CN103146832B - Method for detecting or performing aided detection on sorghum sudanense nationally checked variety - Google Patents
Method for detecting or performing aided detection on sorghum sudanense nationally checked variety Download PDFInfo
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Abstract
The invention discloses a method for detecting or performing aided detection on sorghum sudanense nationally checked variety. The invention provides a primer group for detecting and performing aided detection on sorghum sudanense nationally checked variety, wherein the primer group consists of 20 primer groups comprising the primer group P01-primer group P20. The experiment proves that 20 pairs of SSR primers are screened, the pasture is subjected to polymerase chain reaction (PCR) amplification, the electrophoretogram of 20 products and the electrophoretogram of seven sorghum sudanense nationally checked varieties are subjected to band comparison, and if the electrophoretogram of at least 18 in 20 products is consistent with the band, the pasture can be judged to be in the same variety as the sorghum sudanense in a candidate mode. The sorghum sudanense is identified according to the primers and the method, and whether the pasture to be detected is really in the seven nationally checked varieties can be simply, rapidly, efficiently and accurately identified.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of detection or the careful kind method of auxiliary detection Gao Dancao state.
Background technology
From 2006, connective tissue of the Ministry of Agriculture carries out grass seeds quality surveillance selective examination work, find that some illegal businessmans are under interests drive, change grass seeds title, palm off domestic improved seeds, or with domestic grass seeds personation import grass seeds, upset grass seeds market order, damage the rights and interests of breeding man and peasants and herdsmen's interests.Owing to lacking grass seeds verity Rapid Identification standard, in quality monitoring process, cannot provide scientific verification result, illegal retailer can not get due punishment, and herbage variety owner's rights and interests can not get protection, and grass seeds work of cracking down on the fake difficulty becomes effective.Therefore, in the urgent need to research accurately and reliably, fast and convenient cultivar identification technology.
Gao Dancao is according to hybrid vigour principle, hybridizes and forms, the new herbage being passed through by the up-to-date authorization of the 3rd national herbage variety validation board with Chinese sorghum and arabian cron.High red grass comprehensive the wide and arabian cron ability for tillering of Sorghum Stalk Diameter, leaf, advantage that regeneration power is strong, hybrid vigour is very obvious.In hay, contain crude protein, sugar degree is higher, suitable ensiling.Can be used for the livestock and poultry such as cowboying, sheep, rabbit, goose and fish.Performance good quality and high output in production, benefit is obvious, is a kind of very important herbage aborning.At present state examines kind one and has seven, is respectively that Anhui grass No. 2 (improved variety), day agriculture green grass or young crops are raised No. 1 (improved variety), happy food (introduced variety), Anhui grass No. 3 (improved variety), day agriculture No. 2 (improved variety), No. 1 (improved variety) herded in Ji grass No. 2 (improved variety), Shanxi.
SSR(Simple sequence repeat) be also microsatellite DNA, be the series connection reiterated DNA sequences of a class by several (mostly being 1-5) based composition, its length is generally shorter, is distributed widely in genomic different positions.Because the two ends of SSR sequence are generally conserved sequence, therefore can design amplification SSR sequence by conserved sequence, the SSR sequence amplifying forms collection of illustrative plates different in size, thereby can distinguish different materials.SSR fingerprint is reproducible, simple easy handling, and be codominant marker, be widely used in cultivar identification and purity check.
Set up the method for quick of cultivar identification some staple crops as corn, soybean, wheat, barley etc., but also there is no relevant report at Gao Dancao at present, due to specificity between the kind of SSR primer, every kind of plant all needs to develop new primer and testing process.
Summary of the invention
An object of the present invention is to provide one group for detection of or auxiliary detection grass seeds to be measured be whether the primer sets that Gao Dancao state examines kind, by primer pair P01-primer pair P20 totally 20 primer pairs form;
Described primer pair P01 is made up of the single strand dna shown in sequence 2 in the single strand dna shown in sequence in sequence table 1 and sequence table;
Described primer pair P02 is made up of the single strand dna shown in sequence 4 in the single strand dna shown in sequence in sequence table 3 and sequence table;
Described primer pair P03 is made up of the single strand dna shown in sequence 6 in the single strand dna shown in sequence in sequence table 5 and sequence table;
Described primer pair P04 is made up of the single strand dna shown in sequence 8 in the single strand dna shown in sequence in sequence table 7 and sequence table;
Described primer pair P05 is made up of the single strand dna shown in sequence 10 in the single strand dna shown in sequence in sequence table 9 and sequence table;
Described primer pair P06 is made up of the single strand dna shown in sequence 12 in the single strand dna shown in sequence in sequence table 11 and sequence table;
Described primer pair P07 is made up of the single strand dna shown in sequence 14 in the single strand dna shown in sequence in sequence table 13 and sequence table;
Described primer pair P08 is made up of the single strand dna shown in sequence 16 in the single strand dna shown in sequence in sequence table 15 and sequence table;
Described primer pair P09 is made up of the single strand dna shown in sequence 18 in the single strand dna shown in sequence in sequence table 17 and sequence table;
Described primer pair P10 is made up of the single strand dna shown in sequence 20 in the single strand dna shown in sequence in sequence table 19 and sequence table;
Described primer pair P11 is made up of the single strand dna shown in sequence 22 in the single strand dna shown in sequence in sequence table 21 and sequence table;
Described primer pair P12 is made up of the single strand dna shown in sequence 24 in the single strand dna shown in sequence in sequence table 23 and sequence table;
Described primer pair P13 is made up of the single strand dna shown in sequence 26 in the single strand dna shown in sequence in sequence table 25 and sequence table;
Described primer pair P14 is made up of the single strand dna shown in sequence 28 in the single strand dna shown in sequence in sequence table 27 and sequence table;
Described primer pair P15 is made up of the single strand dna shown in sequence 30 in the single strand dna shown in sequence in sequence table 29 and sequence table;
Described primer pair P16 is made up of the single strand dna shown in sequence 32 in the single strand dna shown in sequence in sequence table 31 and sequence table;
Described primer pair P17 is made up of the single strand dna shown in sequence 34 in the single strand dna shown in sequence in sequence table 33 and sequence table;
Described primer pair P18 is made up of the single strand dna shown in sequence 36 in the single strand dna shown in sequence in sequence table 35 and sequence table;
Described primer pair P19 is made up of the single strand dna shown in sequence 38 in the single strand dna shown in sequence in sequence table 37 and sequence table;
Described primer pair P20 is made up of the single strand dna shown in sequence 40 in the single strand dna shown in sequence in sequence table 39 and sequence table.
