CN106676176A - Method for performing SSR analysis on tetraploid alfalfa by utilizing multiple PCR - Google Patents

Abstract

The invention relates to a method for performing SSR analysis on tetraploid alfalfa by utilizing multiple PCR. The method specifically comprises nine multiple PCR-SSR groups (24 pairs of SSR primers) and corresponding PCR systems and reaction programs. 24 SSR markers are uniformly distributed on chromosomes, the distance between the markers being not smaller than 10Mbp; marker genotypes are easy to identify, and the average genotype accuracy rate is higher than 95%. When the solution is adopted to perform genetic diversity analysis, group structure analysis and variety identification on the tetraploid alfalfa, the method has the advantages of short time, low cost and high accuracy, and the total efficiency can be improved by two to three times.

Description

A kind of method that utilization multiplex PCR carries out ssr analysis to tetraploid alfalfa

Technical field

The method that multiplex PCR carries out ssr analysis to tetraploid alfalfa is the present invention relates to the use of, belongs to biotechnology neck Domain.

Background technology

Herba Medicaginiss (Medicago sativa) are the most important forage crops in global Temperate Region in China, the subspecies of two main cultivations (M.sativa subssp.sativa and M.sativa subssp. × varia) is autotetraploid, natural outcrossing, inbreeding Decline is serious.Genetic diversity is the basis of plant breeding and genetic improvement.Because most alfalfa cultivars are synthesis Kind, is bred as by the fine individual plant and its filial generation continuous random copulation several generations that select.Therefore, analysis of genetic diversity is being carried out When, 20~40 individual plants need to be typically taken in an alfalfa variety.When studying kind and being more, unit of analysis (genotype) is often resulted in Quantity it is very big.Due to the restriction of the factors such as actual workload, funds, time, such project is often difficult to pass through It is thorough.The number of the unit of analysis (genotype) in existing correlational study is usually no more than 1000.

So far, existing different kinds of molecules labelling technique is used in the research of Genetic Diversity of Alfalfa, such as allozyme, kind Sub- storage protein, RFLP, RAPD, SSR, ISSR, EST-SSR etc..Wherein, SSR (Simple sequence repeat, simply Sequence repeats；Also known as Microsatellite DNA, microsatellite DNA) be most widely used, it is more that main cause is that it has Advantage：Codominant inheritance, can be used to differentiate heterozygote and homozygote；Marker chromosome positioning is clear and definite, and quantity is enriched；Using spy Specific primer, simple to operate, reproducible, reliability is high；It is not high to the consumption and purity requirement of DNA.For developing foot For the species of enough SSR markers, its major defect is that test every time is typically only capable of analyzing a SSR marker, obtains a site Genotype information.The scale of test is which greatly limits, for outcrossing plant --- especially it is such for Herba Medicaginiss. Existing genetic diversity Journal of Sex Research mostly using less SSR marker (＜ 50) or (and) the less genotype (＜ of analysis 1000).Additionally, also there is null genotype (being difficult to determine without band or allele dosage) ratio in some SSR markers developed The problems such as rate height is difficult interpretation with collection of illustrative plates, this also limits to a certain extent SSR answering in Genetic Diversity of Alfalfa research With.Therefore, excellent SSR marker is screened, it is to obtain accurate test data, improve test effect to set up high-throughout analysis method The premise of rate.

Multiplex PCR (Multiplex PCR) technology was proposed by Chamberlain equal to 1998, in a PCR reactant Multipair specific primer is added in system, for the zones of different of multiple DNA profilings or same template multiple purpose fragments are expanded Round pcr, be the improvement to regular-PCR technology.Compared with the PCR of term single gene molecular marker, multiplex PCR is in a PCR In can simultaneously detect several target genes, work efficiency is significantly improved, and cost is substantially reduced.Due to multiple PCR technique have with Upper advantage, has been widely used at present the every field of scientific research, such as Viral diagnosis, Bacteria Detection, bird flu detection with And many aspects such as quality trait molecular marker.

At present, research of the multiplex PCR-SSR technologies on plant is deep not enough, and research contents has focused largely on primer Screening, the foundation of multiplex PCR system and optimization；Only have more deep on a few plant such as Radix Betae, Cotton Gossypii, Fructus Myricae rubrae, Fructus Capsici The research for entering.But existing research proves that the method is remarkably improved the quantity of information of single ssr analysis, and reduction is drawn using SSR Thing carries out cost during large scale analysis, the time required to shortening test.At present, there is not yet relevant utilize multiplex PCR-SSR to four The method that times body alfalfa carries out analysis of genetic diversity.This patent is intended to screen excellent Herba Medicaginiss SSR marker, and sets up base In the method for high-flux analysis of multiplex PCR.

