CN106520960A - Sesame microsatellite marker locus development method and length detection method for microsatellite markers in microsatellite marker loci - Google Patents
Sesame microsatellite marker locus development method and length detection method for microsatellite markers in microsatellite marker loci Download PDFInfo
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Abstract
The invention discloses a sesame microsatellite marker locus development method and a length detection method for microsatellite markers in microsatellite marker loci. The development method comprises the steps that a mixing sample is obtained; a genome of the mixing sample is extracted; the genome is fragmented, and genome fragments are obtained; a probe group is hybridized with the genome fragments; multiple genome fragments which are hybridized successfully in a hybridization solution are purified; the multiple purified and hybridized genome fragments are detected through high-throughput sequencing after being mixed; effective high-throughput sequencing fragments are obtained; and the effective high-throughput sequencing fragments are classified. The detection method comprises the steps that the to-be-detected microsatellite marker loci are selected; and the microsatellite markers in the to-be-detected microsatellite marker loci are amplified through multiple amplimers, and the length of the microsatellite markers in the microsatellite marker loci is obtained. The methods are simple, quick, comprehensive and accurate.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of Semen Sesami microsatellite markers development approach and microsatellite
The length detecting method of the microsatellite marker in marker site.
Background technology
Microsatellite marker is also known as STR (short tandem repeats, STR) or simple repeated sequence
(simple sequence repeats, SSR), refers to and is made up of for repetitives tandem sequence repeats more than 2 nucleotide.Microsatellite
Marker site refers to the seat containing microsatellite marker on genome, and microsatellite markers quantity on genome is enriched
And be uniformly distributed, the exploitation of microsatellite markers refers to the process for finding the microsatellite markers on genome.It is different
In sample, the number of repetition of the repetitives of the microsatellite marker in same microsatellite markers may be different, in sample
Between there is length variation, therefore the polymorphism of microsatellite markers refers mainly to the different micro- of same microsatellite markers and defends
The length polymorphism of asterisk note.Microsatellite marker detection technique refers to the microsatellite marker in detection microsatellite markers
The technology of length.The length polymorphism of the microsatellite marker of different samples can be used to identify the identity of sample, therefore,
The application of micro-satellite labeling technique is quite varied, including bio-diversity identification, the identification of plant and animal species fingerprint identification card etc..
The exploitation of traditional Semen Sesami microsatellite markers is comprised the following steps with detection：Genome is extracted, gene pack
The hybridization of Duan Hua, jointing, amplification and simple repeated sequence, purified hybrid product, hybrid product clone, clone products large intestine
Agrobacterium-transformation, picking monoclonal, generation sequencing, analysis sequencing result are carried out to each monoclonal target site obtain and micro- defend
Star marker site, in multiple Semen Sesami samples check microsatellite markers polymorphism, develop the high microsatellite of polymorphism
Marker site, each microsatellite markers to be detected for being expanded in simultaneously each sample to be detected of electrophoresis detection one by one
In microsatellite marker.
During the present invention is realized, inventor has found that prior art at least has problems with：
The exploitation of Semen Sesami microsatellite markers is complicated with testing process, flux is low, extremely time and effort consuming；Secondly, it is micro- to defend
The resolution of the electrophoresis detection of star marker site is low, and testing result is inaccurate, and accurate result needs to refer to sample etc. and carries out school
Just.Thus the problem for deriving from includes：The microsatellite markers for developing are few, within usual 200, account for
1% of microsatellite markers or so；For checking the Semen Sesami sample of microsatellite markers polymorphism also few, generally tens of
It is individual or so, therefore polymorphism probatio inspectionem pecuoarem result is inaccurate；The flanking sequence conservative of microsatellite markers is unknown, affects amplification micro-
The versatility of the primer in satellite markers site；The limited amount of the microsatellite markers of detection, it is typically to be detected at one
Dozens of microsatellite markers are detected in sample, causes the DNA ID card informations of the sample of foundation imperfect, inaccurate.
The content of the invention
In order to solve problem of the prior art, a kind of Semen Sesami microsatellite markers exploitation side is embodiments provided
The length detecting method of the microsatellite marker in method and microsatellite markers.The technical scheme is as follows：
On the one hand, a kind of Semen Sesami microsatellite markers development approach is embodiments provided, methods described includes：
The mass mixings such as the Semen Sesami sample for having polymorphism by n, obtain mixing sample, wherein n ＞ 1；
Extract the genome of the mixing sample；
By the genomic fragment of the mixing sample, genomic fragment is obtained；
Using multiple probes with simple repeated sequence as probe groups, distinguished using each probe in the probe groups
Hybridized with the genomic fragment, obtained multiple hybridization solutions, the gene to successful cross in multiple hybridization solutions
Pack section carries out purification respectively, obtains the hybrid gene pack section of multiple purification；
After the mass mixings such as the hybrid gene pack section by multiple purification, detect mixed using high-flux sequence
The hybrid gene pack section of the purification, obtains the first high-flux sequence fragment；
From the first high-flux sequence fragment, the effective high-flux sequence fragment of screening, the effective height
Flux sequencing fragment includes the microsatellite marker in microsatellite markers；
Had to described according to the homology of the both sides sequence of the microsatellite marker in the effective high-flux sequence fragment
The high-flux sequence fragment of effect is classified, and the of a sort effective high-flux sequence fragment is same microsatellite marker
The described effective high-flux sequence fragment in site, if the described effective high pass of the same microsatellite markers is measured
Bar number >=the α 1 of sequence fragment, then successfully develop the microsatellite markers, wherein, α 1 be the first decision threshold and α 1 >=
(the microsatellite markers number that can be detected on the ratio/genome of high pass sequencing depth × effective high-flux sequence fragment)
× probability ensures.
Generally, for the ease of the genomic fragment of successful cross in hybridization solution described in purification, work(can be carried out to probe
Labelling can be changed, for example can be with
Hybridized using the probe with simple repeated sequence and the genomic fragment of biotin labeling, obtained miscellaneous
Hand over solution；
Using the genomic fragment of successful cross in hybridization solution described in Streptavidin MagneSphere purification, the base of purification is obtained
Because of pack section.
As probe has biotin labeling in above-mentioned steps so that the genomic fragment of successful cross is also by biotin
Labelling, such that it is able to be purified from the hybridization solution using Streptavidin MagneSphere.The utilization biotin labeling and
The technology of Streptavidin MagneSphere purification is known technology.
Specifically, α 1 >=20.
Specifically, the microsatellite marker refers to the sequence being made up of the repetitives tandem sequence repeats of >=2 base compositions.
Specifically, the base number of the both sides sequence of the microsatellite marker in the effective high-flux sequence fragment is equal
At least base number of the sequence of side in the microsatellite marker in >=1, and the effective high-flux sequence fragment
>=10.