Each the equal independent packaging of primer in above-mentioned primer sets;
Described Gao Dancao state examines kind and raises No. 2, No. 1, day agriculture, No. 3, Anhui grass, happy food, No. 2, Anhui grass, No. 2, Ji grass or Shanxi for day agriculture green grass or young crops and herd No. 1.
Another object of the present invention be to provide a kind of for detection of or auxiliary detection grass seeds to be measured be whether the PCR reagent set that Gao Dancao state examines kind.
PCR reagent set provided by the invention, is made up of following 20 kinds of PCR reagent:
1) by the primer pair P01 in above-mentioned primer sets, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;
2) by the primer pair P02 in above-mentioned primer sets, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;
3) by the primer pair P03 in above-mentioned primer sets, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;
4) by the primer pair P04 in above-mentioned primer sets, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;
5) by the primer pair P05 in above-mentioned primer sets, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;
6) by the primer pair P06 in above-mentioned primer sets, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;
7) by the primer pair P07 in above-mentioned primer sets, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;
8) by the primer pair P08 in above-mentioned primer sets, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;
9) by the primer pair P09 in above-mentioned primer sets, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;
10) by the primer pair P10 in above-mentioned primer sets, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;
11) by the primer pair P11 in above-mentioned primer sets, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;
12) by the primer pair P12 in above-mentioned primer sets, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;
13) by the primer pair P13 in above-mentioned primer sets, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;
14) by the primer pair P14 in above-mentioned primer sets, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;
15) by the primer pair P15 in above-mentioned primer sets, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;
16) by the primer pair P16 in above-mentioned primer sets, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;
17) by the primer pair P17 in above-mentioned primer sets, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;
18) by the primer pair P18 in above-mentioned primer sets, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;
19) by the primer pair P19 in above-mentioned primer sets, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;
20) by the primer pair P20 in above-mentioned primer sets, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water.
In above-mentioned PCR reagent set, the final concentration of every primer in each primer pair in the described PCR reagent of correspondence is 20pmol; The equal independent packaging of described PCR reagent; Described Gao Dancao state examines kind and raises No. 2, No. 1, day agriculture, No. 3, Anhui grass, happy food, No. 2, Anhui grass, No. 2, Ji grass or Shanxi for day agriculture green grass or young crops and herd No. 1.
The test kit that contains above-mentioned primer sets or the test kit that contains above-mentioned PCR reagent set are also the scope of protection of the invention.
Whether above-mentioned primer sets, above-mentioned PCR reagent set or above-mentioned test kit are that the application that Gao Dancao state examines in kind is also the scope of protection of the invention in detection or auxiliary detection testing sample.
In above-mentioned application, described Gao Dancao state examines kind and raises No. 2, No. 1, day agriculture, No. 3, Anhui grass, happy food, No. 2, Anhui grass, No. 2, Ji grass or Shanxi for day agriculture green grass or young crops and herd No. 1.
Testing sample is high red grass or doubtful Pacetter varieties.
The 3rd object of the present invention is to provide a kind of detect or whether auxiliary detection grass seeds to be measured is the method that 7 Ge Gaodancao states examine in kind any.
Method provided by the invention, comprises the steps:
1) respectively the careful kind of grass seeds to be measured and 7 Ge Gaodancao states is carried out to SSR amplification with 20 primer pairs in above-mentioned primer sets, above-mentioned PCR reagent set or above-mentioned test kit, electrophoresis detection amplified production, obtains 20 kinds of electrophoretograms of grass seeds to be measured and 7 Ge Gaodancao states and examines each in kinds 20 kinds and contain specific amplified band electrophoretogram;
The each Gao Dancao state that described same primer pair increases that the electrophoretogram of the grass seeds to be measured that obtains obtains with same primer pair amplification examine kind to contain specific amplified band electrophoretogram corresponding;
2) each primer pair the is increased specific amplified band electrophoretogram that contains that the electrophoretogram 7 Ge Gaodancao states corresponding with it of the grass seeds to be measured that obtains examine in kind is compared respectively, if at least 18 primer pairs increase, the electrophoretic band of the electrophoretogram of the grass seeds to be measured that obtains all has the specific amplified band in specific amplified band electrophoretogram that contains that any Gao Dancao state corresponding with it examine kind and forms consistent band, and grass seeds to be measured is or candidate examines kind for corresponding Gao Dancao state; Have the specific amplified band in specific amplified band electrophoretogram that contains that any Gao Dancao state corresponding with it examine in kind and form consistent band if be less than the increase electrophoretic band of electrophoretogram of the grass seeds to be measured that obtains of 18 primer pairs, grass seeds to be measured be or candidate is not the careful kind of corresponding Gao Dancao state;
Described 7 Ge Gaodancao states examine that kind is specially raise No. 2, No. 1, day agriculture, Anhui grass No. 3, happy food, No. 2, Anhui grass, No. 2, Ji grass of day agriculture green grass or young crops and herd No. 1 Shanxi.
The careful kind of each Gao Dancao state can be that the corresponding careful kind of Gao Dancao state also can be examined kind for definite Gao Dancao state.
In the electrophoretogram of 20 pairs of primer amplifications of the careful kind of above-mentioned each Gao Dancao state, the composition of specific amplified band is as shown in table 3.
Above-mentioned grass seeds to be measured is high red grass or doubtful Pacetter varieties.
The 4th object of the present invention is to provide a kind of detection or whether auxiliary detection grass seeds to be measured is the method for same kind with contrasting Pacetter varieties.
