CN103146832A - 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 the careful kind method of the high red careless state of a kind of detection or auxiliary detection.
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 the grass seeds title, palm off domestic improved seeds, or with domestic grass seeds personation import grass seeds, upset the grass seeds market order, damaged the rights and interests of breeding man and peasants and herdsmen's interests.Owing to lacking grass seeds verity Rapid Identification standard, can't provide the scientific verification result in the quality monitoring process, 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 the hybrid vigour principle, forms the new herbage that is passed through by the up-to-date authorization of the 3rd national herbage variety validation board with the hybridization of Chinese sorghum and arabian cron.High red grass comprehensive Sorghum Stalk Diameter, leaf is wide and arabian cron ability for tillering, advantage that regeneration power is strong, hybrid vigour is very obvious.Contain crude protein in hay, 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) also being microsatellite DNA, is a class by the series connection reiterated DNA sequences of several (mostly being 1-5) based composition, and 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 the SSR sequence that increases by conserved sequence, the SSR sequence that amplifies forms collection of illustrative plates different in size, thereby can distinguish different materials.SSR fingerprint good reproducibility, simple easy handling, and be the codominant marker, be widely used in cultivar identification and purity check.
Set up the method for quick of cultivar identification some staple crops such 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 high red careless state examines kind, by primer pair P01-primer pair P20 totally 20 primer pairs form;
Described primer pair P01 is comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised of the single strand dna shown in sequence 40 in the single strand dna shown in sequence in sequence table 39 and sequence table.
The equal independent packaging of each primer in above-mentioned primer sets;
The red careless state of described height 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 high red careless state examines kind.
PCR reagent set provided by the invention is comprised 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; The red careless state of described height 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 the test kit of above-mentioned primer sets or contain above-mentioned PCR reagent set is also the scope of protection of the invention.
Above-mentioned primer sets, above-mentioned PCR reagent set or above-mentioned test kit are detecting or whether the auxiliary detection testing sample is that application during high red careless state examines kind is also the scope of protection of the invention.
In above-mentioned application, described height red careless 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 purpose 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 high pellet careless states examine in kinds any.
Method provided by the invention comprises the steps:
1) respectively grass seeds to be measured and 7 careful kinds of the careless state of high pellet are carried out the SSR amplification with 20 primer pairs in above-mentioned primer sets, above-mentioned PCR reagent set or above-mentioned test kit, the electrophoresis detection amplified production obtains 20 kinds of electrophoretograms of grass seeds to be measured and 7 high pellet careless states and examines in kind each 20 kinds and contain specific amplified band electrophoretogram;
Each careful kind of high red careless state that the electrophoretogram of the grass seeds to be measured that described same primer pair amplification obtains and same primer pair amplification obtain to contain specific amplified band electrophoretogram corresponding;
The specific amplified band electrophoretogram that contains in the electrophoretogram of the grass seeds to be measured that 2) amplification of each primer pair is obtained 7 careful kinds of the careless state of high pellet corresponding with it is compared respectively, contain that in specific amplified band electrophoretogram, the specific amplified band forms consistent band if the electrophoretic band of the electrophoretogram of the grass seeds to be measured that the amplification of at least 18 primer pairs obtains all has with its any corresponding high red careless state examines kind, grass seeds to be measured be or the candidate is the careful kind of corresponding high red careless state; Have to examine with its any corresponding high red careless state and contain that in specific amplified band electrophoretogram, the specific amplified band forms consistent band in kind if be less than the electrophoretic band of the electrophoretogram of the grass seeds to be measured that the amplification of 18 primer pairs obtains, grass seeds to be measured be or the candidate is not the careful kind of corresponding high red careless state;
Described 7 high pellet careless states examine kinds and are specially day agriculture green grass or young crops and raise No. 2, No. 1, day agriculture, No. 3, Anhui grass, happy food, No. 2, Anhui grass, No. 2, Ji grass and Shanxi and herd No. 1.
It can be also that the high red careless state that determines examines kind for the careful kind of the high red careless state of correspondence that each high red careless state examines kind.
In the electrophoretogram of 20 pairs of primer amplifications of above-mentioned each careful kind of high red careless 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 purpose 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 the contrast Pacetter varieties.
