factors in Radix Notoginseng.Horse girls etc. cut Radix Notoginseng by studying different drying means
The influence of piece saponin(e amount, discovery day illumination is shone and 50 DEG C of drying are more suitable for pseudo-ginseng root slices processing；It is high with the development of dry technology
Bright chrysanthemum etc. is dried Radix Notoginseng using micro-wave drying method, the results showed that the microwave radiation of varying strength causes main in Radix Notoginseng
The amount of saponin constituent reduces, and is below " Chinese Pharmacopoeia " version standard in 2010.It can be seen that drying means, which will affect in Radix Notoginseng, to be had
The accumulation of ingredient is imitated, traditional method for airing can keep the amount of Panax notoginseng root effective component well.
Currently, the breeding research of Radix Notoginseng still belongs to the starting stage.Cui Xiuming determines the viability of notogenseng pollen, discovery three
Seven pollen can save 11-13 days at normal temperature.Sun Yuqin etc. observes Radix Notoginseng flowering time and loose powder time.Chen Zhongjian etc. is to green
The Radix Notoginseng Traits changes such as stem, purple stem, green stem tuber and purple stem tuber are studied, it is believed that the character is all that gene controls.
There are the method for scholar's allozyme, RAPD, AFLP to assess the genetic background of Radix Notoginseng.Duan Chengli etc. passes through
RAPD method analyzes 7 character variation types totally 34 Radix Notoginseng samples, finds between the variation type of Radix Notoginseng and same type of
DNA polymorphism between Different Individual is higher, and respectively 75.5% and 75.2%, illustrate Radix Notoginseng from the point of view of genetic background or one
A heterozygosis population has genetic diversity abundant.Hong etc. has found the individual something lost in a plantation by AFLP label
The saponin content for passing composition and Panax notoginseng root has variation, and thinks that the genetic diversity of Radix Notoginseng influences Panax notoginseng root saponin(e and contains
Amount.Zhou etc. studies Radix Notoginseng and Panax stipuleanatus Tsai et Feng using ITS and SFLP, the results showed that the polymorphism for cultivating Radix Notoginseng is less than open country
Non-hibernating eggs Panax stipuleanatus Tsai et Feng.Wang etc. is by carrying out aflp analysis to 92 samples from four population, it is believed that cultivation of pseudo-ginseng kind
Only existing the genetic diversity of moderate, the genetic heterozygosity of four population is respectively as follows: 0.166,0.093,0.094 and 0.125, and
And genetic distance very little (distances < 0.03 Nei genetic) between population.Xiao Hui etc. analyzes Radix Notoginseng using isoenzyme mark
Inbred genetic diversity, it is indicated that its hereditary variation (81.01%) is primarily present between population, and differentiation is smaller in population.And Zhang Jin
Chongqing etc. is made a variation using phenotypic character of the EST-SSR label to 10 cultivation of pseudo-ginseng population in the regional 4 Radix Notoginseng producing regions of mountain of papers and is carried out
Research, it is believed that no matter in population or between population, mountain of papers Radix Notoginseng population all has genetic diversity abundant, and its heredity
Variation is primarily present in population between individual.
The exploitation of SNP site is based primarily upon DNA sequencing and sequence alignment is completed.Currently, no matter being also non-mould for mode
Formula biology, the strategy and technology of SNP exploitation mainly include two classes, that is, have reference sequences (reference-based) and without reference
Sequence (de novo).There is the SNP development strategy of reference sequences mainly to resurvey sequence (Re-sequencing), including high depth and
Two kinds of low depth.Currently, using more widely without reference sequences SNP development strategy including simplifying polymorphic sequence complexity
(complexity reduction of polymorphic sequences, CRoPS), limitation restriction endonuclease association site DNA are surveyed
Sequence (restriction-site-associated DNA sequencing, RAD-seq) and the Genotyping based on sequencing
(genotyping by sequencing, GBS) etc..In addition, carrying out transcript profile to different samples based on the considerations of development cost
Sequencing carries out SNP site to excavate being also effective technological means using common sequence database.