Method provided by the invention, comprises the steps:
1) respectively grass seeds to be measured and contrast Pacetter varieties are carried out to SSR amplification with 20 primer pairs in above-mentioned primer sets, above-mentioned PCR reagent set or above-mentioned test kit, electrophoresis detection amplified production, 20 kinds of electrophoretograms that obtain grass seeds to be measured contain specific amplified band electrophoretogram with 20 kinds that contrast Pacetter varieties;
Described same primer pair the increase electrophoretogram of the contrast Pacetter varieties that obtains of electrophoretogram and the same primer pair of the grass seeds to be measured that obtains that increases is corresponding;
2) each primer pair the is increased electrophoretogram of the grass seeds to be measured that obtains is compared respectively with the electrophoretogram that contrasts Pacetter varieties, all consistent bands of electrophoretogram of the contrast Pacetter varieties corresponding with it of electrophoretic band of the electrophoretogram of the grass seeds to be measured that obtains if at least 18 primer pairs increase, grass seeds to be measured with contrast Pacetter varieties for or candidate be same kind; If it is consistent to be less than the increase electrophoretogram of the electrophoretic band of electrophoretogram of the grass seeds to be measured that the obtains contrast Pacetter varieties corresponding with it of 18 primer pairs, grass seeds to be measured with contrast Pacetter varieties not for or not candidate be same kind;
Above-mentioned contrast Pacetter varieties be Gao Dancao state examine breed standard product or phenotypic evaluation Gao Dancao state examine kind.
The careful kind 20 of above-mentioned Gao Dancao state is as shown in table 3 to the composition of specific amplified band in the electrophoretogram of primer amplification.
In aforesaid method, described electrophoresis adopts denaturing polyacrylamide gel; The template of described PCR is genomic dna, and concrete employing is the genomic dna of the mixed sample of 40 strains in an embodiment of the present invention.
Above-mentioned grass seeds to be measured is high red grass or doubtful Pacetter varieties.
Of the present invention experimental results show that, the present invention filters out 20 pairs of SSR primers, with it, herbage is carried out to pcr amplification, the electrophoretogram that the electrophoretogram of 20 kinds of products that obtain and 7 Zhong Gaodancao states examine kind carries out the bands of a spectrum comparison of object specific band, if in the electrophoretogram of 20 kinds of products at least 18 kinds consistent with the bands of a spectrum of object specific band, can candidate judge this herbage and be same kind.Identify according to primer of the present invention and method, can be simply, fast, whether efficient, precise Identification to go out Gao Dancao to be measured be that 7 states examine kind true or false.
Brief description of the drawings
Fig. 1 is the denaturing polyacrylamide gel electrophoresis result of P17 primer amplification sample 1-9
Fig. 2 is the denaturing polyacrylamide gel electrophoresis result of P01, P02, P03 primer amplification sample 1-11
Fig. 3 is the denaturing polyacrylamide gel electrophoresis result of P04-08 primer amplification sample 1-11
Fig. 4 is the denaturing polyacrylamide gel electrophoresis result of P09-11 primer amplification sample 1-11
Fig. 5 is the denaturing polyacrylamide gel electrophoresis result of P12-17 primer amplification sample 1-11
Fig. 6 is the denaturing polyacrylamide gel electrophoresis result of P18 primer amplification sample 1-9
Fig. 7 is the denaturing polyacrylamide gel electrophoresis result of P18 primer amplification sample 10-11
Fig. 8 is the denaturing polyacrylamide gel electrophoresis result of P19-20 primer amplification sample 1-11
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
In following embodiment, seven Ge Gaodancao states used examine kind: grass No. 2, day agriculture green grass or young crops in Anhui are raised No. 1, happy food, No. 2, Anhui grass No. 3, day agriculture, No. 2, Ji grass, Shanxi and herd No. 1, all can buy from market; Various backgrounds is as shown in table 1:
The grass seeds that in following embodiment, 4 Fei Gaodan grass states of used other examine kinds is as shown in table 1.
Table 1 is the essential information of relevant 11 kinds of experiment
Embodiment 1, examine the screening of SSR primer of kind for detection of Gao Dancao state
From document record the careless primer of the high pellet of 68 couple with polymorphism filter out 20 pairs have polymorphism, amplification stablize, be easy to statistics primer detect for kind, the sequence of 20 pairs of SSR primers is in table 2.
Table 2 be for detection of SSR primer
Embodiment 2, examine the application of SSR primer of kind for detection of Gao Dancao state
One, groping of Sampling Strategy
Respectively seven Ge Gaodancao states are examined to grass seeds sample (No. 3, Ji grass, sweet sorghum, arabian cron and unknown Gao Dancao that kind and other 4 Fei Gaodan grass states examine kind; Source and background are as shown in table 1) each 400 of seed, soak and be placed on 4 DEG C of refrigerators and after 7 days, proceed in wet sandy soil and plant with distilled water;
In the time of height of seedling 2cm, gather respectively the mixed sample of 40 strain blades, the mixed sample of 60 strain blades, the mixed sample of 80 strain blades, the mixed sample of 100 strain blades of each sample; Treat that height of seedling 10cm gathers respectively the mixed sample of 3 single-strain blade samples of each sample, 20 strain blades, liquid nitrogen is preserved or is directly carried out next step analysis.
1, the extraction of total DNA
(1) in the centrifuge tube of 2ml, add 2 × CTAB solution of 850 μ l, add the beta-mercaptoethanol of 10 μ l simultaneously, in 65 DEG C of water-baths, add hot reserve;
(2) blade material of collection is put into mill, add liquid nitrogen, grind rapidly, become Powdered;
(3) ground powder is added in the centrifuge tube of the 2ml in (1), then put into 65 DEG C of water-baths, 85rpm shake incubation 2h;
(4) add isopyknic CI(chloroform: primary isoamyl alcohol=24:1), shake mixes; 4 DEG C, the centrifugal 10min of 12000rpm, takes out water, proceeds to new centrifuge tube;
(5) add 2.5 μ l RNaseA(10mg/ml), 37 DEG C, 2h;
(6) repeating step (4);
(7) add 2/3 times of volume Virahol or 2 times of volume ethanol ,-20 DEG C, 1h;
(8) 4 DEG C, the centrifugal 10min of 12000rpm, removes supernatant liquor;
(9) by 70% ethanol washing and precipitating, 4 DEG C, the centrifugal 10min of 12000rpm, removes supernatant liquor;
(10) air-dry, with appropriate 0.1xTE dissolving, measure concentration, in 4 DEG C of short-term preservations, preserve, obtain genomic dna for-20 DEG C long-term.
2, SSR amplification
(1) reaction system
Taking the mixed sample of 1 each sample obtaining or the genomic dna of individual plant sample as template, use respectively the 20 pairs of SSR primer pairs (P01-P20) in table 2 to carry out pcr amplification.