Specifically, the method for the described n Semen Sesami sample with polymorphism is selected to include：The sesame for selecting formalness different
The wild resource of the different Semen Sesami sample of numb sample, biological classification, the mutually different Semen Sesami sample of labelling or different ecological region
Semen Sesami sample.
Specifically, the quantity of the probe is 12, and the repetitives in the simple repeated sequence of each probe are
CT, GA, TG, AC, TA, TGT, CCA, ATC, CCT, AGA, ATG or CAA, the repetition of the simple repeated sequence of each probe
Number of times is 6～20, preferably 6～15, and such as number of repetition is 8 or 12.
Specifically, SEQ IN NO in the sequence of the probe such as sequence table:1-SEQ IN NO:Shown in 12.
On the other hand, embodiments provide in the microsatellite markers that a kind of above-mentioned development approach is successfully developed
Microsatellite marker length detecting method, the detection method includes：
From the microsatellite markers successfully developed, microsatellite markers to be detected are selected；
The microsatellite marker in the microsatellite markers to be detected is expanded using multiplex amplification primer, is expanded
The amplified production is carried out high-flux sequence by product, obtains the second high-flux sequence fragment, by analyzing second high pass
Sequence fragment is measured, the length of the microsatellite marker in the microsatellite markers is obtained.
Specifically, it is described from the microsatellite markers successfully developed, select the microsatellite mark to be detected
The method in note site includes：
The standard for selecting the microsatellite markers to be detected is the maximum microsatellite markers of H-number, its
In, H-number is the polymorphic sex index of the microsatellite markers,Wherein, i is by the microsatellite marker
When the length of the microsatellite marker in the described effective high-flux sequence fragment in site is classified, i-th classification, i are certainly
So count；Ai is that the number of the effective described high-flux sequence fragment of i-th classification accounts for total effective described high-flux sequence
The ratio of the number of fragment.
Specifically, the method for preparing the multiplex amplification primer includes：
From all described effective high-flux sequence fragment of the microsatellite markers described to be detected for selecting, carry
Take the microsatellite marker and pick out the most long microsatellite marker and defend as the micro- of template sequence of multiplex amplification primer
Asterisk is remembered；
From all described effective high-flux sequence fragment of the microsatellite markers described to be detected for selecting, carry
Take the left side sequence of the microsatellite marker and choose all sequences of the length more than 2 bases of α, it is described all from what is picked out
In sequence, pick out frequency highest sequence, using the frequency highest sequence as reference sequences, by the reference sequences with
The left side sequence of all of microsatellite marker is compared, and in the frequency highest sequence obtains each base
Cover multiple and variation frequency；In the frequency highest sequence, by the covering multiple≤1/ α 3 or described variation frequencies
The base of >=α 3 is changed into after N the left side sequence of the template sequence as the multiplex amplification primer, wherein, N is A, T, C and G tetra-
The base of any one and the above in kind of base；α 2 is the second decision threshold, α 2=(the first high-flux sequence fragment it is flat
The length of the microsatellite markers of equal length-multiplex amplification primer) ÷ 2；α 3 be the 3rd decision threshold, 3 >=5 × (1- of α
The accuracy of the first high-flux sequence fragment)；
According to the left side sequence identical method with the template sequence of the multiplex amplification primer, multiplex amplification primer is obtained
The right flanks of the template sequence of sequence；
By the left side sequence of the template sequence of the multiplex amplification primer, the template sequence of the multiplex amplification primer it is micro-
The right flanks of the template sequence of satellite markers and the multiplex amplification primer are sequentially connected, and obtain the microsatellite markers
Multiplex amplification primer template sequence, using the template sequence of the multiplex amplification primer of the microsatellite markers, obtain
The multiplex amplification primer.
Specifically, the method for the length of the microsatellite marker in the acquisition microsatellite markers is：Remove institute
After stating the microsatellite marker in the second high-flux sequence fragment, the left margin sequence of the second high-flux sequence fragment is obtained
The right border sequence of row and the second high-flux sequence fragment；Using the left margin sequence and the right border sequence by institute
Each fragment stated in the second high-flux sequence fragment is compared on the microsatellite markers to be detected；Intercept each
The microsatellite marker in the second high-flux sequence fragment of the microsatellite markers to be detected；By what is obtained
The microsatellite marker is classified by length, and calculates the validity R of i-th classificationi=Ni/Nmax, wherein, i is by described
When the length of the microsatellite marker in the described effective high-flux sequence fragment of microsatellite markers is classified, i-th
Classification, NiFor the quantity of the second high-flux sequence fragment of i-th classification, NmaxFor described the second of all categories
The maximum of the quantity of high-flux sequence fragment；If the validity Ri>=α 4, then the microsatellite mark of i-th classification
The length of note is the length of the microsatellite marker in the microsatellite markers, if the validity Ri<α 4, then it is described
The length of the microsatellite marker of i-th classification length not for the microsatellite marker in the microsatellite markers, its
In, α 4 is the 4th decision threshold and α 4=0.6.
Specifically, the method for the genomic fragment of the mixing sample is interrupted for machinery or enzyme action.
The beneficial effect that technical scheme provided in an embodiment of the present invention is brought is：The Semen Sesami microsatellite marker that the present invention is provided
The exploitation in site is simple with detection technique, quick and high-flux, comprehensively and accurately.Time loss shortened to 1～2 day by 1～2 year；
The microsatellite markers quantity developed is brought up to close by 1% or so of all microsatellite markers in genome
100%；The quantity of the Semen Sesami sample of the polymorphism of inspection microsatellite markers is lifted to unrestricted, polymorphism by dozens of
The accuracy of product test is greatly improved；The conservative of the flanking sequence of microsatellite markers can be obtained, it is ensured that amplification is micro-
The versatility of the primer in satellite markers site；Using multiple microsatellite markers as a site primer, rather than examine one by one
Survey, multiple Semen Sesami samples to be detected only carry out one-time detection, rather than repeated detection, considerably reduce microsatellite marker position
The workload of point detection, therefore, the quantity of the microsatellite markers of detection is almost unrestricted.Microsatellite markers are detected
Result be base, accuracy is close to 100%；Microsatellite markers detection resolution is promoted to best result rate：Single base；No
Reference cultivars are needed to be corrected testing result again.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, embodiment of the present invention will be made into one below
Step ground is described in detail.
In the embodiment of the present invention, the operating process or working specification of unreceipted or detailed description is common molecular biology
Operation known to technical staff.In the embodiment of the present invention, not marked reagent or biomaterial are on the market conventional
Reagent or biomaterial, are known to common molecular biology technical staff, and commercially can buy.
Embodiment
The development approach of Semen Sesami microsatellite markers：
The Semen Sesami sample mixed in equal amounts for having polymorphism by n, obtains mixing sample, wherein n ＞ 1.