Method provided by the invention comprises the steps:
1) respectively grass seeds to be measured and contrast Pacetter varieties are carried out the SSR amplification with 20 primer pairs in above-mentioned primer sets, above-mentioned PCR reagent set or above-mentioned test kit, the 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;
The electrophoretogram of the grass seeds to be measured that described same primer pair amplification obtains is corresponding with the electrophoretogram of the contrast Pacetter varieties that same primer pair amplification obtains;
The electrophoretogram of the grass seeds to be measured that 2) each primer pair amplification is obtained is compared respectively with the electrophoretogram of contrast Pacetter varieties, if the electrophoretic band of the electrophoretogram of the grass seeds to be measured that the amplification of at least 18 primer pairs obtains is the band consistent with the electrophoretogram of its corresponding contrast Pacetter varieties all, grass seeds to be measured and contrast Pacetter varieties and be or the candidate is same kind; If the electrophoretic band of the electrophoretogram of the grass seeds to be measured that obtains less than the amplification of 18 primer pairs is consistent with the electrophoretogram of its corresponding contrast Pacetter varieties, grass seeds to be measured with contrast Pacetter varieties be not or not the candidate be same kind;
Above-mentioned contrast Pacetter varieties is that high red careless state examines breed standard product or the careful kind of high red careless state of phenotypic evaluation.
In the electrophoretogram of the careful 20 pairs of primer amplifications of kind of the red careless state of above-mentioned height, the composition of specific amplified band is as shown in table 3.
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 pcr amplification, the bands of a spectrum that the electrophoretogram of the electrophoretogram of 20 kinds of products that obtain and 7 kinds of careful kinds of the careless state of high pellet carries out the purpose specific band compare, if in the electrophoretogram of 20 kinds of products, at least 18 kinds of bands of a spectrum with the purpose specific band are consistent, can the 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 the kind true or false.
Description of 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 that uses in following embodiment is ordinary method if no special instructions.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
In following embodiment, seven high pellet careless states used examine kinds: Anhui grass No. 2, day agriculture green grass or young crops 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:
It is as shown in table 1 that in following embodiment, 4 Fei Gaodan grass states of used other examine the grass seeds of kinds.
Table 1 is the essential information of relevant 11 kinds of experiment
Embodiment 1, examine the screening of the SSR primer of kind for detection of height red careless state
Filter out 20 pairs and have primer polymorphism, that amplification is stablized, is easy to add up for the kind detection from the careless primer of the high pellet of 68 couple with polymorphism of document record, the sequence of 20 pairs of SSR primers sees Table 2.
Table 2 be for detection of the SSR primer
Embodiment 2, examine the application of the SSR primer of kind for detection of height red careless state
One, Sampling Strategy gropes
Respectively seven high pellet careless states are examined grass seeds sample (No. 3, Ji grass, sweet sorghum, arabian cron and unknown Gao Dancao that 4 of kinds and other Fei Gaodan grass state examines kinds; Source and background are as shown in table 1) each 400, seed, soak with distilled water and be placed on 4 ℃ of refrigerators and change over to after 7 days in wet sandy soil and plant;
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 when height of seedling 2cm; 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) add 2 * CTAB solution of 850 μ l in the centrifuge tube of 2ml, add simultaneously the beta-mercaptoethanol of 10 μ l, add hot reserve in 65 ℃ of water-baths;
(2) blade material that gathers is put into mill, add liquid nitrogen, grind rapidly, it is become Powdered;
(3) ground powder is added in the centrifuge tube of the 2ml in (1), then put into 65 ℃ of water-baths, 85rpm shakes incubation 2h;
(4) add isopyknic CI(chloroform: primary isoamyl alcohol=24:1), shake mixing; 4 ℃, the centrifugal 10min of 12000rpm takes out water, changes new centrifuge tube over to;
(5) add 2.5 μ l RNaseA(10mg/ml), 37 ℃, 2h;
(6) repeating step (4);
(7) add 2/3 times of volume Virahol or 2 times of volume ethanol ,-20 ℃, 1h;
(8) 4 ℃, the centrifugal 10min of 12000rpm removes supernatant liquor;
(9) with 70% ethanol washing and precipitating, 4 ℃, the centrifugal 10min of 12000rpm removes supernatant liquor;
(10) air-dry, with appropriate 0.1xTE dissolving, measure concentration, in 4 ℃ of short-term preservations ,-20 ℃ of prolonged preservation obtain genomic dna.