The SSR amplification system of 20 μ l of every pair of primer is as follows: 1 μ l genomic dna be template (final concentration in reaction system be 50ng/ μ l), 1 μ l upstream SSR primer, (final concentration of upstream and downstream primer in reaction system is for being 20pmol for 1 μ l downstream SSR primer; Concrete sequence is as shown in table 2), 10 × PCR buffer(TIANGEN Biotech (Beijing) Co., Ltd. of 2.0 μ l, catalog number ET101-02-02), the mixed liquid (final concentration in reaction system is 50umM) of 0.5 μ l4 kind dNTP, (final concentration in reaction system is 0.5U to 0.2 μ lExTaq archaeal dna polymerase, purchased from TIANGEN Biotech (Beijing) Co., Ltd., catalog number ET101-02-02), use ddH
2o complements to 20 μ l.
(2) reaction conditions
94 DEG C of sex change 3min; 94 DEG C of sex change 30s, 50-60 DEG C (each annealing temperature to primer is specifically shown in Table 2) annealing 30s, 72 DEG C are extended 1min, 30 circulations; 72 DEG C are extended 10min.
The SSR amplified production of 20 pairs of primers of the SSR amplified production of the mixed sample 20 that obtains each sample to primer and each sample individual plant sample.
3, denaturing polyacrylamide gel electrophoresis
By SSR amplified production 4.5% denaturing polyacrylamide gel electrophoresis of 20 pairs of primers of the SSR amplified production of 20 pairs of primers of above-mentioned 2 each sample individual plant samples that obtain and the mixed sample of each sample, DNA molecular amount standard used is pUC18DNA/MspI(TIANGEN Biotech (Beijing) Co., Ltd., catalog number MD203-02) and pBR322DNA/MspI(TIANGEN Biotech (Beijing) Co., Ltd., catalog number MD206-01).
The numbering of 11 samples is as follows respectively: sample 1 is raised No. 1, sample 2 for day No. 2, an agriculture, sample 3 are for No. 3, Ji grass, sample 4 are for sweet sorghum, sample 5 are for happy food, sample 6 are for arabian cron, sample 7 are for No. 3, Anhui grass, sample 8 are for unknown Gao Dancao, sample 9 are for No. 2, Anhui grass, sample 10 are for careless No. 2 of Ji, sample 11 are for herding No. 1 Shanxi for day agriculture green grass or young crops.
Part electrophoresis result as shown in Figure 1, for the denaturing polyacrylamide gel electrophoresis result of P17 primer amplification sample 1-9, swimming lane group 1-9 is corresponding sample 1-9 respectively, and each swimming lane group 8 swimming lanes are from left to right followed successively by: single-strain blade, single-strain blade, single-strain blade, the mixed sample of 20 strain blades, the mixed sample of 40 strain blades, the mixed sample of 60 strain blades, the mixed sample of 80 strain blades, the mixed sample of 100 strain blades; Band in frame is corresponding SSR object specific band (specifically in table 3).
Respectively to 3 of 11 samples individual plants, 20, the mixed sample of 40,60,80 and 100 strains is analyzed, found that most samples occurs stable labelling at the mixed sample of 20 strains, when getting after the mixed sample of 40 strains, amplification is in full accord with the mixed sample amplification of 60,80 and 100 strains, therefore,, for cost-saving in the scientific and effective property that ensures grass seeds qualification work, unification detects sample by grass seeds and gets the mixed sample of 40 strains.
Two, specific detection
Respectively 7 Ge Gaodancao states are examined to grass seeds sample (No. 3, Ji grass, sweet sorghum, arabian cron and unknown Gao Dancao that kind and other 4 Fei Gaodan grass states examine kind; Source and background are as shown in table 1) each 400 of seed, soak and be placed on 4 DEG C of refrigerators and after 7 days, proceed in wet sandy soil and plant with distilled water; In the time of height of seedling 2cm, gather respectively the mixed sample of 40 strain blades of each sample, liquid nitrogen is preserved or is directly carried out next step analysis.
1, the extraction of total DNA: with above-mentioned one 1 identical;
2, SSR amplification: with above-mentioned one 2 identical;
3, denaturing polyacrylamide gel electrophoresis: method with above-mentioned one 3 identical;
Result is shown in Fig. 2-8, and the band in frame is corresponding SSR object specific band:
Fig. 2 is the denaturing polyacrylamide gel electrophoresis result of P01, P02, P03 primer amplification sample 1-11; Wherein, figure comprises swimming lane group 5(P01 primer amplification sample 1-11), swimming lane group 35(P02 primer amplification sample 1-11), swimming lane group 67(P03 primer amplification sample 1-11), 11 swimming lanes in each swimming lane group are followed successively by day agriculture green grass or young crops and raise No. 2, No. 1, day agriculture, Ji grass No. 3, sweet sorghum, happy food, arabian cron, No. 3, Anhui grass, unknown Gao Dancao, No. 2, Anhui grass, No. 2, Ji grass, Shanxi are herded No. 1; Frame is corresponding specific amplified pillar location;
Fig. 3 is the denaturing polyacrylamide gel electrophoresis result of P04-08 primer amplification sample 1-11; Wherein, figure comprises swimming lane group 37(P04 primer amplification sample 1-11), swimming lane group 39(P05 primer amplification sample 1-11), swimming lane group 54(P06 primer amplification sample 1-11), swimming lane group 56(P07 primer amplification sample 1-11), swimming lane group 64(P08 primer amplification sample 1-11), 11 swimming lanes in each swimming lane group are followed successively by day agriculture green grass or young crops and raise No. 2, No. 1, day agriculture, Ji grass No. 3, sweet sorghum, happy food, arabian cron, No. 3, Anhui grass, unknown Gao Dancao, No. 2, Anhui grass, No. 2, Ji grass, Shanxi are herded No. 1; Frame is corresponding specific amplified pillar location;
Fig. 4 is the denaturing polyacrylamide gel electrophoresis result of P09-11 primer amplification sample 1-11; Wherein, figure comprises swimming lane group 4(P09 primer amplification sample 1-11), swimming lane group 14(P10 primer amplification sample 1-11), swimming lane group 19(P11 primer amplification sample 1-11), 11 swimming lanes in each swimming lane group are followed successively by day agriculture green grass or young crops and raise No. 2, No. 1, day agriculture, Ji grass No. 3, sweet sorghum, happy food, arabian cron, No. 3, Anhui grass, unknown Gao Dancao, No. 2, Anhui grass, No. 2, Ji grass, Shanxi are herded No. 1; Frame is corresponding specific amplified pillar location;
Fig. 5 is the denaturing polyacrylamide gel electrophoresis result of P12-17 primer amplification sample 1-11; Wherein, figure comprises swimming lane group 33(P12 primer amplification sample 1-11), swimming lane group 40(P13 primer amplification sample 1-11), swimming lane group 11(P14 primer amplification sample 1-11), swimming lane group 18(P15 primer amplification sample 1-11), swimming lane group 28(P16 primer amplification sample 1-11), swimming lane group 34(P17 primer amplification sample 1-11), 11 swimming lanes in each swimming lane group are followed successively by day agriculture green grass or young crops and raise No. 2, No. 1, day agriculture, Ji grass No. 3, sweet sorghum, happy food, arabian cron, No. 3, Anhui grass, unknown Gao Dancao, No. 2, Anhui grass, No. 2, Ji grass, Shanxi are herded No. 1; Frame is corresponding specific amplified pillar location;
Fig. 6 is the denaturing polyacrylamide gel electrophoresis result of P18 primer amplification sample 1-9, swimming lane group 1-9 is corresponding sample 1-9 respectively, and each swimming lane group 8 swimming lanes are from left to right followed successively by: single-strain blade, single-strain blade, single-strain blade, the mixed sample of 20 strain blades, the mixed sample of 40 strain blades, the mixed sample of 60 strain blades, the mixed sample of 80 strain blades, the mixed sample of 100 strain blades; Band in frame is corresponding SSR object specific band; Only see the result of the mixed sample of 40 strain blades.