Semen Sesami sample with polymorphism includes：The different Semen Sesami samples of formalness (form polymorphism), biological classification
(such as different mutation or kind) different Semen Sesami sample, the mutually different Semen Sesami sample of labelling (such as protein labeling) or not syngenesis
The wild resource Semen Sesami sample in state region, wherein, selected Semen Sesami sample is more (n values are bigger), and polymorphism is abundanter, is opened
The suitability of the microsatellite markers for sending is wider.In the present embodiment, the species of microsatellite markers leaved for development are sesame
Fiber crops, selected Semen Sesami are different sesame varieties, and they are respectively：Zhong-zhi No.12 number, Feng Zhi 88, Luo sesames No. 16, Henan sesame No. 4,
Distant product sesame No. 1, excellent sesame No. 5, Yuzhi No.9, Fen sesame No. 5, Henan sesame No. 8, Jin Zhi 8, Henan sesame No. 11, assist Fructificatio Amaurodermatis Rudae 2078, non-irrigated sesame 4
Number, association's agriculture one, Zhengzhi97C01, Henan sesame No. 8, middle sesame 16, Zheng H03, Zhengzazhi H03 and middle sesame 17, totally 20 kinds, these
Sesame variety is Chinese widely used kind, open and known, purchases and obtain on market.Wherein, microsatellite marker refer to by
The sequence that the repetitives tandem sequence repeats of >=2 base compositions are constituted.
Take 20 sesame varieties of the above etc. quality blade mix, extract the genome of mixing sample, extracting method is pressed
According to TIANGEN Biotech (Beijing) Co., Ltd. article No. for DP320 plant genes group extracts kit workbook
Carry out.Semen Sesami sample selected in the present embodiment is blade, and used as common knowledge, the Semen Sesami sample can also take from seed etc.
Position.
By the genomic fragment of mixing sample, genomic fragment is obtained.Specifically, by the genomic fragment of mixing sample
The method of change includes：Machinery is interrupted or enzyme action.The length of genomic fragment controls the detectable fragment in high-flux sequence
In length range.In the present embodiment, PI chip of the high-flux sequence using PROTON high-flux sequence instrument, its detection length are about
200bp, therefore, the peak value of the length of the genomic fragment of acquisition is also controlled near 200bp as far as possible.The present embodiment is using automatic
The genome of the broken mixing sample of the broken instrument Covaris S220 of sound wave focusing (U.S. Covaris is produced, model S220), breaks
Workbook of the broken method by the instrument《DNA Shearing with S220/E220Focused-ultrasonicator》
(version number:010308Rev G) in the method for described acquisition 200bp (peak value) target fragment carry out, it is broken after obtain
The genomic fragment of mixing sample, presses the journey of its double-stranded DNA using the Q5000 spectrophotometers that Quawell companies of the U.S. produce
After ordered pair genomic fragment is detected, by concentration dilution or 100ng/ μ L are concentrated to, obtain genomic fragment.
Using the probe with simple repeated sequence of multiple biotin labelings as probe groups, using probe groups and genome
Fragment is hybridized, and obtains hybridization solution.Base number >=2 of the repetitives in the probe with simple repeated sequence.Tool
Body ground, the repetitives in the simple repeated sequence of probe be CT, GA, TG, AC, TA, TGT, CCA, ATC, CCT, AGA, ATG or
CAA, this 12 probes can be hybridized for the microsatellite marker of repetitives with all possible 2 base and 3 bases, because
This, the fishing of the microsatellite marker in the genomic fragment that can be used in all species takes.In previous experiments, we have detected not
The efficiency of microsatellite marker is hooked up with probe length, when the number of repetition of the simple repeated sequence of discovery probe is 6～20, efficiency
Higher, preferred number of repetition is 6～15, such as 8 or 12.In the present embodiment, the probe groups include 12 probes, this 12
The sequence of probe is respectively such as SEQ IN NO in sequence table:1-SEQ IN NO:Shown in 12.Above probe holds up the new industry of section by Beijing
Bioisystech Co., Ltd synthesizes and carries out 5 ' end biotin labelings.The experiment of early stage shows, different probe is fished respectively and takes base
Because the efficiency of the microsatellite marker in pack section takes the microsatellite marker in genomic fragment better than fishing after all probes are mixed
Efficiency, therefore, fish the microsatellite marker taken in genomic fragment in the present embodiment using different probe respectively, specifically,
By each of the above probe respectively with without the solution that enzyme water dissolution is equimolar concentration (10pM/ μ L), 12 in above probe groups are taken
The each 1 μ L of individual probe are hybridized after being mixed with the genomic fragment of 5 μ g mixing samples respectively, obtain 12 kinds of hybridization solutions respectively.Hybridization
Program be：95 DEG C 10 minutes, 65 DEG C 10 minutes, 37 DEG C 10 minutes.
Using the genomic fragment of successful cross in Streptavidin MagneSphere purified hybrid solution, the genome of purification is obtained
Fragment.Specifically, 12 kinds of hybridization solutions of purification are distinguished using Streptavidin MagneSphere, its purge process is：12 kinds for obtaining
In a kind of hybridization solution be placed on magnetic frame (production of Invitrogen companies of the U.S.), to hybridization solution clarification after, suck molten
Liquid, is cleaned magnetic bead 2 times with without enzyme water, takes 10 μ L and mixed with Streptavidin MagneSphere without enzyme water, and in PCR instrument, 95 DEG C are heated 5 points
Clock, is immediately placed on magnetic frame, and the solution of acquisition is the hybrid gene pack section of the purification of first probe.By with obtain
The hybrid gene pack section identical method of the purification of first probe, obtains the hybrid gene pack of all 12 purification successively
Section, they is mixed, i.e., finally obtains the hybrid gene pack section of the purification of all probes.In order to successful purification
Hybrid gene pack section, in the present embodiment, coordinates strepto- parent using the probe with simple repeated sequence of biotin labeling
With the mode of biscuit porcelain pearl, in other embodiments, it would however also be possible to employ other modes carry out the hybridization of genomic fragment and purification.
The hybrid gene pack section of purification is detected using secondary high-flux sequence, the first high-flux sequence fragment is obtained.Profit
Build with DNA library reagent preparation box (being produced by NEB companies of Britain, article No. is E6270L) and by the workbook of the test kit
Secondary high-throughput sequencing library, using the secondary high-throughput sequencing library and test kit Ion PI Template OT2 that obtain
(invirtrigen companies of the U.S. produce 200Kit v2, and article No. is the ePCR (Emulsion before 4485146) being sequenced
PCR, emulsion polymerization enzyme chain reaction) expand, operational approach is carried out by the workbook of the test kit, obtains ePCR amplified productions.
Using ePCR amplified productions and test kit Ion PI Sequencing 200Kit v2 (invirtrigen companies of the U.S. produce,
Article No. is that high-flux sequence 4485149) is carried out on the secondary high-flux sequence instrument of Proton, behaviour of the operational approach by the test kit
Carry out as handbook.In the present embodiment, high-flux sequence amount is set to 10M sequencings fragment (1M=,100 ten thousand), and sequencing length is arranged
(circulate) for 500cycle, after sequencing terminates, obtain the first high-flux sequence fragment.