The developmental research of Radix Notoginseng SNP marker is started late, but increasingly by the attention of researchers.The benefits such as Chen Zhongjian
Go out multiple anti-root rot associated SNP positions with RAD-seq technology screening, and successfully selects first Radix Notoginseng disease-resistant varieties.Wei
Chen etc. completes the genome sequencing of Radix Notoginseng for the first time, while finding the candidate gene of a large amount of saponin(e biosynthesis.Root conduct
One of main medicinal part of Radix Notoginseng, total root directly affects the yield of Radix Notoginseng again, and yield is to restrict the master of Radix Notoginseng industry development
Want factor.According to country mark, the grade of Radix Notoginseng is with the differentiation of head number.The head number of Radix Notoginseng refers to Radix Notoginseng in every 500 grams (1 jin, a traditional unit of weight) Radix Notoginseng
(opposite) number.It is sold in the market in terminal, the price of Radix Notoginseng is different because of head number, and head number is fewer, and higher grade, and price is also got over
It is high.For popular, head number is fewer, then Radix Notoginseng head is bigger, and Growing years are longer, and nutriment is abundanter, and on the contrary then quality is slightly
It is weaker.Roots of Panax Notoginseng size (total root weight), although having important economic value, rare people is using Molecular tools to this character
It is parsed.Current technology can only substantially judge root size (root weight) according to plant type in the maturity period and be had after excavation
Body is clear, cannot angularly explain from genetic background and physiological mechanism.The complement mark of Radix Notoginseng genome sequencing open
The Breeding Efficiency that hair high-precision SNP marker is possibly realized, while will improve Radix Notoginseng, accelerates the process of Radix Notoginseng molecular breeding.
Summary of the invention
The object of the present invention is to provide a kind of SNP markers and its application with the total root weight close linkage of Radix Notoginseng.
In a first aspect, following SNP site or for detecting in Radix Notoginseng genome in claimed Radix Notoginseng genome
Answering in the total root principal characteristic shape for identifying Radix Notoginseng to be measured is being identified or assisted to the substance of the single nucleotide polymorphism of following SNP site
With:
The SNP site are as follows: using Radix Notoginseng genomic DNA as template, using the single stranded DNA as shown in SEQ ID No.1 and
Single stranded DNA shown in SEQ ID No.2 composition primer pair carry out PCR amplification obtained by amplified production the 199th from 5 ' ends
Position nucleotide；Nucleotide at the SNP site is C or T.
Further, described " the single stranded DNA group shown in the single stranded DNA as shown in SEQ ID No.1 and SEQ ID No.2
At primer pair carry out PCR amplification obtained by amplified production " be SEQ ID No.3 shown in nucleotide sequence (full length sequence 1001bp)
303-876 from 5 ' ends, the SNP site is the 501st of sequence shown in SEQ ID No.3, is indicated with Y, and Y is represented
C or T.
Second aspect, claimed reagent for identifying or assisting to identify total root principal characteristic shape of Radix Notoginseng to be measured or
Kit.
It is provided by the present invention to be used to identify or assist to identify that the reagent of total root principal characteristic shape of Radix Notoginseng to be measured is for detecting
The substance of the single nucleotide polymorphism of following SNP site in Radix Notoginseng genome；The kit contains the reagent.The SNP
Site is same as above.
It is described " for detecting the mononucleotide of following SNP site in Radix Notoginseng genome in first aspect and second aspect
The substance of polymorphism " can for following (a) or (b) or (c) shown in primer pair:
(a) primer pair that single stranded DNA shown in the single stranded DNA shown in SEQ ID No.1 and SEQ ID No.2 forms；
(b) by by SEQ ID No.1 and SEQ ID No.2 by one or several nucleotide substitution and/or missing and/
Or two single strand dnas composition shown in gained sequence after addition, and primer identical with primer pair function described in (a)
It is right.
(c) as designing resulting primer pair through nucleotide sequence shown in SEQ ID No.3, and with primer pair described in (a)
The identical primer pair of function.
The third aspect, the claimed molecule mark for being used to identify or assist to identify total root principal characteristic shape of Radix Notoginseng to be measured
Note.
It is provided by the present invention to be used to identify or assist to identify that the molecular labeling of total root principal characteristic shape of Radix Notoginseng to be measured is with three
Seven genomic DNAs are template, expand resulting amplified production using primer pair A.
Wherein, the primer pair A meets following condition: carrying out PCR amplification institute by template of the genomic DNA of Radix Notoginseng to be measured
It obtains amplified production and contains the nucleotide at following SNP site.The SNP site is same as above.