Fig. 7 is the denaturing polyacrylamide gel electrophoresis result of P18 primer amplification sample 10-11; Swimming lane group 2 is from left to right followed successively by P18 amplified sample 10 swimming lane groups, P18 amplified sample 11 swimming lane groups; Eight lanes in swimming lane group are followed successively by single-strain blade, single-strain blade, single-strain blade, the mixed sample of 20 strain blades, the mixed sample of 40 strain blades, the mixed sample of 60 strain blades, the mixed sample of 80 strain blades, the mixed sample of 100 strain blades; Band in frame is corresponding SSR object specific band; Only see the result of the mixed sample of 40 strain blades.
Fig. 8 is the denaturing polyacrylamide gel electrophoresis result of P19-20 primer amplification sample 1-11; Wherein, figure comprises swimming lane group 31(P19 primer amplification sample 1-11), swimming lane group 62(P20 primer amplification sample 1-11), 11 swimming lanes in each swimming lane group are followed successively by day agriculture green grass or young crops and raise No. 2, No. 1, day agriculture, Ji grass No. 3, sweet sorghum, happy food, arabian cron, No. 3, Anhui grass, unknown Gao Dancao, No. 2, Anhui grass, No. 2, Ji grass, Shanxi are herded No. 1; Frame is corresponding specific amplified pillar location.Above-mentioned experiment is repeated after 5 times, and the bands of a spectrum that the object specific band sequencing result in 20 pairs of primer SSR amplified productions of the careful kind of 7 states is added up are as shown in table 3.And other 4 Fei Gaodan grass states examine kinds grass seeds 20 pairs of SSR primer extension products all not with the on all four spectrum of object specific band of 20 pairs of SSR primers of 7 states shown in table 3 careful kinds
Band.Table 3 is the bands of a spectrum (unit: bp) that 7 states examine the SSR object specific band of kind
-expression does not have object specific band.
Therefore, aforesaid method can have following purposes:
1) be used for detecting or whether auxiliary detection grass seeds to be measured is that Gao Dancao state examines kind, concrete grammar is as follows:
A, respectively grass seeds to be measured and 7 Ge Gaodancao states are examined to kind with 20 pairs of SSR primers and carry out SSR amplification, electrophoresis detection amplified production, obtains 20 kinds of electrophoretograms of grass seeds to be measured and 7 Ge Gaodancao states and examines each in kinds 20 kinds and contain specific amplified band electrophoretogram; The each Gao Dancao state that described same primer pair increases that the electrophoretogram of the grass seeds to be measured that obtains obtains with same primer pair amplification examine kind to contain specific amplified band electrophoretogram corresponding;
B, each primer pair specific amplified band electrophoretogram that contains that the electrophoretogram 7 Ge Gaodancao states corresponding with it of the grass seeds to be measured that obtains examine in kind that increases is compared respectively, if at least 18 primer pairs increase, the electrophoretic band of the electrophoretogram of the grass seeds to be measured that obtains all has the specific amplified band in specific amplified band electrophoretogram that contains that any Gao Dancao state corresponding with it examine kind and forms consistent band, and grass seeds to be measured is or candidate examines kind for corresponding Gao Dancao state; Have the specific amplified band in specific amplified band electrophoretogram that contains that any Gao Dancao state corresponding with it examine in kind and form consistent band if be less than the increase electrophoretic band of electrophoretogram of the grass seeds to be measured that obtains of 18 primer pairs, grass seeds to be measured be or candidate is not the careful kind of corresponding Gao Dancao state;
7 Ge Gaodancao states examine kind: day agriculture green grass or young crops is raised No. 2, No. 1, day agriculture, No. 3, Anhui grass, happy food, No. 2, Anhui grass, No. 2, Ji grass and Shanxi and herds No. 1.
2) can be used for detect or auxiliary detection grass seeds to be measured whether be same kind with contrasting Pacetter varieties, concrete grammar is as follows:
A) respectively grass seeds to be measured and contrast Pacetter varieties are carried out to SSR amplification with 20 primer pairs in above-mentioned primer sets, above-mentioned PCR reagent set or above-mentioned test kit, electrophoresis detection amplified production, obtains 20 kinds of electrophoretograms of grass seeds to be measured and 20 kinds of electrophoretograms of contrast Pacetter varieties;
Described same primer pair the increase electrophoretogram of the contrast Pacetter varieties that obtains of electrophoretogram and the same primer pair of the grass seeds to be measured that obtains that increases is corresponding;
B) each primer pair the is increased electrophoretogram of the grass seeds to be measured that obtains is compared respectively with the electrophoretogram that contrasts Pacetter varieties, the electrophoretogram that the electrophoretic band of the electrophoretogram of the grass seeds to be measured that obtains all has a contrast Pacetter varieties corresponding with it if at least 18 primer pairs increase forms consistent band, grass seeds to be measured with contrast Pacetter varieties for or candidate be same kind; Form consistent band if be less than 18 primer pairs electrophoretogram that the electrophoretic band of electrophoretogram of the grass seeds to be measured that obtains has a contrast Pacetter varieties corresponding with it that increases, grass seeds to be measured with contrast Pacetter varieties not for or candidate be not same kind;
Above-mentioned contrast Pacetter varieties be Gao Dancao state examine kind or phenotypic evaluation Gao Dancao state examine kind.