From the first high-flux sequence fragment, effective high-flux sequence fragment is screened.Wherein, effective high-flux sequence
Fragment includes the microsatellite marker in microsatellite markers, the both sides of the microsatellite marker in effective high-flux sequence fragment
The base number of sequence >=1, and the effectively at least sequence of side in the microsatellite marker in high-flux sequence fragment
Base number >=10.Whether each bar segment analyzed in the first high-flux sequence fragment contains microsatellite marker, removes and does not contain
There is the first high-flux sequence fragment of microsatellite marker.In the first high-flux sequence fragment for remaining, microsatellite is analyzed
The base number of the both sides sequence of labelling whether >=1, if it is, showing microsatellite marker in the first high-flux sequence fragment
In be complete, this point is necessary, because the polymorphism of microsatellite marker refers to the length polymorphism of microsatellite marker, only
Having guarantees that microsatellite marker is complete, could correctly obtain the length polymorphism of microsatellite marker, after correctly carrying out
Continuous analysis.After first high-flux sequence fragment of the both sides sequence of microsatellite marker respectively less than 10 bases cannot be carried out exactly
Continuous homology analysis, can introduce error because sequence is too short, therefore, the both sides sequence for further removing microsatellite marker is equal
Less than the first high-flux sequence fragment of 10 bases.Through above flow process, the first high pass sequencing fragment for finally remaining
As effective high-flux sequence fragment.
Wherein, whether each bar segment in the first high-flux sequence fragment is analyzed during containing microsatellite marker, can be with
Molecule is carried out using analysis software commonly used in the prior art, it is also possible to simply by manually to each the first high-flux sequence
Fragment is judged.
According to the homology of the both sides sequence of the microsatellite marker in effective high-flux sequence fragment to effective high pass
Measure sequence fragment to be classified, of a sort effective high-flux sequence fragment is the effective of same microsatellite markers
High-flux sequence fragment, if the bar number >=α 1 of the effective high-flux sequence fragment of same microsatellite markers, success
One microsatellite markers of exploitation, wherein, α 1 is the first decision threshold and 1 >=(high pass sequencing depth × effective high flux of α
The microsatellite markers number that can be detected on the ratio/genome of sequencing fragment) guarantee of × probability.The concrete value of α 1 can root
It is adjusted according to the depth of high-flux sequence.Microsatellite marker in effective high-flux sequence fragment is removed, will be remaining
Both sides sequence is merged into a complete sequence, using software Megablast (version 2 .2.26) complete sequences after merging
Paired comparison analysis is carried out between row, each parameter of comparison is set to：Parameter-e is set to 1e-5；Parameter-p is set to 0；Ginseng
Number v is set to 5000；Parameter-m is set to 1.Will be with the effective height of homology (homology refers to similar in DNA sequence)
Flux sequencing fragment is classified as same class, while the quantity of the effective high-flux sequence fragment included in classification is calculated, if comprising
Effective high-flux sequence fragment quantity >=α 1 when, then the effective high-flux sequence fragment of the category is a success
The high-flux sequence fragment of the microsatellite markers of exploitation.Its concrete principle is as follows：Same microsatellite marker on genome
Site is likely to be detected repeatedly during high-flux sequence, as the object of high-flux sequence multiple has to contain
The mixing sample of the sample of polymorphism, therefore, the length of the microsatellite marker in same microsatellite markers exists polymorphic
Property variation, the both sides sequence of microsatellite markers there is also variation, it is thus impossible to require same microsatellite markers
The sequence of all effective high-flux sequence fragments is identical, can only be judged according to the homology of high-flux sequence fragment be
It is no for same microsatellite markers, wherein, high-flux sequence fragment includes two lateral orders of microsatellite marker and microsatellite marker
Row, therefore, the homology of high-flux sequence fragment can be two lateral orders of the homology or microsatellite marker of microsatellite marker
The homology of row.Microsatellite marker in different microsatellite markers may be identical, it is thus impossible to according to microsatellite marker
Homology judge whether effective high-flux sequence fragment belongs to same microsatellite markers, can only be according to microsatellite marker
The homology of the both sides sequence in site judges whether effective high-flux sequence fragment belongs to same microsatellite markers.Institute
To require that the quantity >=α 1 of the effective high-flux sequence fragment of same microsatellite markers is to prevent such as sample
The false positive that the unmanageable factor such as contaminated is caused.When α 1 >=3 is to confirm a fragment necessary being in bioinformatics,
The general threshold value for adopting, the concrete value of α 1 can be adjusted according to the depth of high-flux sequence, can calculate as follows：α1
>=(the microsatellite marker position that can be detected on the ratio/genome of high pass sequencing depth × effective high-flux sequence fragment
Points) guarantee of × probability, wherein, statistically, probability ensures that general value is 5% or 1%.In the present embodiment, high pass is measured
The depth of sequence is 10,000,000, rule of thumb, the ratio generally 40% or so of effective high-flux sequence fragment, general gene
The microsatellite markers that can be detected in group are 10,000 or so, therefore, what average each microsatellite markers were included has
The high-flux sequence fragment of effect is 400, and the sequencing depth of actual microsatellite markers is presented normal distribution, so, one
As less than average effective high-flux sequence segments 1/20 (5%) distribution it is less.Therefore, in the present embodiment, 1 values of α are
20.By above flow process and standard, in the present embodiment, 13001 microsatellite markers are successfully developed altogether.
The standard for selecting microsatellite markers to be detected is the maximum microsatellite markers of H-number, wherein, H-number is
The polymorphic sex index of microsatellite markers,Wherein, i is the effective high pass by microsatellite markers
When the length of the microsatellite marker in measurement sequence fragment is classified, i-th classification, i are natural number；Ai is i-th classification
Effectively the number of high-flux sequence fragment accounts for the ratio of the number of total effective high-flux sequence fragment.Such as the hypothesis of table 1
Microsatellite markers classified by the length of the microsatellite marker in effective high-flux sequence fragment, have 3 kinds：
(TG) 20, (TG) 21 and (TG) 22, therefore S=3；The number of total effective high-flux sequence fragment of the microsatellite markers
Mesh is 40, wherein, the number of the 1st kind of microsatellite marker (TG) 20 is 3 articles, therefore a1=3/40=7.50%, equally calculates a2
=32/40=80%, a3=5/40=12.50%.The computing formula that values above substitutes into H is obtained into the microsatellite markers
H-number is 0.98.