Further, the primer pair A can for following (a) or (b) or (c) shown in primer pair:
(a) primer pair that single stranded DNA shown in the single stranded DNA shown in SEQ ID No.1 and SEQ ID No.2 forms；
(b) by by SEQ ID No.1 and SEQ ID No.2 by one or several nucleotide substitution and/or missing and/
Or two single strand dnas composition shown in gained sequence after addition, and primer identical with primer pair function described in (a)
It is right.
(c) as designing resulting primer pair through nucleotide sequence shown in SEQ ID No.3, and with primer pair described in (a)
The identical primer pair of function.
Fourth aspect, the claimed reagent or the kit or the molecular labeling are being identified or are being assisted
Identify the application in total root principal characteristic shape of Radix Notoginseng to be measured.
5th aspect, a kind of claimed method identified or assist identifying total root principal characteristic shape of Radix Notoginseng to be measured.
The method that identification provided by the present invention or auxiliary identify total root principal characteristic shape of Radix Notoginseng to be measured, specifically may include as follows
Step: detecting the nucleotide in the genome of Radix Notoginseng to be measured at following SNP site, with the genotype of the determination Radix Notoginseng to be measured,
Total root principal characteristic shape of Radix Notoginseng to be measured: to be measured the three of TT genotype is determined according to following rule according to the genotype of the Radix Notoginseng to be measured
Seven total root it is great in or the candidate Radix Notoginseng to be measured for being greater than CC genotype, total root dense medium of the Radix Notoginseng to be measured of CT genotype is in the two
Between.The SNP site is same as above.
The TT genotype be using Radix Notoginseng genomic DNA as template, using the single stranded DNA as shown in SEQ ID No.1 and
The primer pair of the composition of single stranded DNA shown in SEQ ID No.2 carry out the resulting amplified production of PCR amplification the from 5 ' ends
199 nucleotide are the homozygous of T.
The CC genotype be using Radix Notoginseng genomic DNA as template, using the single stranded DNA as shown in SEQ ID No.1 and
The primer pair of the composition of single stranded DNA shown in SEQ ID No.2 carry out the resulting amplified production of PCR amplification the from 5 ' ends
199 nucleotide are the homozygous of C.
The CT genotype be using Radix Notoginseng genomic DNA as template, using the single stranded DNA as shown in SEQ ID No.1 and
The primer pair of the composition of single stranded DNA shown in SEQ ID No.2 carry out the resulting amplified production of PCR amplification the from 5 ' ends
The heterozygous that 199 nucleotide are C and T.
Further, the method for described " detecting the nucleotide in the genome of Radix Notoginseng to be measured at following SNP site " can be
Sequencing analysis；The sequencing analysis may include PCR amplification and carry out two steps of sequencing to the PCR amplification products therefrom；Institute
The template for stating PCR amplification is the genomic DNA of the Radix Notoginseng to be measured, and primer pair used meets following condition: with Radix Notoginseng to be measured
Genomic DNA be template carry out PCR amplification obtained by amplified production contain the nucleotide at the SNP site.
Further, the primer pair concretely following (a) or (b) or (c) shown in primer pair:
(a) primer pair that single stranded DNA shown in the single stranded DNA shown in SEQ ID No.1 and SEQ ID No.2 forms；
(b) by by SEQ ID No.1 and SEQ ID No.2 by one or several nucleotide substitution and/or missing and/
Or two single strand dnas composition shown in gained sequence after addition, and primer identical with primer pair function described in (a)
It is right；
(c) as designing resulting primer pair through nucleotide sequence shown in SEQ ID No.3, and with primer pair described in (a)
The identical primer pair of function.
6th aspect, the claimed reagent or the kit or the molecular labeling or the method exist
Application in Radix Notoginseng breeding.
In the application, Radix Notoginseng high yield excellent variety can be cultivated by the method for molecular mark.