The test kit (primer pair independent packaging) that contains all 20 pairs of SSR primers that above-mentioned SSR amplification adopts or the test kit that contains the PCR reagent set (the equal independent packaging of each PCR reagent in PCR reagent set) that above-mentioned SSR amplification adopts all can be used to detect or whether auxiliary detection is that Gao Dancao state examines kind or object Gao Dancao state examines kind;
Above-mentioned PCR reagent set is made up of 20 PCR reagent, and each PCR reagent is grouped into by following one-tenth: 1 pair of SSR primer pair (any in table 2 in 20 pairs of SSR primer pairs), 10 × PCR buffer, dNTP, ExTaq archaeal dna polymerase and water form; Wherein, every SSR primer final concentration in PCR reagent is 20pmol; The final concentration of each dNTP in PCR reagent is 50umM; The final concentration of archaeal dna polymerase in PCR reagent is 0.5U.
Whether embodiment 3, detection sample to be tested are that Gao Dancao state examines kind
8 samples to be tested are that phenotypic evaluation is the grass seeds that following 7 Ge Gaodancao states examine kind and No. 1 arabian cron of Nei Nong:
No. 2, Anhui grass: blade hypertrophy, stem stalk is sturdy, and appearance is like Chinese sorghum, and seed is less than normal, and look purple brown.
It agriculture green grass or young crops is raised No. 1: seedling stage purple, blade is open and flat roomy, shell chocolate, seed redness.
Happy food: blade long strip shape, vein white, the flat oval of seed.
No. 3, Anhui grass: the similar Chinese sorghum of strain shape, blade hypertrophy, seed is less than normal, puce.
No. 2, it agriculture: seedling stage purple, shell black, seed redness.
No. 2, Ji grass: bud scale purple, seedling green, leaf amount is abundant, and blade is roomy, La Mai, stem stalk is more sturdy, succulence, plant type compactness.
Herd No. 1 Shanxi: leaf sheath green, and plant type compactness, well developed root system, stem stalk is sturdy, succulence.
No. 1 arabian cron of interior agriculture (negative findings): bunch type dogstail, leaf sheath is without hair, and planting shell color is black (yellow, red) look.
The mixed sample of 40 strain blades of collecting each sample after the seed plantation of above-mentioned each sample to be tested is tested as follows.
1, the extraction of total DNA:
Extract respectively sample to be tested and 7 Zhong Gaodancao states and examine total DNA of the mixed sample blade of 40 strains of kind according to one 1 the method for embodiment 2.
2, SSR amplification: with according to embodiment 2 above-mentioned one 2 identical;
3, denaturing polyacrylamide gel electrophoresis: method with above-mentioned one 3 identical;
What obtain that 20 kinds of electrophoretograms of each sample to be tested and each Gao Dancao state examine kind contains specific amplified band electrophoretogram.The each Gao Dancao state that same primer pair increases that the electrophoretogram of the grass seeds to be measured that obtains obtains with same primer pair amplification examine kind to contain specific amplified band electrophoretogram corresponding;
Each primer pair specific amplified band electrophoretogram that contains that the electrophoretogram 7 Ge Gaodancao states corresponding with it of the grass seeds to be measured that obtains examine in kind that increases is compared respectively, if at least 18 primer pairs increase, the electrophoretic band of the electrophoretogram of the grass seeds to be measured that obtains all has the specific amplified band in specific amplified band electrophoretogram that contains that any Gao Dancao state corresponding with it examine kind and forms consistent band, and grass seeds to be measured is or candidate examines kind for corresponding Gao Dancao state; Have the specific amplified band in specific amplified band electrophoretogram that contains that any Gao Dancao state corresponding with it examine in kind and form consistent band if be less than the increase electrophoretic band of electrophoretogram of the grass seeds to be measured that obtains of 18 primer pairs, grass seeds to be measured be or candidate is not the careful kind of corresponding Gao Dancao state.
The specific amplified band electrophoretogram (bands of a spectrum of specific amplified band are shown in the table 3 obtaining of embodiment 2) that contains of 20 kinds of electrophoretograms of each sample to be tested and each Gao Dancao state being examined to kind compares, and result is as follows:
In electrophoretic band in the electrophoretogram that SSR primer P01, P02, P03, P04, P05, P07, P08, P09, P10, P11, P12, P13, P14, P15, P16, P17, P18, P19, the P20 that No. 2, sample to be tested Anhui grass obtains, all have with specific amplified band in the electrophoretogram of No. 2, the Anhui grass shown in the table 3 of corresponding embodiment 2 and form consistent band (the sample to be tested electrophoretogram that same primer obtains and Gao Dancao state examine the electrophoretogram comparison of kind), illustrate that No. 2, sample to be tested Anhui grass is careless No. 2 of the careful kind of state Anhui really;
Agriculture green grass or young crops in sample to be tested sky is raised the SSR primer P01 of No. 1, P02, P03, P04, P05, P06, P07, P08, P09, P10, P11, P12, P13, P14, P15, P16, P17, P18, P19, in electrophoretic band in the electrophoretogram that P20 obtains, all have and raise specific amplified band in the electrophoretogram of No. 1 with the sky agriculture green grass or young crops shown in the table 3 of corresponding embodiment 2 and form the consistent band electrophoretogram comparison of the careful kind of Gao Dancao state (the sample to be tested electrophoretogram that same primer obtains with), illustrate sample to be tested sky agriculture green grass or young crops raise No. 1 really for state examine kind sky agriculture green grass or young crops raise No. 1,
In electrophoretic band in the electrophoretogram that the SSR primer P01 of the happy food of sample to be tested, P02, P03, P04, P05, P06, P07, P08, P10, P11, P12, P13, P14, P15, P16, P17, P18, P19, P20 obtain, all have with specific amplified band in the electrophoretogram of the happy food shown in the table 3 of corresponding embodiment 2 and form consistent band (the sample to be tested electrophoretogram that same primer obtains and Gao Dancao state examine the electrophoretogram comparison of kind), illustrate that the happy food of sample to be tested is really for the careful kind pleasure of state is eaten;