The content of the invention

The purpose of the present invention is for the deficiencies in the prior art, there is provided one kind is pale reddish brown to tetraploid using multiple PCR technique The method that Herba Medicaginiss carry out ssr analysis.

Technical solution of the present invention is summarized as follows：24 pairs of SSR primers are filtered out first, by optimum organization into 9 PCR groups； Then add in a PCR reaction tube and belong to the different 2-3 of a group but concentration together to SSR specific primers pair, using different Reaction system amplifies multiple purpose fragments to the zones of different of same template simultaneously；Difference is drawn finally by an electrophoresis Multiple amplified productions of thing are separated.

Specifically, technical solution of the present invention is comprised the following steps：

1st, 24 pairs of SSR primers are filtered out

According to existing documents and materials, the amplification condition of PCR reactions, primer distribution feelings on chromosome are considered The factors such as condition, the size of band, definition and polymorphism, the accuracy rate and stability of genotype, filter out 24 pairs of SSR primers. Following table is the SSR primer SEQ ID NO for filtering out：1~SEQ ID NO：48 (wherein SEQ ID NO：1/SEQ ID NO：2 are A pair forward and reverse primer pairs, sequentially analogize according to this).

Table 1 is applied to the SSR primers of alfalfa multiplex PCR

2nd, multiplex PCR

According to the respective feature of above-mentioned primer, optimum organization is into 9 PCR groups.Add in a PCR reaction tube and belong to one together The 2-3 of group constitutes multiple differential responses systems to SSR specific primers pair for same DNA profiling, and according to same PCR Amplification program completes multiplex PCR.The packet of multi-PRC reaction and primer concentration are shown in Table 2.

The packet of the multiplex PCR of table 2

Note：Primer concentration refers both to the respective concentration of forward and reverse primer

Further, the reaction system includes following components：Template DNA 1.0~2.5ng/ μ L, Taq archaeal dna polymerases 0.03~0.10U/ μ L, 0.20~0.35mM of dNTPs, 1 × PCR Buffer (MgCl2 containing 1.5mM), primer concentration 0.085 ~0.285 μM, deionized water is mended to 15 μ L.

Further, the PCR amplification programs are comprised the following steps：

(1) 24 pairs of primers in table 1 are synthesized；

(2) 9 PCR groups are set according to table 2, and enter performing PCR amplification as follows：

94 DEG C of denaturations 4min；

94 DEG C of degeneration 30s, 56 DEG C of annealing 45s, 72 DEG C of extension 1min, totally 34 circulations；

72 DEG C of extension 8min, 4 DEG C of preservations.

3rd, electrophoresis detection

Amplified production is detected using conventional denaturing polyacrylamide gel electrophoresis.

The advantages of the present invention are：

1) reacted by 9 PCR, obtained the spectrum data of 24 SSR markers.During analysis, template DNA, Taq The consumption of the medicines such as archaeal dna polymerase, dNTPs, PCR reaction tube and consumptive material is averagely reduced to the 37.5% (9/24) of single primer PCR, from Perform PCR operations to start to the time of electrophoresis sample-adding also to shorten to the 37.5% (9/24) of single primer PCR.

2) accuracy：The genotype of labelling is readily discernible, reads data fast；Selected SSR marker is divided on chromosome Cloth is uniform, and average gene type accuracy rate is higher than 95%, the reliable results of analysis of genetic diversity.

Description of the drawings

Fig. 1：The AFLP system of the WL323 Herba Medicaginiss individual plants of primer pair 12 of A groups 3 pairs；Wherein：M is Marker, and 1-12 is difference The amplification of Herba Medicaginiss individual plant.

Fig. 2：The AFLP system of the Algonquin Herba Medicaginiss individual plant of primer pair 12 of H groups 2 pairs；Wherein：M is Marker, and 1-12 is The amplification of Different Alfalfa individual plant.

Fig. 3：The AFLP system of No. 1 Herba Medicaginiss individual plant of middle lucerne of primer pair 12 of D groups 3 pairs；Wherein：M is Marker, and 1-12 is not for With the amplification of Herba Medicaginiss individual plant.

Fig. 4：The AFLP system of the pastoral song Herba Medicaginiss individual plant of primer pair 12 of B groups 3 pairs；Wherein：M is Marker, and 1-12 is difference The amplification of Herba Medicaginiss individual plant.

Fig. 5：The primer pair 12 three of C groups 3 pairs is got profit the AFLP system of Herba Medicaginiss individual plant；Wherein：M is Marker, and 1-12 is not for With the amplification of Herba Medicaginiss individual plant.