2, SSR amplification
(1) reaction system
Take the genomic dna of the mixed sample of 1 each sample that obtains or 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 is template (final concentration in reaction system is 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), 0.5 μ l4 kind dNTP mixes liquid (final concentration in reaction system is 50umM), (final concentration in reaction system is 0.5U to 0.2 μ lExTaq archaeal dna polymerase, available from TIANGEN Biotech (Beijing) Co., Ltd., catalog number ET101-02-02), use ddH
2O complements to 20 μ l.
(2) reaction conditions
94 ℃ of sex change 3min; 94 ℃ of sex change 30s, 50-60 ℃ (each annealing temperature to primer specifically is shown in Table 2) annealing 30s, 72 ℃ are extended 1min, 30 circulations; 72 ℃ are extended 10min.
Obtain the SSR amplified production of 20 pairs of primers of the SSR amplified production of 20 pairs of primers of mixed sample of each sample and each sample individual plant sample.
3, denaturing polyacrylamide gel electrophoresis
SSR amplified production 4.5% denaturing polyacrylamide gel electrophoresis with 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 agriculture green grass or young crops and is herded No. 1 for the Shanxi for No. 2, Ji grass, sample 11 for No. 2, Anhui grass, sample 10 for unknown Gao Dancao, sample 9 for No. 3, Anhui grass, sample 8 for arabian cron, sample 7 for happy food, sample 6 for sweet sorghum, sample 5 for No. 3, Ji grass, sample 4 for day No. 2, an agriculture, sample 3.
The part electrophoresis result as shown in Figure 1, denaturing polyacrylamide gel electrophoresis result for 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 from left to right 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 purpose specific band (specifically seeing Table 3).
Respectively to 3 individual plants of 11 samples, 20, the mixed sample of 40,60,80 and 100 strains is analyzed, the sample that found that the overwhelming majority stable labelling namely occurs at the mixed sample of 20 strains, after getting the mixed sample of 40 strains, amplification is namely in full accord with the mixed sample amplification of 60,80 and 100 strains, therefore, in order to save cost in the scientific and effective property that guarantees grass seeds evaluation work, unification is got the mixed sample of 40 strains with the grass seeds test sample.
Two, specific detection
Respectively 7 high pellet careless states are examined grass seeds sample (No. 3, Ji grass, sweet sorghum, arabian cron and unknown Gao Dancao that 4 of kinds and other Fei Gaodan grass state examines kinds; Source and background are as shown in table 1) each 400, seed, soak with distilled water and be placed on 4 ℃ of refrigerators and change over to after 7 days in wet sandy soil and plant; Gather respectively the mixed sample of 40 strain blades of each sample when height of seedling 2cm, 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 purpose 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 from left to right 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 purpose specific band; The result of only seeing the mixed sample of 40 strain blades gets final product.
Fig. 7 is the denaturing polyacrylamide gel electrophoresis result of P18 primer amplification sample 10-11; Swimming lane group 2 from left to right is followed successively by 11 swimming lane groups of P18 amplified sample 10 swimming lane groups, P18 amplified sample; 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 purpose specific band; The result of only seeing the mixed sample of 40 strain blades gets final product.
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.After above-mentioned experiment is repeated 5 times, that the bands of a spectrum that the purpose 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 purpose 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 purpose specific band of kind
-expression does not have the purpose 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 high red careless state examines kind, concrete grammar is as follows:
A, respectively grass seeds to be measured and 7 high pellet careless states are examined kinds with 20 pairs of SSR primers and carry out the SSR amplification, the electrophoresis detection amplified production obtains 20 kinds of electrophoretograms of grass seeds to be measured and 7 high pellet careless states and examines in kind each 20 kinds and contain specific amplified band electrophoretogram; Each careful kind of high red careless state that the electrophoretogram of the grass seeds to be measured that described same primer pair amplification obtains and same primer pair amplification obtain to contain specific amplified band electrophoretogram corresponding;
The specific amplified band electrophoretogram that contains in the electrophoretogram of B, grass seeds to be measured that the amplification of each primer pair is obtained 7 careful kinds of the careless state of high pellet corresponding with it is compared respectively, contain that in specific amplified band electrophoretogram, the specific amplified band forms consistent band if the electrophoretic band of the electrophoretogram of the grass seeds to be measured that the amplification of at least 18 primer pairs obtains all has with its any corresponding high red careless state examines kind, grass seeds to be measured be or the candidate is the careful kind of corresponding high red careless state; Have to examine with its any corresponding high red careless state and contain that in specific amplified band electrophoretogram, the specific amplified band forms consistent band in kind if be less than the electrophoretic band of the electrophoretogram of the grass seeds to be measured that the amplification of 18 primer pairs obtains, grass seeds to be measured be or the candidate is not the careful kind of corresponding high red careless state;
7 careful kinds of the careless state of high pellet: day agriculture green grass or young crops is raised No. 2, No. 1, day agriculture, No. 3, Anhui grass, happy food, careless No. 2 of Anhui, No. 2, Ji grass and Shanxi and herds No. 1.