In electrophoretic band in the electrophoretogram that SSR primer P01, P02, P03, P04, P05, P06, P07, P08, P09, P10, P11, P12, P13, P14, P15, P16, P17, P18, P19, the P20 that No. 3, sample to be tested Anhui grass obtains, all have with specific amplified band in the electrophoretogram of No. 3, the Anhui grass shown in the table 3 of corresponding embodiment 2 and form consistent band (the sample to be tested electrophoretogram that same primer obtains and Gao Dancao state examine the electrophoretogram comparison of kind), illustrate that No. 3, sample to be tested Anhui grass is careless No. 3 of the careful kind of state Anhui really;
In electrophoretic band in the electrophoretogram that the SSR primer P01 that No. 2, the agriculture of sample to be tested sky, P02, P03, P04, P05, P06, P07, P08, P09, P10, P11, P12, P13, P14, P15, P16, P17, P18, P19, P20 obtain, all have with specific amplified band in the electrophoretogram of No. 2, the sky agriculture shown in the table 3 of corresponding embodiment 2 and form consistent band (the sample to be tested electrophoretogram that same primer obtains and Gao Dancao state examine the electrophoretogram comparison of kind), illustrate that the agriculture of sample to be tested sky is for No. 2 No. 2, the careful kind sky agriculture of state really;
In electrophoretic band in the electrophoretogram that SSR primer P01, P02, P05, P06, P07, P08, P09, P10, P11, P12, P13, P14, P15, P16, P17, P18, P19, the P20 that No. 2, sample to be tested Ji grass obtains, all have with specific amplified band in the electrophoretogram of No. 2, the Ji grass shown in the table 3 of corresponding embodiment 2 and form consistent band (the sample to be tested electrophoretogram that same primer obtains and Gao Dancao state examine the electrophoretogram comparison of kind), illustrate that No. 2, sample to be tested Ji grass is careless No. 2 of the careful kind of state Ji really;
Sample to be tested Shanxi is herded all to have in the electrophoretic band in the electrophoretogram that the SSR primer P01, P02, P03, P04, P05, P06, P07, P08, P09, P10, P11, P12, P13, P15, P16, P17, P18, P19, P20 of No. 1 obtain and is herded specific amplified band in the electrophoretogram of No. 1 with the Shanxi shown in the table 3 of corresponding embodiment 2 and form consistent band (the sample to be tested electrophoretogram that same primer obtains and Gao Dancao state examine the electrophoretogram comparison of kind), illustrates that sample to be tested Shanxi herds No. 1 really for herd No. 1 the careful kind of state Shanxi; Electrophoretic band in the electrophoretogram that the SSR primer P01-P20 of sample to be tested arabian cron obtains is not on all four with specific amplified band composition in the electrophoretogram of 7 Gao Dancao shown in the table 3 of corresponding embodiment 2 (the sample to be tested electrophoretogram that same primer obtains and Gao Dancao state examine the electrophoretogram comparison of kind) all, illustrates that sample to be tested arabian cron is not that 7 kinds of states examine any in kind really: Anhui grass No. 2, day agriculture green grass or young crops raise No. 1, happyly eat, No. 2, careless No. 3, day agriculture in Anhui, careless No. 2 of Ji, Shanxi herd No. 1.
The above results explanation, method of the present invention is correct, can verify whether sample to be tested is that 7 kinds of states examine kind, thereby whether checking sample to be tested is the verity of Gao Dancao.
Claims (10)
- One group for detection of or auxiliary detection grass seeds to be measured be whether the primer sets that Gao Dancao state examines kind, by primer pair P01-primer pair P20 totally 20 primer pairs form;Described primer pair P01 is made up of the single strand dna shown in sequence 2 in the single strand dna shown in sequence in sequence table 1 and sequence table;Described primer pair P02 is made up of the single strand dna shown in sequence 4 in the single strand dna shown in sequence in sequence table 3 and sequence table;Described primer pair P03 is made up of the single strand dna shown in sequence 6 in the single strand dna shown in sequence in sequence table 5 and sequence table;Described primer pair P04 is made up of the single strand dna shown in sequence 8 in the single strand dna shown in sequence in sequence table 7 and sequence table;Described primer pair P05 is made up of the single strand dna shown in sequence 10 in the single strand dna shown in sequence in sequence table 9 and sequence table;Described primer pair P06 is made up of the single strand dna shown in sequence 12 in the single strand dna shown in sequence in sequence table 11 and sequence table;Described primer pair P07 is made up of the single strand dna shown in sequence 14 in the single strand dna shown in sequence in sequence table 13 and sequence table;Described primer pair P08 is made up of the single strand dna shown in sequence 16 in the single strand dna shown in sequence in sequence table 15 and sequence table;Described primer pair P09 is made up of the single strand dna shown in sequence 18 in the single strand dna shown in sequence in sequence table 17 and sequence table;Described primer pair P10 is made up of the single strand dna shown in sequence 20 in the single strand dna shown in sequence in sequence table 19 and sequence table;Described primer pair P11 is made up of the single strand dna shown in sequence 22 in the single strand dna shown in sequence in sequence table 21 and sequence table;Described primer pair P12 is made up of the single strand dna shown in sequence 24 in the single strand dna shown in sequence in sequence table 23 and sequence table;Described primer pair P13 is made up of the single strand dna shown in sequence 26 in the single strand dna shown in sequence in sequence table 25 and sequence table;Described primer pair P14 is made up of the single strand dna shown in sequence 28 in the single strand dna shown in sequence in sequence table 27 and sequence table;Described primer pair P15 is made up of the single strand dna shown in sequence 30 in the single strand dna shown in sequence in sequence table 29 and sequence table;Described primer pair P16 is made up of the single strand dna shown in sequence 32 in the single strand dna shown in sequence in sequence table 31 and sequence table;Described primer pair P17 is made up of the single strand dna shown in sequence 34 in the single strand dna shown in sequence in sequence table 33 and sequence table;Described primer pair P18 is made up of the single strand dna shown in sequence 36 in the single strand dna shown in sequence in sequence table 35 and sequence table;Described primer pair P19 is made up of the single strand dna shown in sequence 38 in the single strand dna shown in sequence in sequence table 37 and sequence table;Described primer pair P20 is made up of the single strand dna shown in sequence 40 in the single strand dna shown in sequence in sequence table 39 and sequence table.