Fig. 6：The AFLP system of Victoria's Herba Medicaginiss individual plant of primer pair 12 of E groups 3 pairs；Wherein：M is Marker, and 1-12 is The amplification of Different Alfalfa individual plant.

Fig. 7：The AFLP system of the reinder Herba Medicaginiss individual plant of primer pair 12 of F groups 3 pairs；Wherein：M is Marker, and 1-12 is difference The amplification of Herba Medicaginiss individual plant.

Fig. 8：The AFLP system of the primer pair 12 of G groups 2 pairs Baoding Herba Medicaginiss individual plant；Wherein：M is Marker, and 1-12 is difference The amplification of Herba Medicaginiss individual plant.

Fig. 9：The AFLP system of I groups 2 pairs of primer pairs, 12 Longdong alfalfa individual plants；Wherein：M is Marker, and 1-12 is difference The amplification of Herba Medicaginiss individual plant.

Specific embodiment

Below by the specific embodiment narration present invention.Unless stated otherwise, technological means used in the present invention It is method known in those skilled in the art.In addition, embodiment be interpreted as it is illustrative, and it is unrestricted the present invention Scope, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this On the premise of invention spirit and scope, various changes that the material component and consumption in these embodiments is carried out or change Belong to protection scope of the present invention.

The alfalfa variety selected in following examples is interpreted as exemplary, and the inventive method is for the institute being currently known There are alfalfa cultivars to be suitable for.

Embodiment 1：

1st, the extraction of genomic DNA and primer synthesize

With alfalfa cultivars WL323 as test material, 12 individual plants, the enough young leaflet tablets of clip, liquid nitrogen are randomly selected After freeze grinding, genomic DNA is extracted using the CTAB methods of Doyle et al., using λ DNA as reference standard, use 0.8% agar Sugared gel carries out electrophoresis detection to the quality and quantity of genomic DNA.

According to primer sequence synthesis A group primer BBI131F, GBG230 and E776153 in table 1.

2nd, multiplex PCR

(1) reaction system includes following components：

Template DNA 1.5ng/ μ L, Taq archaeal dna polymerase 0.035U/ μ L, dNTPs 0.25mM, 1 × PCR Buffer (contain 1.5mM MgCl2), the concentration of primer BBI131F, GBG230 and E776153 is respectively 0.145 μM, 0.170 μM and 0.135 μM (forward and reverse primer concentration is identical), deionized water is mended to 15 μ L.

(2) performing PCR amplification is entered as follows：

94 DEG C of denaturations 4min；

94 DEG C of degeneration 30s, 56 DEG C of annealing 45s, 72 DEG C of extension 1min, totally 34 circulations；

72 DEG C of extension 8min, 4 DEG C of preservations.

3rd, electrophoresis detection

Detected using conventional denaturing polyacrylamide gel electrophoresis.Fig. 1 be 3 pairs of primers of A groups (BBI131, GBG230 and E776153) AFLP system.As can be seen from Figure 1 the amplified production of 3 pairs of primers is clear, polymorphic site enriches, phase It is easily discriminated between mutually.By taking a3 as an example, 4 bands are had from top to bottom, equivalent to 4 allele, be respectively designated as a, b, C, d, then the genotype of 12 samples be followed successively by：bccc、aadd、abcd、cccd、bbdd、aaab、abbd、abcd、accd、 addd、cccc、bccd。

Embodiment 2：

1st, the extraction of genomic DNA and primer synthesize

With alfalfa cultivars Algonquin as test material, 12 individual plants, the enough young leaflet tablets of clip, liquid are randomly selected After chilled nitrogen grinding, genomic DNA is extracted using the CTAB methods of Doyle et al., using λ DNA as reference standard, use 0.8% fine jade Sepharose carries out electrophoresis detection to the quality and quantity of genomic DNA.

According to primer sequence synthesis H group primer CBF96 and GAW212 in table 1.

2nd, multiplex PCR

(1) reaction system includes following components：

Template DNA 1.5ng/ μ L, Taq archaeal dna polymerase 0.035U/ μ L, dNTPs 0.25mM, 1 × PCR Buffer (contain 1.5mM MgCl2), primer CBF96 and GAW212 be respectively 0.160 μM with 0.140 μM (forward and reverse primer concentration is identical), go from Sub- water is mended to 15 μ L.