2) can be used for detecting or whether auxiliary detection grass seeds to be measured is same kind with the contrast Pacetter varieties, concrete grammar is as follows:
A) respectively grass seeds to be measured and contrast Pacetter varieties are carried out the SSR amplification with 20 primer pairs in above-mentioned primer sets, above-mentioned PCR reagent set or above-mentioned test kit, the electrophoresis detection amplified production obtains 20 kinds of electrophoretograms of grass seeds to be measured and 20 kinds of electrophoretograms of contrast Pacetter varieties;
The electrophoretogram of the grass seeds to be measured that described same primer pair amplification obtains is corresponding with the electrophoretogram of the contrast Pacetter varieties that same primer pair amplification obtains;
The electrophoretogram of the grass seeds to be measured that B) each primer pair amplification is obtained is compared respectively with the electrophoretogram of contrast Pacetter varieties, form consistent band if the electrophoretic band of the electrophoretogram of the grass seeds to be measured that the amplification of at least 18 primer pairs obtains all has with the electrophoretogram of its corresponding contrast Pacetter varieties, grass seeds to be measured with the contrast Pacetter varieties is or the candidate is same kind; Have with the electrophoretogram of its corresponding contrast Pacetter varieties and form consistent band if be less than the electrophoretic band of the electrophoretogram of the grass seeds to be measured that the amplification of 18 primer pairs obtains, grass seeds to be measured with the contrast Pacetter varieties be or the candidate is not same kind;
Above-mentioned contrast Pacetter varieties is that high red careless state examines kind or the careful kind of high red careless state of phenotypic evaluation.
The test kit that contains the test kit (primer pair independent packaging) of all 20 pairs of SSR primers that above-mentioned SSR amplification adopts or contain the PCR reagent set (the equal independent packaging of each PCR reagent in the PCR reagent set) that above-mentioned SSR amplification adopts all can be used to detect or whether auxiliary detection is that high red careless state examines kind or purpose high red careless state examines kind;
Above-mentioned PCR reagent set is comprised of 20 PCR reagent, and each PCR reagent is grouped into by following one-tenth: the 1 pair of SSR primer pair (any in table 2 in the 20 pairs of SSR primer pairs), 10 * PCR buffer, dNTP, ExTaq archaeal dna polymerase and water form; Wherein, the final concentration of every SSR primer 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 high red careless state examines kind
8 samples to be tested are that phenotypic evaluation is the grass seeds of following 7 careful kinds of the careless state of high pellet and No. 1 arabian cron of Nei Nong:
No. 2, Anhui grass: blade is loose, and the 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, the shell chocolate, seed is red.
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 is loose, and seed is less than normal, puce.
No. 2, it agriculture: seedling stage purple, shell black, seed is red.
No. 2, Ji grass: bud scale purple, seedling green, the leaf amount is abundant, and blade is roomy, La Mai, the stem stalk is more sturdy, succulence, plant type is compact.
Herd No. 1 the Shanxi: leaf sheath is green, and plant type is compact, well developed root system, and the stem stalk is sturdy, succulence.
No. 1 arabian cron of interior agriculture (negative findings): bunch type dogstail, leaf sheath are planted the shell color and are black (yellow, red) look without hair.
The mixed sample of 40 strain blades of collecting each sample after seed plantation with above-mentioned each sample to be tested carries out following experiment.