It is by the microsatellite markers identical computational methods with the hypothesis of table 1, all in calculating the present embodiment successfully to open
The H-number of all microsatellite markers for sending, the descending arrangement of H-number of the microsatellite markers of all acquisitions are selected
The microsatellite markers of first 50 of sorting are the microsatellite markers for needing in sample to be detected in this enforcement to detect.Ginseng
Number 50 is fixed according to actual needs, for example, in Semen Sesami Purity, 1 microsatellite markers, in Semen Sesami finger printing
During structure, the microsatellite markers of 50 or so are typically chosen, when substantive derived relation is analyzed between kind, then require choosing
Select general 300 microsatellite markers and could meet requirement.Why select the microsatellite markers that H-number is maximum, be because
Separating capacity for them is most strong, can distinguish more samples as far as possible with minimum microsatellite markers and offer to the greatest extent may be used
Information more than energy, and it is the most crucial task of micro-satellite labeling technique to distinguish sample.
The microsatellite marker in all effective high-flux sequence fragment of microsatellite markers is extracted, as listed in table 1
Hypothesis microsatellite markers in microsatellite marker for 3 (TG) 20,32 (TG) 21 and 5 (TG) 22 set.
Extract the left side sequence composition microsatellite of the microsatellite marker in all effective high-flux sequence fragment of microsatellite markers
The left side sequence of the left side sequence of marker site, the such as microsatellite markers of the hypothesis of table 1 is 3 (A) 2G (A) 2,5 (A)
The set of 87G (A) 3,27 (A) 86G (A), 3 and 5 (A) 81G (A) 4.Same method, obtains the right side of microsatellite markers
Lateral order is arranged, and such as the right flanks of the microsatellite marker of the hypothesis of table 1 are 3 (A) 4G (A), 80,5 (A) 3G (A) 2,27 (A)
The set of 81 and 5 2G (A) 85 of 3G (A).
The detection method of the length of the microsatellite marker in Semen Sesami microsatellite markers：
From the microsatellite markers successfully developed, microsatellite markers to be detected are selected, design amplification is to be checked
The multiplex amplification primer of the microsatellite markers of survey.Below by taking the microsatellite markers of the hypothesis in table 1 as an example, introduce such as
What selects microsatellite markers and designs multiplex amplification primer.
Design expands the method for the multiplex amplification primer of the microsatellite markers of selection to be included：From the microsatellite mark for selecting
In all effective high-flux sequence fragment in note site, microsatellite marker is extracted, most long microsatellite marker is selected,
As the microsatellite marker of multiplex amplification design of primers；In the microsatellite markers of the hypothesis of table 1, (TG) 22 is most long
Microsatellite marker, therefore, (TG) 22 is the microsatellite of the template sequence of the multiplex amplification design of primers of the microsatellite markers
Labelling.Why microsatellite locus that the microsatellite marker most grown be guarantee designed multiplex amplification primer expanded are selected
Amplification ability of the length not over multiplex PCR, so as to reduce shortage of data when microsatellite is detected.
From all effective high-flux sequence fragment of the microsatellite markers for selecting, a left side for microsatellite marker is extracted
Lateral order is arranged, and therefrom chooses all sequences of the length more than 2 bases of α, and α 2 is the second decision threshold, and (secondary high pass is measured α 2=
The length of the microsatellite markers of the average length-multiplex amplification primer of the first high-flux sequence fragment that sequence technology can be detected
Degree) ÷ 2.In the present embodiment, the average length of the first high-flux sequence fragment is 200bp, and the micro- of multiplex amplification design of primers defends
The length of asterisk note is that 44 (TG repeats 22 times, and length is 44) therefore α 2=78bp.Therefore, the left side sequence of microsatellite marker
In, the length chosen is 5 (A) 87G (A), 3,27 (A) 86G (A), 3 and 5 (A) 81G (A) 4 more than all sequences of α 2
Set.From all sequences picked out, frequency highest sequence is picked out, such as the above-mentioned length chosen is all more than α 2
In sequence, frequency highest sequence is (A) 86G (A) 3.Using frequency highest sequence as reference sequences and all of microsatellite
The left side sequence of labelling is compared, and obtains the covering multiple and variation frequency of each base in frequency highest sequence.Example
Such as, in the microsatellite markers of the hypothesis of table 1, it should carry out by reference sequences of (A) 86G (A) 3 with all of left side sequence
Compare, during comparison, the 5 ' of reference sequences (A) 86G (A) 3 the 1st base held is A, and which has been capped 5+27=32 times, due to
The position is A entirely, therefore, variation frequency is 0, and (A) the 87th base that the 5 ' of 86G (A) 3 are held is G, and which has equally been capped 5+
27=32 times, wherein, the base for G does not have 5 (in 5 (A) 81G (A) 4), thus variation frequency be 5 ÷ (5+27+5)=
0.175, by the above process, calculate the covering multiple and variation frequency for obtaining each base in (A) 86G (A) 3.By frequency most
The base of covering multiple≤1/ α 3 or variation frequency >=α 3 in high sequence is changed into after N the left side as multiplex amplification design of primers
Lateral order is arranged, wherein, N is the base of any one and the above in tetra- kinds of bases of A, T, C and G；α 3 be the 3rd decision threshold, α 3 >=5
× (accuracy of 1- the first high-flux sequence fragments), the occurrence of α 3 is according to the tight of the requirement of the versatility of multiplex amplification primer
The depth of lattice degree and high-flux sequence is adjusted, it is desirable to which versatility is stronger or high-flux sequence depth is deeper, then the value of α 3
It is less.In the present embodiment, the accuracy of high-flux sequence is 99%, therefore 3 >=5 × (1-99%)=5% of α, the present embodiment
The highly versatile of designed multiplex amplification primer is required, therefore, 3 values of α are 5%.Therefore, table 1 hypothesis microsatellite
In marker site, the covering multiple≤1/5%=20 that will go out in frequency highest sequence ((A) 86G (A) 3) or variation frequency >=
0.05 base is designated as being changed into after N the left side sequence of the template sequence as multiplex amplification primer.For (A) 86G (A) 3 the 1st
Base, which has been capped 32 times and variation frequency is 0, therefore, N is not changed into, for 3 the 86th bases of (A) 86G (A), which covers
32 times of lid multiple but variation frequency are 0.175 >=0.05, therefore, the base is changed into into N, by this rule, table 1 hypothesis it is micro-
In satellite markers site, the left side sequence of the template sequence of multiplex amplification primer is (A) 85NNN (A) 2.The high alkali of variation frequency
In base mixing sample, degree of variation is big, therefore, designed multiplex amplification primer poor universality covers the low base error of multiple
Greatly, they are all become after N, in follow-up multi-primerses design cycle, it is possible to avoid these base positions, to guarantee to set
Meter multiplex amplification primer out can be general between different samples.Traditional microsatellite marker is developed due to the limit of workload
System, the border sequence of a microsatellite marker can only often be detected once or minority several times, it is impossible to obtain and avoid primer
The change isobase in design area, it is difficult to ensure the versatility of amplimer, easily cause shortage of data.