(2) performing PCR amplification is entered as follows：

94 DEG C of denaturations 4min；

94 DEG C of degeneration 30s, 56 DEG C of annealing 45s, 72 DEG C of extension 1min, totally 34 circulations；

72 DEG C of extension 8min, 4 DEG C of preservations

3rd, electrophoresis detection

Detected using conventional denaturing polyacrylamide gel electrophoresis.Fig. 2 is H group primer CBF96 and GAW212 AFLP system.As can be seen from Figure 2 the amplified production of these two pair primer is clear, polymorphic site enriches, and area is easy to each other Point.By taking h1 as an example, 6 bands are had from top to bottom, equivalent to 6 allele, be respectively designated as a, b, c, d, e, f, then 12 The genotype of sample is followed successively by：ccce、abee、bbee、eeee、eeef、adee、abde、ddee、aabd、acde、abee、 bbdd。

Embodiment 3：

1st, the extraction of genomic DNA and primer synthesize

Lucerne 1 randomly selects 12 individual plants, the enough young leaflet tablets of clip, liquid as test material with alfalfa cultivars After chilled nitrogen grinding, genomic DNA is extracted using the CTAB methods of Doyle et al., using λ DNA as reference standard, use 0.8% fine jade Sepharose carries out electrophoresis detection to the quality and quantity of genomic DNA.

According to primer sequence synthesis D group primer GBF56, CAW306 and BBG28 in table 1.

2nd, multiplex PCR

(1) reaction system includes following components：

Template DNA 1.5ng/ μ L, Taq archaeal dna polymerase 0.035U/ μ L, dNTPs 0.25mM, 1 × PCR Buffer (contain 1.5mM MgCl2), primer GBF56, CAW306 and BBG28 are respectively 0.285 μM, 0.115 μM and 0.125 μM (forward and reverse primer Concentration is identical), deionized water is mended to 15 μ L.

(2) performing PCR amplification is entered as follows：

94 DEG C of denaturations 4min；

94 DEG C of degeneration 30s, 56 DEG C of annealing 45s, 72 DEG C of extension 1min, totally 34 circulations；

72 DEG C of extension 8min, 4 DEG C of preservations

3rd, electrophoresis detection

Detected using conventional denaturing polyacrylamide gel electrophoresis.Fig. 3 is D groups 3 couples of primers GBF56, CAW306 With the AFLP system of BBG28.As can be seen from the figure the amplified production of this 3 pairs of primers is clear, polymorphic site enriches, each other It is easily discriminated.By taking d2 as an example, 5 bands are had from top to bottom, equivalent to 5 allele, be respectively designated as a, b, c, d, e, Then the genotype of 12 samples is followed successively by：bcde、acde、bcde、ccdd、abce、dddd、ccde、abbd、cddd、acdd、 bdde、bcde。

Embodiment 4：

1st, the extraction of genomic DNA and primer synthesize

With alfalfa cultivars pastoral song as test material, 12 individual plants are randomly selected, the enough young leaflet tablets of clip, liquid nitrogen is cold After freezing grinding, genomic DNA is extracted using the CTAB methods of Doyle et al., using λ DNA as reference standard, use 0.8% agarose Gel carries out electrophoresis detection to the quality and quantity of genomic DNA.

According to primer sequence synthesis B group primer DMt1H10, CBF156 and DBI28 in table 1.

2nd, multiplex PCR

(1) reaction system includes following components：

Template DNA 1.5ng/ μ L, Taq archaeal dna polymerase 0.035U/ μ L, dNTPs 0.25mM, 1 × PCR Buffer (contain 1.5mM MgCl2), primer DMt1H10, CBF156 and DBI28 be respectively 0.225 μM, 0.105 μM and 0.120 μM it is (forward and reverse to draw Thing concentration is identical), deionized water is mended to 15 μ L.

(2) performing PCR amplification is entered as follows：

94 DEG C of denaturations 4min；

94 DEG C of degeneration 30s, 56 DEG C of annealing 45s, 72 DEG C of extension 1min, totally 34 circulations；

72 DEG C of extension 8min, 4 DEG C of preservations

3rd, electrophoresis detection

Detected using conventional denaturing polyacrylamide gel electrophoresis.Fig. 4 be 3 couples of primer DMt1H10 of B groups, The AFLP system of CBF156 and DBI28.As can be seen from the figure the amplified production of this 3 pairs of primers is clear, polymorphic site enriches, phase It is easily discriminated between mutually.By taking b3 as an example, 8 bands are had from top to bottom, equivalent to 8 allele, be respectively designated as a, b, C, d, e, f, g, h, then the genotype of 12 samples be followed successively by：dggg、eeef、eefg、abgh、ccee、ddef、efhh、adde、 aabg、eeeh、deeh、bdee。

Embodiment 5：

1st, the extraction of genomic DNA and primer synthesize

Got profit as test material with alfalfa cultivars three, randomly select 12 individual plants, the enough young leaflet tablets of clip, liquid nitrogen After freeze grinding, genomic DNA is extracted using the CTAB methods of Doyle et al., using λ DNA as reference standard, use 0.8% agar Sugared gel carries out electrophoresis detection to the quality and quantity of genomic DNA.