1, the extraction of total DNA:
Extract respectively according to one 1 the method for embodiment 2 total DNA that sample to be tested and 7 kinds of high pellet careless states examine the mixed sample blade of 40 strains of kinds.
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 high red careless state examine kinds contains specific amplified band electrophoretogram.Each careful kind of high red careless state that the electrophoretogram of the grass seeds to be measured that the amplification of same primer pair obtains and same primer pair amplification obtain to contain specific amplified band electrophoretogram corresponding;
The specific amplified band electrophoretogram that contains in the electrophoretogram of the grass seeds to be measured that the amplification of each primer pair is obtained 7 careful kinds of the careless state of high pellet corresponding with it is compared respectively, contain that in specific amplified band electrophoretogram, the specific amplified band forms consistent band if the electrophoretic band of the electrophoretogram of the grass seeds to be measured that the amplification of at least 18 primer pairs obtains all has with its any corresponding high red careless state examines kind, grass seeds to be measured be or the candidate is the careful kind of corresponding high red careless state; Contain that in specific amplified band electrophoretogram, the specific amplified band forms consistent band if the electrophoretic band of the electrophoretogram of the grass seeds to be measured that obtains less than the amplification of 18 primer pairs has to examine with its any corresponding high red careless state in kind, grass seeds to be measured be or the candidate is not the careful kind of corresponding high red careless state.
The specific amplified band electrophoretogram (bands of a spectrum of specific amplified band are shown in the table 3 that obtains of embodiment 2) that contains of 20 kinds of electrophoretograms of each sample to be tested and each high red careless state being examined kinds compares, and result is as follows:
All have specific amplified band in the electrophoretogram of No. 2, the Anhui grass shown in the table 3 with corresponding embodiment 2 in electrophoretic band in the electrophoretogram that the SSR primer P01 that No. 2, sample to be tested Anhui grass, P02, P03, P04, P05, P07, P08, P09, P10, P11, P12, P13, P14, P15, P16, P17, P18, P19, P20 obtain and form consistent band (electrophoretogram that the sample to be tested electrophoretogram that the same primer obtains and high red careless state examine kind relatively), 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, all having the sky agriculture green grass or young crops shown in the table 3 with corresponding embodiment 2 in electrophoretic band in the electrophoretogram that P20 obtains raises specific amplified band in the electrophoretogram of No. 1 and forms consistent band (electrophoretogram that the sample to be tested electrophoretogram that the same primer obtains and high red careless state examine kind relatively), illustrate that sample to be tested sky agriculture green grass or young crops raises No. 1 and really raise No. 1 for state examines kind sky agriculture green grass or young crops,
All have specific amplified band in the electrophoretogram of the happy food shown in the table 3 with corresponding embodiment 2 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 and form consistent band (electrophoretogram that the sample to be tested electrophoretogram that the same primer obtains and high red careless state examine kind relatively), illustrate that the happy food of sample to be tested eats for state examines the kind pleasure really;
All have specific amplified band in the electrophoretogram of No. 3, the Anhui grass shown in the table 3 with corresponding embodiment 2 in electrophoretic band in the electrophoretogram that the SSR primer P01 that No. 3, sample to be tested Anhui grass, P02, P03, P04, P05, P06, P07, P08, P09, P10, P11, P12, P13, P14, P15, P16, P17, P18, P19, P20 obtain and form consistent band (electrophoretogram that the sample to be tested electrophoretogram that the same primer obtains and high red careless state examine kind relatively), illustrate that No. 3, sample to be tested Anhui grass is careless No. 3 of the careful kind of state Anhui really;
All have specific amplified band in the electrophoretogram of No. 2, the sky agriculture shown in the table 3 with corresponding embodiment 2 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 and form consistent band (electrophoretogram that the sample to be tested electrophoretogram that the same primer obtains and high red careless state examine kind relatively), illustrate that sample to be tested sky agriculture is for No. 2 No. 2, the careful kind sky agriculture of state really;
All have specific amplified band in the electrophoretogram of No. 2, the Ji grass shown in the table 3 with corresponding embodiment 2 in electrophoretic band in the electrophoretogram that the SSR primer P01 that No. 2, sample to be tested Ji grass, P02, P05, P06, P07, P08, P09, P10, P11, P12, P13, P14, P15, P16, P17, P18, P19, P20 obtain and form consistent band (electrophoretogram that the sample to be tested electrophoretogram that the same primer obtains and high red careless state examine kind relatively), illustrate that No. 2, sample to be tested Ji grass is careless No. 2 of the careful kind of state Ji really;