By the identical method of left side sequence with the template sequence of multiplex amplification primer, multiplex amplification primer is obtained
The right flanks of template sequence.In the microsatellite markers of the hypothesis of table 1, the right side of the template sequence of multiplex amplification primer
Sequence is (A) 2NNN (A) 80.By the left side sequence of the template sequence of multiplex amplification primer, the template sequence of multiplex amplification primer
Microsatellite marker and the right flanks of template sequence of multiplex amplification primer be sequentially connected, obtain many of microsatellite markers
The template sequence of weight amplimer, using the template sequence of the multiplex amplification primer of microsatellite markers, obtains multiplex amplification
Primer.The template sequence of the multiplex amplification primer of the microsatellite markers of the hypothesis in table 1 is 2 (TG) 22 of (A) 85NNN (A)
(A)2NNN(A)80。
By method and parameter same as described above, obtain final choice in this enforcement 50 microsatellite markers it is many
The template sequence of weight amplimer.
First high-flux sequence fragment of the microsatellite markers of 1 one hypothesis of table
In the first high-flux sequence clip types shown in table 1, the part with underscore represents microsatellite marker, in bracket
Letter representation microsatellite marker repetitives, the number of repetition of the digitized representation repetitives after bracket.
The microsatellite mark of selection is expanded using the template sequence design of the multiplex amplification primer of all microsatellite markers
The multiplex amplification primer in note site.Concrete grammar is as follows：By the multiplex amplification primer of 50 microsatellite markers for obtaining
Template sequence is coupled together with 100 N, is built into an artificial reference gene group.Log in multiple PCR primer Photographing On-line webpage
https://ampliseq.com/, selects " DNA Hotspot designs in " Application type " option
(single-pool)”.And after selecting " Custom " in " Select the genome you wish to use " option, on
Pass and build artificial reference genome." DNA Type " option selects " Standard DNA ".In " Add Hotspot " option,
The original position and final position of each microsatellite marker in the artificial reference genome of structure are inserted, is finally clicked on
" Submit targets " button is submitted to and obtains the sequence of multiplex amplification primer.In the present embodiment, selected 50 micro- to defend
In star marker site, the microsatellite markers of successful design multiplex amplification primer are 41, this 41 microsatellite marker
Site is microsatellite markers to be detected.The present embodiment matches the multiplex PCR skill that Mo Feishier companies provide using the U.S.
Art, which can expand up to 12000 test zones simultaneously, therefore, the present invention has the ability disposably to detect 12000 microsatellites
Marker site, this is 12000 times of traditional microsatellite markers power of test.
Microsatellite marker in microsatellite markers to be detected is expanded by multiplex amplification primer, amplification is obtained and is produced
Amplified production is carried out high-flux sequence by thing, obtains the second high-flux sequence fragment.In the present embodiment, sample to be detected is
The 100 plants of Semen Sesami blades taken in kind " Jin Zhi 7 ", will obtain mixing sample after 100 plants of Semen Sesami blade mixed in equal amounts, using plant
Thing genome DNA extracting reagent kit (article No.：DP305, produces company：TIANGEN Biotech (Beijing) Co., Ltd.) by its operation
The method that handbook is provided extracts the genomic DNA for obtaining mixing sample.Using 41 pairs of designed multiplex amplification primers and library
Build test kit 2.0 (to be produced by LifeTechnology companies of the U.S., article No. is 4475345) and by the manipulator of the test kit
Volume is expanded to the genomic DNA of mixing sample, builds high-throughput sequencing library, using the high-throughput sequencing library for obtaining and
(invirtrigen companies of the U.S. produce test kit Ion PI Template OT2 200Kit v2, and article No. is 4485146) to enter
EPCR (Emulsion PCR, emulsion polymerization enzyme chain reaction) amplifications before row sequencing, manipulator of the operational approach by the test kit
Volume is carried out, and obtains ePCR products.Using ePCR products and the test kit Ion PI Sequencing 200Kit v2 (U.S.
Invirtrigen companies produce, and article No. is 4485149) to carry out high-flux sequence on the secondary high-flux sequence instrument of Proton, behaviour
Making method is carried out by the workbook of the test kit.In the present embodiment, high-flux sequence amount is set to 1M sequencing fragment (1M=
100 ten thousand), and high-flux sequence length is set to 500cycle (circulation), after sequencing terminates, obtain the second high-flux sequence product.
By analyzing the second high-flux sequence product, the length of the microsatellite marker in microsatellite markers is obtained.Tool
Body method is：After removing the microsatellite marker in the second high-flux sequence fragment, the left side of the second high-flux sequence fragment is obtained
The right border sequence of boundary's sequence and the second high-flux sequence fragment；Using left margin sequence and right border sequence by the second high flux
Each fragment in sequencing fragment is compared on microsatellite markers to be detected；Intercept each microsatellite mark to be detected
Microsatellite marker in the second high-flux sequence fragment in note site；The microsatellite marker of acquisition is classified by length, and
Calculate the validity R of i-th classificationi=Ni/Nmax, wherein, NiFor the quantity of the second high-flux sequence fragment of i-th classification,
NmaxFor the maximum of the quantity of the second high-flux sequence fragment of all categories；If validity Ri>=α 4, then i-th classification
The length of microsatellite marker is the length of the microsatellite marker in microsatellite markers, if validity Ri<α 4, then i-th class
The length of other microsatellite marker length not for the microsatellite marker in microsatellite markers, wherein, α 4 is judged for the 4th
Threshold value.The polymorphism of the microsatellite marker in microsatellite markers is due to the weight of the simple repeated sequence in microsatellite marker
Again the inconsistent length polymorphism for causing is counted, therefore, the detection of microsatellite markers is primarily referred to as detecting microsatellite marker
The length of the microsatellite marker in site.General species are diploid, if sample is homozygosis, then, it is same micro- to defend
A kind of loci of microsatellite marker should only be included in star marker site, it is if sample is heterozygosis, same micro- to defend
Star marker site has the loci of 2 different microsatellite markers.If sample is polyploid, such as Semen Tritici aestivi and Cotton Gossypii then judge
Standard should also be adjusted accordingly.When multiplex amplification is carried out, microsatellite marker amplification may be produced microsatellite markers
Slide, therefore, in the second high-flux sequence fragment, partially due to the length and mixing sample of the microsatellite marker for producing that slides
In the length of real microsatellite marker differ, so as to form interfering noise, validity RiInterfering noise can have been reacted
Power, RiValue is bigger, then disturb less.It is necessary to set the decision threshold α 4 of a validity is determining i-th kind of classification
In microsatellite marker whether necessary being.In the case where existing reference material is lacked and for homozygote, (site is only
Can have a kind of genotype) when, 4 general values of α are 0.6；If during heterozygote, then using 0.6/X as α 4 value, wherein, X
For the ploidy level of species to be detected, if such as 4 times bodies, then the value of α 4 is 0.6/4=0.15.If known slide what is produced
The size of microsatellite marker interference, then can formulate more specifically standard.For example, when known certain microsatellite markers exist
In 100 detections, there is the ratio for disturbing microsatellite marker that the slip of more than 95 times is produced to be respectively less than 0.3, then, we can be with
The value of α 4 is defined as into 0.3, then, we have 95% confidence level to ensure the microsatellite mark of i-th classification of our acquisitions
The genotype of note is necessary being.If it is noted that 4 values of α are larger, judging to violate during microsatellite marker necessary being
Wrong probability is just relatively low, but may be mistaken for not existing the microsatellite marker of part necessary being；If conversely, 4 values of α compared with
Little, then the microsatellite marker of more necessary beings will be judged out, but judge during SSR necessary beings the probability made a mistake just compared with
It is high.Therefore, in the present embodiment, the value of α 4 is one way in which, is needed according to actual needs or existing result of study
It is adjusted.In the present embodiment, determine that in default of reference material the value and sample to be tested of α 4 is diploid, be homozygosis
Body, so, 4 values of α are 0.6.As the microsatellite marker of the falseness for producing of sliding is produced with the amplification of real microsatellite marker
Thing difference in length is little, and the detection method of traditional microsatellite marker mostly is electrophoresis, it is impossible to distinguish less difference in length,
Allow to distinguish, also cannot accurate quantitative analysis, therefore, when traditional microsatellite marker is detected, it is impossible to calculate or accurately cannot count
Calculate RiValue, cause substantial amounts of inaccurate or even wrong conclusion.