According to primer sequence synthesis C group primer ABE93, CIC338 and AMTIC95 in table 1.

2nd, multiplex PCR

(1) reaction system includes following components：

Template DNA 1.5ng/ μ L, Taq archaeal dna polymerase 0.035U/ μ L, dNTPs 0.25mM, 1 × PCR Buffer (contain 1.5mM MgCl2), primer ABE93, CIC338 and AMTIC95 be respectively 0.235 μM, 0.150 μM and 0.140 μM it is (forward and reverse to draw Thing concentration is identical), deionized water is mended to 15 μ L.

(2) performing PCR amplification is entered as follows：

94 DEG C of denaturations 4min；

94 DEG C of degeneration 30s, 56 DEG C of annealing 45s, 72 DEG C of extension 1min, totally 34 circulations；

72 DEG C of extension 8min, 4 DEG C of preservations

3rd, electrophoresis detection

Detected using conventional denaturing polyacrylamide gel electrophoresis.Fig. 5 is C groups 3 couples of primers ABE93, CIC338 With the AFLP system of AMTIC95.As can be seen from the figure the amplified production of this 3 pairs of primers is clear, polymorphic site enriches, mutually it Between be easily discriminated.By taking c3 as an example, 5 bands are had from top to bottom, equivalent to 5 allele, be respectively designated as a, b, c, d, E, then the genotype of 12 samples be followed successively by：beee、aace、aaae、bdde、bcce、bbbe、abee、bccc、ccee、abcd、 bcee、ccce。

Embodiment 6：

1st, the extraction of genomic DNA and primer synthesize

With alfalfa cultivars Victoria as test material, 12 individual plants, the enough young leaflet tablets of clip, liquid are randomly selected After chilled nitrogen grinding, genomic DNA is extracted using the CTAB methods of Doyle et al., using λ DNA as reference standard, use 0.8% fine jade Sepharose carries out electrophoresis detection to the quality and quantity of genomic DNA.

According to primer sequence synthesis E group primer HAW178, AAW365 and HAL82 in table 1.

2nd, multiplex PCR

(1) reaction system includes following components：

Template DNA 1.5ng/ μ L, Taq archaeal dna polymerase 0.035U/ μ L, dNTPs 0.25mM, 1 × PCR Buffer (contain 1.5mM MgCl2), primer HAW178, AAW365 and HAL82 be respectively 0.205 μM, 0.100 μM and 0.145 μM it is (forward and reverse to draw Thing concentration is identical), deionized water is mended to 15 μ L.

(2) performing PCR amplification is entered as follows：

94 DEG C of denaturations 4min；

94 DEG C of degeneration 30s, 56 DEG C of annealing 45s, 72 DEG C of extension 1min, totally 34 circulations；

72 DEG C of extension 8min, 4 DEG C of preservations

3rd, electrophoresis detection

Detected using conventional denaturing polyacrylamide gel electrophoresis.Fig. 6 is E groups 3 couples of primers HAW178, AAW365 With the AFLP system of HAL82.As can be seen from the figure the amplified production of this 3 pairs of primers is clear, polymorphic site enriches, each other It is easily discriminated.By taking e1 as an example, 7 bands are had from top to bottom, equivalent to 7 allele, be respectively designated as a, b, c, d, e, F, g, then the genotype of 12 samples be followed successively by：gggg、fggg、fggg、cfgg、bggg、dfgg、aefg、bdff、bdfg、 ffgg、bdgg、bggg。

Embodiment 7：

1st, the extraction of genomic DNA and primer synthesize

With alfalfa cultivars reinder as test material, 12 individual plants are randomly selected, the enough young leaflet tablets of clip, liquid nitrogen is cold After freezing grinding, genomic DNA is extracted using the CTAB methods of Doyle et al., using λ DNA as reference standard, use 0.8% agarose Gel carries out electrophoresis detection to the quality and quantity of genomic DNA.

According to primer sequence synthesis F group primer DAW289, DBE84 and BBG280 in table 1.

2nd, multiplex PCR

(1) reaction system includes following components：

Template DNA 1.5ng/ μ L, Taq archaeal dna polymerase 0.035U/ μ L, dNTPs 0.25mM, 1 × PCR Buffer (contain 1.5mM MgCl2), primer DAW289, DBE84 and BBG280 are respectively 0.160 μM, 0.205 μM and 0.160 μM forward and reverse primer Concentration is identical), deionized water is mended to 15 μ L.