- 2. primer sets according to claim 1, is characterized in that: each the equal independent packaging of primer in described primer sets;Described Gao Dancao state examines kind and raises No. 2, No. 1, day agriculture, No. 3, Anhui grass, happy food, No. 2, Anhui grass, No. 2, Ji grass or Shanxi for day agriculture green grass or young crops and herd No. 1.
- For detection of or auxiliary detection grass seeds to be measured be whether the PCR reagent set that Gao Dancao state examines kind, formed by following 20 kinds of PCR reagent:1) by the primer pair P01 in primer sets described in claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;2) by the primer pair P02 in primer sets described in claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;3) by the primer pair P03 in primer sets described in claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;4) by the primer pair P04 in primer sets described in claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;5) by the primer pair P05 in primer sets described in claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;6) by the primer pair P06 in primer sets described in claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;7) by the primer pair P07 in primer sets described in claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;8) by the primer pair P08 in primer sets described in claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;9) by the primer pair P09 in primer sets described in claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;10) by the primer pair P10 in primer sets described in claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;11) by the primer pair P11 in primer sets described in claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;12) by the primer pair P12 in primer sets described in claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;13) by the primer pair P13 in primer sets described in claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;14) by the primer pair P14 in primer sets described in claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;15) by the primer pair P15 in primer sets described in claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;16) by the primer pair P16 in primer sets described in claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;17) by the primer pair P17 in primer sets described in claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;18) by the primer pair P18 in primer sets described in claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;19) by the primer pair P19 in primer sets described in claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;20) by the primer pair P20 in primer sets described in claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water.
- 4. PCR reagent set according to claim 3, is characterized in that: the final concentration of every primer in each primer pair in the described PCR reagent of correspondence is 20pmol; The equal independent packaging of described PCR reagent; Described Gao Dancao state examines kind and raises No. 2, No. 1, day agriculture, No. 3, Anhui grass, happy food, No. 2, Anhui grass, No. 2, Ji grass or Shanxi for day agriculture green grass or young crops and herd No. 1.
- 5. the test kit that contains the test kit of primer sets described in claim 1 or 2 or contain PCR reagent set described in claim 3 or 4.
- Described in claim 1 or 2 described in primer sets, claim 3 or 4 PCR reagent set or test kit claimed in claim 5 detecting or whether auxiliary detection testing sample is that Gao Dancao state examines the application in kind.
- 7. application according to claim 6, is characterized in that: described Gao Dancao state examines kind and raises No. 2, No. 1, day agriculture, No. 3, Anhui grass, happy food, No. 2, Anhui grass, No. 2, Ji grass or Shanxi for day agriculture green grass or young crops and herd No. 1.
- 8. detect or whether auxiliary detection grass seeds to be measured is the method that 7 Ge Gaodancao states examine in kind any, comprise the steps:1) with PCR reagent set described in 20 primer pairs in primer sets described in claim 1 or 2, claim 3 or 4 or test kit claimed in claim 5, the careful kind of grass seeds to be measured and 7 Ge Gaodancao states is carried out to SSR amplification, electrophoresis detection amplified production, obtains 20 kinds of electrophoretograms of grass seeds to be measured and 7 Ge Gaodancao states and examines each in kinds 20 kinds and contain specific amplified band electrophoretogram;The each Gao Dancao state that described same primer pair increases that the electrophoretogram of the grass seeds to be measured that obtains obtains with same primer pair amplification examine kind to contain specific amplified band electrophoretogram corresponding;2) each primer pair the is increased specific amplified band electrophoretogram that contains that the electrophoretogram 7 Ge Gaodancao states corresponding with it of the grass seeds to be measured that obtains examine in kind is compared respectively, if at least 18 primer pairs increase, the electrophoretic band of the electrophoretogram of the grass seeds to be measured that obtains all has the specific amplified band in specific amplified band electrophoretogram that contains that any Gao Dancao state corresponding with it examine kind and forms consistent band, and grass seeds to be measured is or candidate examines kind for corresponding Gao Dancao state; Have the specific amplified band in specific amplified band electrophoretogram that contains that any Gao Dancao state corresponding with it examine in kind and form consistent band if be less than the increase electrophoretic band of electrophoretogram of the grass seeds to be measured that obtains of 18 primer pairs, grass seeds to be measured be or candidate is not the careful kind of corresponding Gao Dancao state;Described 7 Ge Gaodancao states examine that kind is specially raise No. 2, No. 1, day agriculture, Anhui grass No. 3, happy food, No. 2, Anhui grass, No. 2, Ji grass of day agriculture green grass or young crops and herd No. 1 Shanxi.
- 9. whether detection or auxiliary detection grass seeds to be measured are a method for same kind with contrasting Pacetter varieties, comprise the steps:1) with PCR reagent set described in 20 primer pairs in primer sets described in claim 1 or 2, claim 3 or 4 or test kit claimed in claim 5, grass seeds to be measured and contrast Pacetter varieties are carried out to SSR amplification, electrophoresis detection amplified production, 20 kinds of electrophoretograms that obtain grass seeds to be measured contain specific amplified band electrophoretogram with 20 kinds that contrast Pacetter varieties;Described same primer pair increase electrophoretogram and the same primer pair of the grass seeds to be measured that obtains increase the contrast Pacetter varieties that obtains to contain specific amplified band electrophoretogram corresponding;2) each primer pair the is increased electrophoretogram of the grass seeds to be measured that obtains is compared respectively with the specific amplified band electrophoretogram that contains contrasting in Pacetter varieties, the specific amplified band in specific amplified band electrophoretogram that contains that the electrophoretic band of the electrophoretogram of the grass seeds to be measured that obtains all has a contrast Pacetter varieties corresponding with it if at least 18 primer pairs increase forms consistent band, grass seeds to be measured with contrast Pacetter varieties for or candidate be same kind; Form consistent band if be less than 18 primer pairs specific amplified band in specific amplified band electrophoretogram that contains that the electrophoretic band of electrophoretogram of the grass seeds to be measured that obtains has a contrast Pacetter varieties corresponding with it that increases, grass seeds to be measured with contrast Pacetter varieties not for or not candidate be same kind.
- 10. method according to claim 8 or claim 9, is characterized in that: described electrophoresis adopts denaturing polyacrylamide gel; The template of described PCR is genomic dna.
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