Assume again that table 1 is the microsatellite markers for detecting below, treat in illustrating how to detect mixing sample
The microsatellite markers of detection.In second high-flux sequence fragment of the microsatellite markers assumed in table 1, intercepting
Set of the microsatellite marker for 3 (TG) 20,32 (TG) 21 and 5 (TG) 22, the microsatellite marker for intercepting is single by repeating
Unit's classification, is TG, retains the microsatellite marker of frequency of occurrences highest repetitives, and they are 3 (TG) 20,32 (TG)
The set of 21 and 5 (TG) 22；The microsatellite marker for remaining further is classified by length, 3 classifications are obtained altogether,
Respectively (TG) 20, (TG) 21 and (TG) 22.In this 3 classifications, occupy the quantity of the second most high-flux sequence fragments
Classification be the 2nd classification (TG) 21, i.e. Nmax=N2=32.The second high-flux sequence fragment that 1st classification (TG) 20 is occupied
Quantity be 3, i.e. N1=3, then, R1=3/32<α 4=0.6, therefore, judge that the 1st classification (TG) 20 is not true
Exist, caused by slip.Equally, calculate R2=1, R3=5/32, according to same standard, judge, the 2nd classification is
Necessary being, the 3rd classification is not necessary being.Therefore, in microsatellite markers to be detected in mixing sample
Length of the length of microsatellite marker for the microsatellite marker of classification 2, i.e., the microsatellite markers to be detected for assuming in table 1
The length of interior microsatellite marker is 42bp (TG repeats 21 times, therefore its length is 21 × 2bp=42bp).
By being detected with identical method in the embodiment of above-mentioned hypothesis and parameter again, the present embodiment is successfully have detected
In, the length of the microsatellite marker in 41 microsatellite markers to be detected.
The development approach of microsatellite markers provided in an embodiment of the present invention is quick with detection method, simple, comprehensive, accurate
Really.The development approach of traditional microsatellite markers, as workload is big, can only find about 1% or so in genome
Microsatellite markers, also can only verify the polymorphism of microsatellite markers in less than 100 samples.For the present invention comes
Say, in theory it can be found that all microsatellite markers on genome, in the embodiment developed for the microsatellite of Semen Sesami, send out
Show more than 10,000 microsatellite markers, substantially the 50% of all microsatellite markers of Semen Sesami, therefore, in microsatellite mark
On the ability of discovery in note site, 50 times are improve, if increasing high-throughout sequencing amount (this is easy to accomplish), can be with
The ability of discovery of microsatellite markers is brought up to into 80 times or even 100 times are close to, is all easier to realize.It is of the invention real
Apply example be by the exploitation of microsatellite markers (it was found that) unite two into one with polymorphic detection, do not pay extra work,
But for the polymorphic detection work of traditional microsatellite markers, take and be difficult to, such as in 20 sesames
The polymorphism of 13001 microsatellite markers is detected in numb kind, equivalent to 20*13001=being in traditional detection
260020 PCR amplifications and electrophoresis, this workload is inconceivable.In addition, traditional microsatellite markers
Development technique is big due to workload, has no ability to detect multiple sequences of same microsatellite markers, so, it is impossible to analyze
The conservative of multiplex amplification primer, causes the poor universality of the multiplex amplification primer of the microsatellite marker for developing, and this
Bright embodiment solves this problem.With the detection of the length of the microsatellite marker in the Semen Sesami microsatellite markers of the present invention
In method as a example by 41 microsatellite markers of one-time detection, for traditional detection method, then 41 PCR are needed to expand
Increase and electrophoresis.For the present invention, even 10,000 microsatellite markers of detection, its workload will not also increase, but right
For traditional detection method, workload then increased 10,000 times.Traditional detection method is to judge microsatellite mark by electrophoresis
The length of note, but there is error in electrophoresis, accordingly, it would be desirable to Reference cultivars are contrasted, so as to increased the workload of detection,
And, seldom there is laboratory there be the Reference cultivars of complete set, and the embodiment of the present invention uses high-flux sequence, obtains
What is obtained is base sequence, as acquired results are absolute values, so no error, therefore, it is no longer necessary to Reference cultivars.Additionally,
Electrophoresis detection cannot differentiate different individual plants, and such as, sample of the present invention in Semen Sesami detection is the mixing of 100 individual plants, in electricity
In swimming result, it is impossible to accurately calculate the ratio of the different microsatellite markers of same microsatellite markers, therefore, it is impossible to differentiate
Individual plant, so as to the important indicators such as hybrid strain rate cannot be calculated.
The foregoing is only presently preferred embodiments of the present invention, not to limit the present invention, all spirit in the present invention and
Within principle, any modification, equivalent substitution and improvements made etc. should be included within the scope of the present invention.