(2) performing PCR amplification is entered as follows：

94 DEG C of denaturations 4min；

94 DEG C of degeneration 30s, 56 DEG C of annealing 45s, 72 DEG C of extension 1min, totally 34 circulations；

72 DEG C of extension 8min, 4 DEG C of preservations

3rd, electrophoresis detection

Detected using conventional denaturing polyacrylamide gel electrophoresis.Fig. 7 is F groups 3 couples of primers DAW289, DBE84 With the AFLP system of BBG280.As can be seen from the figure the amplified production of this 3 pairs of primers is clear, polymorphic site enriches, mutually it Between be easily discriminated.By taking f2 as an example, 5 bands are had from top to bottom, equivalent to 5 allele, be respectively designated as a, b, c, d, E, then the genotype of 12 samples be followed successively by：abbd、aabe、aaae、aaaa、aaad、ccce、accc、aaaa、aaac、aadd、 bbcc、aabc。

Embodiment 8：

1st, the extraction of genomic DNA and primer synthesize

With alfalfa cultivars Baoding as test material, 12 individual plants are randomly selected, the enough young leaflet tablets of clip, liquid nitrogen is cold After freezing grinding, genomic DNA is extracted using the CTAB methods of Doyle et al., using λ DNA as reference standard, use 0.8% agarose Gel carries out electrophoresis detection to the quality and quantity of genomic DNA.

According to primer sequence synthesis G group primer HMt1G03 and FAW115 in table 1.

2nd, multiplex PCR

(1) reaction system includes following components：

Template DNA 1.5ng/ μ L, Taq archaeal dna polymerase 0.035U/ μ L, dNTPs 0.25mM, 1 × PCR Buffer (contain 1.5mM MgCl2), primer HMt1G03 and FAW115 be respectively 0.195 μM with 0.105 μM (forward and reverse primer concentration is identical), go Ionized water is mended to 15 μ L.

(2) performing PCR amplification is entered as follows：

94 DEG C of denaturations 4min；

94 DEG C of degeneration 30s, 56 DEG C of annealing 45s, 72 DEG C of extension 1min, totally 34 circulations；

72 DEG C of extension 8min, 4 DEG C of preservations

3rd, electrophoresis detection

Detected using conventional denaturing polyacrylamide gel electrophoresis.Fig. 8 be 2 couples of primer HMt1G03 of G groups with The AFLP system of FAW115.As can be seen from the figure the amplified production of this 3 pairs of primers is clear, polymorphic site enriches, each other It is easily discriminated.By taking g2 as an example, 7 bands are had from top to bottom, equivalent to 7 allele, be respectively designated as a, b, c, d, e, F, g, then the genotype of 12 samples be followed successively by：ddfg、ccgg、accc、bddd、aade、cccd、cceg、abbd、addd、 ddfg、adee、dddd。

Embodiment 9：

1st, the extraction of genomic DNA and primer synthesize

With alfalfa cultivars Long Dong as test material, 12 individual plants are randomly selected, the enough young leaflet tablets of clip, liquid nitrogen is cold After freezing grinding, genomic DNA is extracted using the CTAB methods of Doyle et al., using λ DNA as reference standard, use 0.8% agarose Gel carries out electrophoresis detection to the quality and quantity of genomic DNA.

According to primer sequence synthesis I group primer GBG288 and BBG238 in table 1.

2nd, multiplex PCR

(1) reaction system includes following components：

Template DNA 1.5ng/ μ L, Taq archaeal dna polymerase 0.035U/ μ L, dNTPs 0.25mM, 1 × PCR Buffer (contain 1.5mM MgCl2), primer GBG288 and BBG238 be respectively 0.085 μM with 0.215 μM (forward and reverse primer concentration is identical), go Ionized water is mended to 15 μ L.

(2) performing PCR amplification is entered as follows：

94 DEG C of denaturations 4min；

94 DEG C of degeneration 30s, 56 DEG C of annealing 45s, 72 DEG C of extension 1min, totally 34 circulations；

72 DEG C of extension 8min, 4 DEG C of preservations

3rd, electrophoresis detection

Detected using conventional denaturing polyacrylamide gel electrophoresis.Fig. 9 be 2 couples of primer GBG288 of I groups with The AFLP system of BBG238.As can be seen from the figure the amplified production of this 2 pairs of primers is clear, polymorphic site enriches, each other It is easily discriminated.By taking i1 as an example, 5 bands are had from top to bottom, equivalent to 7 allele, be respectively designated as a, b, c, d, e, F, g, then the genotype of 12 samples be followed successively by：ccdf、ccef、dddf、deeg、dddd、eeeg、addf、bdef、bcdd、 abdd、cdef、deeg。

SEQUENCE LISTING

<110>TanJin Agricultural College

<120>A kind of method that utilization multiplex PCR carries out ssr analysis to tetraploid alfalfa