7th aspect, the method for claimed following (A) or (B):
(A) method for cultivating the increased pseudoginseny of total root weight, including selecting the Radix Notoginseng of TT or CT genotype to carry out breeding
The step of；
The TT genotype be using Radix Notoginseng genomic DNA as template, using the single stranded DNA as shown in SEQ ID No.1 and
The primer pair of the composition of single stranded DNA shown in SEQ ID No.2 carry out the resulting amplified production of PCR amplification the from 5 ' ends
199 nucleotide are the homozygous of T；
The CT genotype be using Radix Notoginseng genomic DNA as template, using the single stranded DNA as shown in SEQ ID No.1 and
The primer pair of the composition of single stranded DNA shown in SEQ ID No.2 carry out the resulting amplified production of PCR amplification the from 5 ' ends
The heterozygous that 199 nucleotide are C and T (heterozygous can carry out selfing purifying in offspring).
(B) method for identifying and filtering the small pseudoginseny of total root weight, including CC is filtered out from Radix Notoginseng group to be identified
The Radix Notoginseng step of genotype；The CC genotype is using Radix Notoginseng genomic DNA as template, using as shown in SEQ ID No.1
The primer pair of the composition of single stranded DNA shown in single stranded DNA and SEQ ID No.2 carry out the resulting amplified production of PCR amplification from 5 '
It is the homozygous of C that the 199th nucleotide is played in end.
In one embodiment of the invention, total root principal characteristic shape is embodied as fresh total root weight.
The present invention is accurately positioned total root weight on the basis of studying different weight Radix Notoginseng germ plasm resource by sequencing technologies
Gene loci, and then develop the SNP marker closely coupled with it, it is intended to the genetic mechanisms for analyzing its behind, from molecular layer
Face positioning and this heavy important character of the total root of explanation Radix Notoginseng.And then the screening of high yield strain is realized in notoginseng planting early stage, improve three
Seven planting benefits.
Beneficial effects of the present invention embody are as follows:
According to an embodiment of the invention, the method that the pcr amplification product is sequenced is not particularly limited, as long as energy
Enough sequences for effectively obtaining the segment where pcr amplification product, that is, SNP marker.Some specific examples according to the present invention,
Can using selected from HISEQ2000, SOLiD, 454 and single-molecule sequencing method it is at least one to the pcr amplification product into
Row sequencing.Thereby, it is possible to it is high-throughput, quickly, efficiently and accurately obtain sequencing result.
According to an embodiment of the invention, being based on sequencing result, genome sequence is referred to by comparing Radix Notoginseng, it can effectively really
The genotype of the fixed SNP marker to be measured for carrying Radix Notoginseng is TT or CC.
According to an embodiment of the invention, fresh total root of the TT genotype individuals of the SNP marker is significantly higher than CC gene again
Type individual.Fresh total root of mentioned-above SNP marker i.e. of the invention and Radix Notoginseng is closely related again.As a result, based on determining to be measured
The genotype of the SNP marker of Radix Notoginseng, can accurately and effectively prejudge fresh total root principal characteristic shape of Radix Notoginseng, such as the SNP site
When genotype is TT, then the sample belongs to the biggish individual of total root fresh weight.And then method of the invention can be effective for Radix Notoginseng
Molecular mark, so as to assist early stage to realize short time, low cost, the high accuracy ground excellent product of breeding Radix Notoginseng
Kind.It should be noted that SNP marker provided by the invention is not limited by Radix Notoginseng plant size, it can be used for the early stage choosing of Radix Notoginseng
It educates, remarkably promotes breeding process.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1, with the exploitation of the SNP marker scaffold6086_33242 of Radix Notoginseng total root weight close linkage and its
Using
One, the exploitation of the SNP marker scaffold6086_33242 of close linkage is weighed with the total root of Radix Notoginseng
1, the building of Radix Notoginseng sample sources group
Used Radix Notoginseng sample populations pick up from the different counties and cities of Zhuang-Miao Autonomous Prefecture of Wenshan, Yunnan Province, wherein Maguan
County, Qiubei County, Yanshan County and other have 179,150,386 and 803 parts of materials respectively, totally 1518 single plants.
2, simplify gene order-checking (Restriction site Associated DNA sequencing, RAD-seq)
Based on Hiseq2000 high-flux sequence platform, it is individual to 1518 that gene order-checking method is simplified using RAD
DNA sample is sequenced, and is generated the data volume of average each individual 0.4G or so, is averagely covered 1.6% Radix Notoginseng genome.