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<120>The length detection of the microsatellite marker in Semen Sesami microsatellite markers development approach and microsatellite markers
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Claims (10)
1. a kind of Semen Sesami microsatellite markers development approach, it is characterised in that the development approach includes：
The mass mixings such as the Semen Sesami sample for having polymorphism by n, obtain mixing sample, wherein n ＞ 1；
Extract the genome of the mixing sample；
By the genomic fragment of the mixing sample, genomic fragment is obtained；
Using multiple probes with simple repeated sequence as probe groups, using each probe in the probe groups respectively with institute
State genomic fragment to be hybridized, obtain multiple hybridization solutions, the gene pack to successful cross in multiple hybridization solutions
Section carries out purification respectively, obtains the hybrid gene pack section of multiple purification；
It is after the mass mixings such as the hybrid gene pack section by multiple purification, mixed described using high-flux sequence detection
The hybrid gene pack section of purification, obtains the first high-flux sequence fragment；
From the first high-flux sequence fragment, the effective high-flux sequence fragment of screening, the effective high flux
Sequencing fragment includes the microsatellite marker in microsatellite markers；
According to the homology of the both sides sequence of the microsatellite marker in the effective high-flux sequence fragment to it is described effectively
High-flux sequence fragment is classified, and the of a sort effective high-flux sequence fragment is same microsatellite markers
Described effective high-flux sequence fragment, if the described effective high-flux sequence piece of the same microsatellite markers
Bar number >=the α 1 of section, then successfully develop the microsatellite markers, wherein, α 1 is the first decision threshold and 1 >=(height of α
The microsatellite markers number that can be detected on the ratio/genome of logical sequencing depth × effective high-flux sequence fragment) ×
Probability ensures.
2. development approach according to claim 1, it is characterised in that α 1 >=20.
3. development approach according to claim 1, it is characterised in that described in the effective high-flux sequence fragment
The base number of the both sides sequence of microsatellite marker >=1, and the effective high-flux sequence fragment in the microsatellite
At least base number >=10 of the sequence of side in labelling.
4. development approach according to claim 1, it is characterised in that select the described n Semen Sesami sample with polymorphism
Method include：The different Semen Sesami sample of the different Semen Sesami sample of selection formalness, biological classification, the mutually different sesame of labelling
The Semen Sesami sample of the wild resource in numb sample or different ecological region.
5. development approach according to claim 1, it is characterised in that the quantity of the probe is 12, each described spy
Repetitives in the simple repeated sequence of pin be CT, GA, TG, AC, TA, TGT, CCA, ATC, CCT, AGA, ATG or CAA, often
The number of repetition of the simple repeated sequence of the individual probe is 6～20；Preferably, the simple repeated sequence of each probe
Number of repetition is 6～15.
6. development approach according to claim 1, it is characterised in that SEQ IN in the sequence of the probe such as sequence table
NO:1-SEQ IN NO:Shown in 12.
7. the microsatellite mark in microsatellite markers that the development approach described in a kind of any one of claim 1-6 is successfully developed
The length detecting method of note, it is characterised in that the detection method includes：
From the microsatellite markers successfully developed, microsatellite markers to be detected are selected；
The microsatellite marker in the microsatellite markers to be detected is expanded using multiplex amplification primer, amplification is obtained and is produced
The amplified production is carried out high-flux sequence by thing, obtains the second high-flux sequence fragment, by analyzing second high flux
Sequencing fragment, obtains the length of the microsatellite marker in the microsatellite markers.
8. detection method according to claim 7, it is characterised in that described from the microsatellite marker position successfully developed
In point, the method for microsatellite markers to be detected is selected to include：
The standard for selecting the microsatellite markers to be detected is the maximum microsatellite markers of H-number, wherein, H
It is worth the polymorphic sex index for the microsatellite markers,Wherein, i is by the microsatellite markers
Described effective high-flux sequence fragment in the length of microsatellite marker when being classified, i-th classification, i are natural number；
Ai is that the number of the effective described high-flux sequence fragment of i-th classification accounts for total effective described high-flux sequence fragment
The ratio of number.
9. detection method according to claim 8, it is characterised in that obtain described micro- in the microsatellite markers
The method of the length of satellite markers is：After removing the microsatellite marker in the second high-flux sequence fragment, institute is obtained
State the right border sequence of the left margin sequence and the second high-flux sequence fragment of the second high-flux sequence fragment；Using described
Each fragment in the second high-flux sequence fragment is compared described to be checked by left margin sequence and the right border sequence
On the microsatellite markers of survey；Intercept second high-flux sequence of each microsatellite markers to be detected
The microsatellite marker in fragment；The microsatellite marker for obtaining is classified by length, and is calculated i-th classification
Validity Ri=Ni/Nmax, wherein, i is micro- in the described effective high-flux sequence fragment by the microsatellite markers
When the length of satellite markers is classified, i-th classification, NiFor the second high-flux sequence fragment of i-th classification
Quantity, NmaxFor the maximum of the quantity of the second high-flux sequence fragment of all categories；If the validity Ri≥α
4, then the length of the microsatellite marker of i-th classification is the microsatellite marker in the microsatellite markers
Length, if the validity Ri<α 4, then the length of the microsatellite marker of i-th classification is not the microsatellite marker
The length of the microsatellite marker in site, wherein, α 4 is the 4th decision threshold and α 4=0.6.
10. detection method according to claim 8, it is characterised in that the method for preparing the multiplex amplification primer includes：
From all described effective high-flux sequence fragment of the microsatellite markers described to be detected for selecting, institute is extracted
State microsatellite marker and the most long microsatellite marker is picked out as the microsatellite mark of the template sequence of multiplex amplification primer
Note；
From all described effective high-flux sequence fragment of the microsatellite markers described to be detected for selecting, institute is extracted
State the left side sequence of microsatellite marker and choose all sequences of the length more than 2 bases of α, from all sequences picked out
In, pick out frequency highest sequence, using the frequency highest sequence as reference sequences, by the reference sequences with it is all
The left side sequence of the microsatellite marker compare, the covering of each base is obtained in the frequency highest sequence
Multiple and variation frequency；In the frequency highest sequence, by the covering multiple≤1/ α 3 or described variation frequencies >=α 3
Base be changed into after N the left side sequence of the template sequence as the multiplex amplification primer, wherein, N is tetra- kinds of alkali of A, T, C and G
The base of any one and the above in base；α 2 be the second decision threshold, α 2=(the average length of the first high-flux sequence fragment
The length of the microsatellite markers of the degree-multiplex amplification primer) ÷ 2；α 3 is the 3rd decision threshold, α 3 >=5 × (described in 1-
The accuracy of the first high-flux sequence fragment)；
According to the left side sequence identical method with the template sequence of the multiplex amplification primer, multiplex amplification primer sequence is obtained
Template sequence right flanks；
By the left side sequence of the template sequence of the multiplex amplification primer, the microsatellite of the template sequence of the multiplex amplification primer
The right flanks of the template sequence of labelling and the multiplex amplification primer are sequentially connected, and obtain many of the microsatellite markers
The template sequence of weight amplimer, using the template sequence of the multiplex amplification primer of the microsatellite markers, obtains described
Multiplex amplification primer.
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| CN103667480A (en) * | 2013-12-06 | 2014-03-26 | 中国农业科学院油料作物研究所 | SSR core primer group developed based on sesame complete genomic sequence and application |
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| Title |
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| 柯涛 等: "芝麻EST-SSR引物的开发与应用", 《中国油料作物学报》 * |
| 魏利斌 等: "芝麻EST-SSR标记的开发和初步研究", 《作物学报》 * |
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