<130> 2017

<160> 48

<170> PatentIn version 3.5

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<211> 20

<212> DNA

<213>Artificial sequence

<400> 24

gcaactccag ctgcatcttt 20

<210> 25

<211> 22

<212> DNA

<213>Artificial sequence

<400> 25

ttgcctcaac ctctgctaat tc 22

<210> 26

<211> 22

<212> DNA

<213>Artificial sequence

<400> 26

gccgaagagc ctttgatagt aa 22

<210> 27

<211> 22

<212> DNA

<213>Artificial sequence

<400> 27

caccactatc tcttccctca cc 22

<210> 28

<211> 22

<212> DNA

<213>Artificial sequence

<400> 28

tgttggtaat gttcaagctc ca 22

<210> 29

<211> 20

<212> DNA

<213>Artificial sequence

<400> 29

ccgaatgaga gcaaccattt 20

<210> 30

<211> 20

<212> DNA

<213>Artificial sequence

<400> 30

ttgatcaaca gcgaatcgag 20

<210> 31

<211> 22

<212> DNA

<213>Artificial sequence

<400> 31

acgaggcaca cactctctct ct 22

<210> 32

<211> 22

<212> DNA

<213>Artificial sequence

<400> 32

ggtgctttca ttacatccca ta 22

<210> 33

<211> 22

<212> DNA

<213>Artificial sequence

<400> 33

tccgaaccct acttccaaat ta 22

<210> 34

<211> 22

<212> DNA

<213>Artificial sequence

<400> 34

tgggatactg attttctgct tc 22

<210> 35

<211> 22

<212> DNA

<213>Artificial sequence

<400> 35

tcagcagtta gttttggtat gc 22

<210> 36

<211> 22

<212> DNA

<213>Artificial sequence

<400> 36

tgttgaagtt ggagttttgg tg 22

<210> 37

<211> 20

<212> DNA

<213>Artificial sequence

<400> 37

aaagagattg ggtcggtgaa 20

<210> 38

<211> 21

<212> DNA

<213>Artificial sequence

<400> 38

tggttgatca atgttcctcc t 21

<210> 39

<211> 20

<212> DNA

<213>Artificial sequence

<400> 39

accaaaccca cttcccatct 20

<210> 40

<211> 20

<212> DNA

<213>Artificial sequence

<400> 40

ttgaagttgg tggaacagca 20

<210> 41

<211> 20

<212> DNA

<213>Artificial sequence

<400> 41

cgatctggct tgaggatagg 20

<210> 42

<211> 20

<212> DNA

<213>Artificial sequence

<400> 42

ggcagggcta agcttctcat 20

<210> 43

<211> 22

<212> DNA

<213>Artificial sequence

<400> 43

gtcgaaatgg ttgcttctct tt 22

<210> 44

<211> 21

<212> DNA

<213>Artificial sequence

<400> 44

ggttagggtt ttgggtttga a 21

<210> 45

<211> 22

<212> DNA

<213>Artificial sequence

<400> 45

gaaggagaaa aggtgagggt tt 22

<210> 46

<211> 22

<212> DNA

<213>Artificial sequence

<400> 46

tcggatgatg atgaagaagt gt 22

<210> 47

<211> 18

<212> DNA

<213>Artificial sequence

<400> 47

ccggctcaac gatccagt 18

<210> 48

<211> 19

<212> DNA

<213>Artificial sequence

<400> 48

agtgggaatt ggagggtca 19

Claims (5)

1. a kind of method that utilization multiple PCR technique carries out ssr analysis to tetraploid alfalfa, it is characterised in that include as Lower step：

1) 24 pairs of SSR primers are filtered out, optimum organization is as follows into 9 PCR groups：