3, the heavy relevant SNP marker of the fresh total root of Radix Notoginseng is obtained
The roots growth traits phenotypic evaluation such as again is carried out to this 1518 individuals.Data are handled using PLINK software
Then Screening Treatment uses the EMMAX software based on mixed linear model to carry out GWAS analysis, finds from 254,083 SNP
1 to the significant relevant SNP site of fresh total root weight.The SNP site is located at 33, the 242bp of scaffold6086, this site
Base be C or T (SNP site is denoted as scaffold6086_33242).Genotype is the Radix Notoginseng of homozygosis TT at the site
Fresh total root be significantly higher than the Radix Notoginseng that genotype herein is homozygosis CC again, total root dense medium of CT genotype in homozygous CC and TT it
Between.
Further, SNP site scaffold6086_33242 is sequence shown in SEQ ID No.3 in Radix Notoginseng genomic DNA
The 501st of column.
Two, the application of the SNP marker scaffold6086_33242 of close linkage is weighed with the total root of Radix Notoginseng
1, the extraction of Radix Notoginseng sample genomic dna
Radix Notoginseng sample to be measured includes the sources group of 1518 samples in above-mentioned elaboration, randomly selects 300 samples,
Genomic DNA is extracted respectively with CTAB method, and the specific method is as follows:
(1) blade for taking 20 DEG C of freezen protectives, is put into mortar, with 1.5 × CTAB of 3mL is added after liquid nitrogen grinding, is ground into
Homogenate is transferred in the centrifuge tube of 15mL, and 1.5 × CTAB of 1mL flushing is then added into mortar and is transferred in centrifuge tube again.After mixing
In 65 DEG C of water-bath 30min, during which slowly shake up frequently.
Wherein 1.5 × CTAB is formulated following (1L): CTAB 15g；Tris.Cl (pH 8.0) 75mL of 1mol/L；
The EDTA 30mL of 0.5mol/L；NaCl 61.4g；Add deionized water to be settled to 1L, is added final concentration of 0.2% using preceding
The mercaptoethanol of (2ml).
(2) it is cooled to room temperature, isometric chloroform/isoamyl alcohol (24:1, volume ratio) is added, mixes gently, until subnatant
Become bottle green.
(3) 4200rpm is centrifuged 10min, and upper strata aqueous phase is moved on to new 15mL centrifuge tube, adds the anhydrous of 2 times of volume pre-coolings
Ethyl alcohol mixes static 5min.DNA is precipitated in -20 DEG C of placement 30min.
(4) 4200rpm is centrifuged 10min, discards supernatant, and 75% ethanol washing of 1mL is added and precipitates 1 time, it is dry to be inverted centrifuge tube
200 μ L TE dissolving DNAs are added in dry DNA.
(5) genomic DNA is detected with 0.8% Ago-Gel.
(6) by obtained genomic DNA be stored in -20 DEG C it is spare.
2, the nucleotide fragments containing SNP site are expanded
Using the aforementioned genomic DNA for extracting each Radix Notoginseng to be measured obtained as template, forward primer F and reverse primer R are utilized
Amplify the nucleotide fragments where SNP marker to be measured.
Forward primer F:5 '-ACCCGTTGCTCTAATGCCTC-3 ' (SEQ ID No.1)；
Reverse primer R:5 '-ACTCTTCCGGACTCGAACAC-3 ' (SEQ ID No.2).
Amplified production is 303-876 of nucleotide sequence shown in SEQ ID No.3 from 5 ' ends, SEQ ID No.3
501st as SNP site scaffold6086_33242 of the shown nucleotide sequence from 5 ' ends.
PCR reaction system (25 μ l) is as follows: 20.2 μ l of sterile water；10 × Buffer (contains Mg2+)2.5μl；dNTPs(25mM)
0.15μl；0.15 μ l of Taq enzyme (5U/ μ l)；0.5 μ l of forward primer；0.5 μ l of reverse primer；1.0 μ l of template.
PCR response procedures are as follows: 94 DEG C initial denaturation 5 minutes；94 DEG C are denaturalized 30 seconds, and 60 DEG C are annealed 30 seconds, and 72 DEG C extend 40
Second, run 35 circulations；Last 72 DEG C extend 3 minutes.Pcr amplification product can be saved at 4 DEG C.
3, sequencing identification SNP site genotype
By each pcr amplification product obtained in above-mentioned steps in being unidirectionally sequenced on ABI3730 sequenator, SNP is identified
Mark the genotype of scaffold6086_33242.
Using Radix Notoginseng genomic DNA as template, using forward primer F and reverse primer R (SEQ ID No.1 and SEQ ID
No.2) carrying out the 199th nucleotide from 5 ' ends of amplified production obtained by PCR amplification is SNP site scaffold6086_
33242；Nucleotide at the SNP site is C or T.
4, the association analysis of SNP site genotype and fresh total root weight
Phenotype (the fresh total root weight) distribution situation for counting different genotype (CC, TT and CT), obtains table 1.CC genotype is
Using Radix Notoginseng genomic DNA as template, carried out using forward primer F and reverse primer R (SEQ ID No.1 and SEQ ID No.2)
(the 501st of corresponding SEQ ID No.3) nucleotide the 199th from 5 ' ends of the resulting amplified production of PCR amplification is C's
It is homozygous.TT genotype be using Radix Notoginseng genomic DNA as template, using forward primer F and reverse primer R (SEQ ID No.1 and
SEQ ID No.2) carry out (the of correspondence SEQ ID No.3 the 199th from 5 ' ends ing of the resulting amplified production of PCR amplification
501) nucleotide be T it is homozygous.CT genotype is to draw using forward primer F and reversely using Radix Notoginseng genomic DNA as template
Object R (SEQ ID No.1 and SEQ ID No.2) carry out the resulting amplified production of PCR amplification from 5 ' ends the 199th it is (right
The 501st for answering SEQ ID No.3) nucleotide be C and T heterozygous.
As shown in Table 1, the fresh total root weight-average value of the homozygous individual of the fresh total root weight-average value ratio CC of the homozygous individual of TT is big, and CT is miscellaneous
Fresh total root weight-average value of mould assembly individual falls between.
The Phenotype Distribution situation of 1 different genotype of table
| Genotype | Mean value | Standard deviation | Sample number |
| CC | 29.3595 | 8.22357 | 160 |
| TT | 46.2548 | 15.02478 | 58 |
| CT | 39.0117 | 11.04394 | 82 |
| It amounts to | 35.2642 | 12.56897 | 300 |
It is based on the above table 1 as a result, using SPSS software generalized linear model single factor analysis method analyze SNP
The relevance of the genotype of point and fresh total root weight, wherein representing phenotypic number again when analysis with fresh total root.
The genotype of SNP site and the association analysis result of fresh total root weight see the table below 2.
The association analysis result of 2 SNP genotype of table and fresh total root weight
Association analysis shown in table 2 the result shows that, the correlation of the mean value of genotype and fresh total root weight reaches extremely significant property water
Flat (P < 0.01).In turn, it was demonstrated that the SNP site, it is significant related to the fresh total root weight of Radix Notoginseng, it is the heavy relevant SNP of the fresh total root of Radix Notoginseng
Label, fresh total root of the TT genotype individuals of the SNP marker are significantly higher than CC genotype individuals again, CT genotype individuals it is fresh total
Root dense medium is between the two.
<110>Shenzhen's Hua Da agricultural application study institute, Shenzhen Hua Da life science institute, Yunnan Hua Da Gene science have
Limit company, Wenshan Miaoxiang Notoginseng Industry Co., Ltd.