A groups：Primer pair BBI131 such as SEQ ID NO：1 and SEQ ID NO：Shown in 2,

Primer pair GBG230 such as SEQ ID NO：3 and SEQ ID NO：Shown in 4,

Primer pair E776153 such as SEQ ID NO：5 and SEQ ID NO：Shown in 6；

B groups：Primer pair DMt1H10 such as SEQ ID NO：7 and SEQ ID NO：Shown in 8,

Primer pair CBF156 such as SEQ ID NO：9 and SEQ ID NO：Shown in 10,

Primer pair DBI28 such as SEQ ID NO：11 and SEQ ID NO：Shown in 12；

C groups：Primer pair ABE93 such as SEQ ID NO：13 and SEQ ID NO：Shown in 14,

Primer pair CIC338 such as SEQ ID NO：15 and SEQ ID NO：Shown in 16,

Primer pair AMTIC95 such as SEQ ID NO：17 and SEQ ID NO：Shown in 18；

D groups：Primer pair GBF56 such as SEQ ID NO：19 and SEQ ID NO：Shown in 20,

Primer pair CAW306 such as SEQ ID NO：21 and SEQ ID NO：Shown in 22,

Primer pair BBG28 such as SEQ ID NO：23 and SEQ ID NO：Shown in 24；

E groups：Primer pair HAW178 such as SEQ ID NO：25 and SEQ ID NO：Shown in 26,

Primer pair AAW365 such as SEQ ID NO：27 and SEQ ID NO：Shown in 28,

Primer pair HAL82 such as SEQ ID NO：29 and SEQ ID NO：Shown in 30；

F groups：Primer pair DAW289 such as SEQ ID NO：31 and SEQ ID NO：Shown in 32,

Primer pair DBE84 such as SEQ ID NO：33 and SEQ ID NO：Shown in 34,

Primer pair BBG280 such as SEQ ID NO：35 and SEQ ID NO：Shown in 36；

G groups：Primer pair HMt1G03 such as SEQ ID NO：37 and SEQ ID NO：Shown in 38,

Primer pair FAW115 such as SEQ ID NO：39 and SEQ ID NO：Shown in 40；

H groups：Primer pair CBF96 such as SEQ ID NO：41 and SEQ ID NO：Shown in 42,

Primer pair GAW212 such as SEQ ID NO：43 and SEQ ID NO：Shown in 44；

I groups：Primer pair GBG288 such as SEQ ID NO：45 and SEQ ID NO：Shown in 46,

Primer pair BBG238 such as SEQ ID NO：47 and SEQ ID NO：Shown in 48；

2) multiplex PCR：The 2-3 for belonging to one group together is added in a PCR reaction tube to SSR specific primers pair, for same DNA profiling constitutes multiple differential responses systems, and completes multiplex PCR according to same PCR amplification programs；

3) amplified production is separated by electrophoresis detection.

2. the method that a kind of utilization multiple PCR technique as claimed in claim 1 carries out ssr analysis to tetraploid alfalfa, Characterized in that, the reaction system includes following components：Template DNA 1.0~2.5ng/ μ L, Taq archaeal dna polymerases 0.03~ 0.10U/ μ L, 0.20~0.35mM of dNTPs, 1 × PCR Buffer (MgCl containing 1.5mM₂), primer concentration 0.085~0.285 μM, deionized water is mended to 15 μ L.

3. the method that a kind of utilization multiple PCR technique as claimed in claim 2 carries out ssr analysis to tetraploid alfalfa, Characterized in that, the primer concentration is as follows respectively：

A groups：The concentration of primer pair BBI131 is 0.145 μM, and the concentration of primer pair GBG230 is 0.170 μM, primer pair E776153 Concentration be 0.135 μM；

B groups：The concentration of primer pair DMt1H10 is 0.225 μM, and the concentration of primer pair CBF156 is 0.105 μM, primer pair DBI28 Concentration be 0.120 μM；

C groups：The concentration of primer pair ABE93 is 0.235 μM, and the concentration of primer pair CIC338 is 0.150 μM, primer pair AMTIC95 Concentration be 0.140 μM；

D groups：The concentration of primer pair GBF56 is 0.285 μM, and the concentration of primer pair CAW306 is 0.115 μM, primer pair BBG28 Concentration is 0.125 μM；

E groups：The concentration of primer pair HAW178 is 0.205 μM, and the concentration of primer pair AAW365 is 0.100 μM, primer pair HAL82 Concentration is 0.145 μM；

F groups：The concentration of primer pair DAW289 is 0.160 μM, and the concentration of primer pair DBE84 is 0.205 μM, primer pair BBG280 Concentration is 0.160 μM；

G groups：The concentration of primer pair HMt1G03 is 0.195 μM, and the concentration of primer pair FAW115 is 0.105 μM；

H groups：The concentration of primer pair CBF96 is 0.160 μM, and the concentration of primer pair GAW212 is 0.140 μM；

I groups：The concentration of primer pair GBG288 is 0.085 μM, and the concentration of primer pair BBG238 is 0.215 μM；

Concentrations above refers both to the respective concentration of forward and reverse primer.

4. a kind of utilization multiple PCR technique as described in claim 1 or 2 or 3 carries out ssr analysis to tetraploid alfalfa Method, it is characterised in that the PCR amplification programs are comprised the following steps：

1) 94 DEG C of denaturations 4min；

2) 94 DEG C of degeneration 30s, 56 DEG C of annealing 45s, 72 DEG C of extension 1min, totally 34 circulations；

3) 72 DEG C of extension 8min, 4 DEG C of preservations.

5. the method described in claim 1-4 any claim is being carried out in analysis of genetic diversity to tetraploid alfalfa Application.