<120>SNP marker and its application of close linkage are weighed with the total root of Radix Notoginseng
<130> GNCLN180721
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence
<400> 1
acccgttgct ctaatgcctc 20
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence
<400> 2
actcttccgg actcgaacac 20
<210> 3
<211> 1001
<212> DNA
<213> Panax notoginseng
<220>
<221> misc_feature
<222> (501)..(501)
<223> y is c or t
<400> 3
ccggatccag acagaatggg aatatacacc tctggatgac cgtgctcttt gtagtctatc 60
agattagggt tatactcagg catgcgataa agcccttatt taagcccttt gggagcagat 120
aagggtatct cagttggatg cccaacgtga gtcaagccaa gataactcgg acaagagtga 180
gaccgaggat actcacatgg gagctctcca tttgcttaat gtcattcatt aaaggctatg 240
ccgatagcaa ggcaacaaaa caagcaacga ctttcgcgcg ctagtctagt agtagtacgc 300
tcacccgttg ctctaatgcc tcgttacgta aacaatggta accttgtcgg tttgccgaca 360
atacaccgag aattcacggt gtttcgatgt tacgcgtaac aaaagtgttt taggctaatc 420
acctaacagt acttcatgta gagtttcata gtctgtgact aatgaacact tggaaaagtt 480
tttaacattg tttaagaatt yattacctta tggtcattca gttatttgat attattcata 540
tcattggaaa gttgagccac aactgatgaa aatcatttca ttttgagtta tttactcttt 600
tattagttgt cgagttaaga gacaaagatt caagctttta ttattcctta gatccgggat 660
tctgaaatgt tacgatatat tttaaacgat aaattgtatt aattgttaat tattgagcta 720
taaaacttga tgaacaaatt tgttgctcac ttatacttat aggacctctt tcgagcaatt 780
tcagaatagg gtagagcact acccaacaga gataggtcag acaagttaca ctatcagcaa 840
gggtctgtct agctttgtgt tcgagtccgg aagagtacct agatgccata agagaagacc 900
tatgagactt ttgtgtgtgt gtgtgtgtgt gtctatatat atatatatat atggaaaatg 960
atctatagca agtatttgct atggatgaca aatatccatt g 1001
Claims (10)
1. following SNP site or the mononucleotide polymorphic for detecting following SNP site in Radix Notoginseng genome in Radix Notoginseng genome
Property substance identifying or assisting to identify the application in total root principal characteristic shape of Radix Notoginseng to be measured；
The SNP site are as follows: using Radix Notoginseng genomic DNA as template, using single stranded DNA and SEQ as shown in SEQ ID No.1
The primer pair of the composition of single stranded DNA shown in ID No.2 carries out the 199th core from 5 ' ends of amplified production obtained by PCR amplification
Thuja acid；Nucleotide at the SNP site is C or T.
2. being used to identify or assist to identify the reagent or kit of total root principal characteristic shape of Radix Notoginseng to be measured, it is characterised in that: the examination
Agent is the substance for detecting the single nucleotide polymorphism of following SNP site in Radix Notoginseng genome；The kit contains described
Reagent；
The SNP site are as follows: using Radix Notoginseng genomic DNA as template, using single stranded DNA and SEQ as shown in SEQ ID No.1
The primer pair of the composition of single stranded DNA shown in ID No.2 carries out the 199th core from 5 ' ends of amplified production obtained by PCR amplification
Thuja acid；Nucleotide at the SNP site is C or T.
It is using Radix Notoginseng genomic DNA as mould 3. being used to identify or assist to identify the molecular labeling of total root principal characteristic shape of Radix Notoginseng to be measured
Plate expands resulting amplified production using primer pair A；
The primer pair A meets following condition: carrying out PCR amplification gained amplification as template using the genomic DNA of Radix Notoginseng to be measured and produces
Object contains the nucleotide at following SNP site；
The SNP site are as follows: using Radix Notoginseng genomic DNA as template, using single stranded DNA and SEQ as shown in SEQ ID No.1
The primer pair of the composition of single stranded DNA shown in ID No.2 carries out the 199th core from 5 ' ends of amplified production obtained by PCR amplification
Thuja acid；Nucleotide at the SNP site is C or T.
4. application according to claim 1 or reagent as claimed in claim 2 or kit or as claimed in claim 3 point
Son label, it is characterised in that: " for the detecting the substance of the single nucleotide polymorphism of following SNP site in Radix Notoginseng genome "
With the primer pair A be following (a) or (b) or (c) shown in primer pair:
(a) primer pair that single stranded DNA shown in the single stranded DNA shown in SEQ ID No.1 and SEQ ID No.2 forms；
(b) by by the substitution of one or several nucleotide and/or missing and/or adding SEQ ID No.1 and SEQ ID No.2
Two single strand dnas shown in rear gained sequence are added to form, and primer pair identical with primer pair function described in (a)；
(c) as designing resulting primer pair through nucleotide sequence shown in SEQ ID No.3, and with primer pair function described in (a)
Identical primer pair.
5. reagent or kit described in Claims 2 or 3 or molecular labeling as claimed in claim 4 identify or assist identification to
Survey the application in total root principal characteristic shape of Radix Notoginseng.
6. a kind of method that identification or auxiliary identify total root principal characteristic shape of Radix Notoginseng to be measured, includes the following steps: to detect Radix Notoginseng to be measured
Genome in nucleotide at following SNP site, with the genotype of the determination Radix Notoginseng to be measured, according to the Radix Notoginseng to be measured
Genotype determines total root principal characteristic shape of Radix Notoginseng to be measured according to following rule: total root of the Radix Notoginseng to be measured of TT genotype it is great in or wait
Choosing is greater than the Radix Notoginseng to be measured of CC genotype, and total root of CT genotype falls between again；
The SNP site are as follows: using Radix Notoginseng genomic DNA as template, using single stranded DNA and SEQ as shown in SEQ ID No.1
The primer pair of the composition of single stranded DNA shown in ID No.2 carries out the 199th core from 5 ' ends of amplified production obtained by PCR amplification
Thuja acid；Nucleotide at the SNP site is C or T；
The TT genotype is using Radix Notoginseng genomic DNA as template, using single stranded DNA and SEQ as shown in SEQ ID No.1
Single stranded DNA shown in ID No.2 composition primer pair carry out the resulting amplified production of PCR amplification the 199th from 5 ' ends
Nucleotide is the homozygous of T；
The CC genotype is using Radix Notoginseng genomic DNA as template, using single stranded DNA and SEQ as shown in SEQ ID No.1
Single stranded DNA shown in ID No.2 composition primer pair carry out the resulting amplified production of PCR amplification the 199th from 5 ' ends
Nucleotide is the homozygous of C；
The CT genotype is using Radix Notoginseng genomic DNA as template, using single stranded DNA and SEQ as shown in SEQ ID No.1
Single stranded DNA shown in ID No.2 composition primer pair carry out the resulting amplified production of PCR amplification the 199th from 5 ' ends
Nucleotide is the heterozygous of C and T.
7. according to the method described in claim 6, it is characterized by: described " detect SNP following in the genome of Radix Notoginseng to be measured
The method of nucleotide at point " is sequencing analysis；The sequencing analysis includes PCR amplification and to the PCR amplification products therefrom
Carry out two steps of sequencing；The template of the PCR amplification is the genomic DNA of the Radix Notoginseng to be measured, and primer pair used meets
Following condition: PCR amplification gained amplified production is carried out as template using the genomic DNA of Radix Notoginseng to be measured and is contained at the SNP site
Nucleotide.
8. according to the method described in claim 7, it is characterized by: the primer pair be following (a) or (b) or (c) shown in
Primer pair:
(a) primer pair that single stranded DNA shown in the single stranded DNA shown in SEQ ID No.1 and SEQ ID No.2 forms；
(b) by by the substitution of one or several nucleotide and/or missing and/or adding SEQ ID No.1 and SEQ ID No.2
Two single strand dnas shown in rear gained sequence are added to form, and primer pair identical with primer pair function described in (a)；
(c) as designing resulting primer pair through nucleotide sequence shown in SEQ ID No.3, and with primer pair function described in (a)
Identical primer pair.
9. appointing in reagent or kit described in Claims 2 or 3 or molecular labeling as claimed in claim 4 or claim 6-8
Application of the method described in one in Radix Notoginseng breeding.
The method of following 10. (A) or (B):
(A) method for cultivating the increased pseudoginseny of total root weight, the Radix Notoginseng including selection TT genotype or CT genotype are educated
The step of kind；The TT genotype be using Radix Notoginseng genomic DNA as template, using the single stranded DNA as shown in SEQ ID No.1 and
The primer pair of the composition of single stranded DNA shown in SEQ ID No.2 carry out the resulting amplified production of PCR amplification the from 5 ' ends
199 nucleotide are the homozygous of T；The CT genotype is using Radix Notoginseng genomic DNA as template, using by SEQ ID No.1
Shown in the primer pair of single stranded DNA composition shown in single stranded DNA and SEQ ID No.2 carry out the resulting amplified production of PCR amplification
The 199th nucleotide from 5 ' ends be C and T heterozygous；
(B) method for identifying and filtering the small pseudoginseny of total root weight, including CC gene is filtered out from Radix Notoginseng group to be identified
The step of Radix Notoginseng of type；The CC genotype is using Radix Notoginseng genomic DNA as template, using single as shown in SEQ ID No.1
The primer pair of the composition of single stranded DNA shown in chain DNA and SEQ ID No.2 carry out the resulting amplified production of PCR amplification from 5 ' ends
Having held the 199th nucleotide is the homozygous of C.
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