The sample to be tested Shanxi is herded the Shanxi shown in the table 3 that all has in 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 with corresponding embodiment 2 and is herded specific amplified band in the electrophoretogram of No. 1 and form consistent band (electrophoretogram that the sample to be tested electrophoretogram that the same primer obtains and high red careless state examine kind relatively), illustrate that the sample to be tested Shanxi herds No. 1 and really herd No. 1 for 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 all not with the electrophoretogram of 7 Gao Dancao shown in the table 3 of corresponding embodiment 2 in the specific amplified band form on all four (electrophoretogram that the sample to be tested electrophoretogram that the same primer obtains and high red careless state examine kind relatively), illustrate that the 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 are raised No. 1, happy food, No. 2, careless No. 3, day agriculture in Anhui, careless No. 2 of Ji, Shanxi and 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 the 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 high red careless state examines kind, by primer pair P01-primer pair P20 totally 20 primer pairs form;Described primer pair P01 is comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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 comprised 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: the equal independent packaging of each primer in described primer sets;The red careless state of described height 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.
- One kind for detection of or auxiliary detection grass seeds to be measured be whether the PCR reagent set that high red careless state examines kind, formed by following 20 kinds of PCR reagent:1) by the primer pair P01 in the described primer sets of claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;2) by the primer pair P02 in the described primer sets of claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;3) by the primer pair P03 in the described primer sets of claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;4) by the primer pair P04 in the described primer sets of claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;5) by the primer pair P05 in the described primer sets of claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;6) by the primer pair P06 in the described primer sets of claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;7) by the primer pair P07 in the described primer sets of claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;8) by the primer pair P08 in the described primer sets of claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;9) by the primer pair P09 in the described primer sets of claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;10) by the primer pair P10 in the described primer sets of claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;11) by the primer pair P11 in the described primer sets of claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;12) by the primer pair P12 in the described primer sets of claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;13) by the primer pair P13 in the described primer sets of claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;14) by the primer pair P14 in the described primer sets of claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;15) by the primer pair P15 in the described primer sets of claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;16) by the primer pair P16 in the described primer sets of claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;17) by the primer pair P17 in the described primer sets of claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;18) by the primer pair P18 in the described primer sets of claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;19) by the primer pair P19 in the described primer sets of claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water;20) by the primer pair P20 in the described primer sets of claim 1 or 2, pcr amplification damping fluid, dNTP, archaeal dna polymerase and water.
- 4. PCR reagent set according to claim 3, it 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; The red careless state of described height 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. contain the test kit of claim 1 or 2 described primer sets or contain the test kit of the described PCR reagent set of claim 3 or 4.
- 6. the described primer sets of claim 1 or 2, the described PCR reagent set of claim 3 or 4 or test kit claimed in claim 5 are detecting or whether the auxiliary detection testing sample is application during high red careless state examines kind.
- 7. application according to claim 6 is characterized in that: described height red careless 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. one kind is detected or whether auxiliary detection grass seeds to be measured is the method that 7 high pellet careless states examine in kinds any, comprises the steps:1) with the described PCR reagent set of 20 primer pairs, claim 3 or 4 in the described primer sets of claim 1 or 2 or test kit claimed in claim 5, grass seeds to be measured and 7 careful kinds of the careless state of high pellet are carried out the SSR amplification, the electrophoresis detection amplified production obtains 20 kinds of electrophoretograms of grass seeds to be measured and 7 high pellet careless states and examines in kind each 20 kinds and contain specific amplified band electrophoretogram;Each careful kind of high red careless state that the electrophoretogram of the grass seeds to be measured that described same primer pair amplification obtains and same primer pair amplification obtain to contain specific amplified band electrophoretogram corresponding;The specific amplified band electrophoretogram that contains in the electrophoretogram of the grass seeds to be measured that 2) amplification of each primer pair is obtained 7 careful kinds of the careless state of high pellet corresponding with it is compared respectively, contain that in specific amplified band electrophoretogram, the specific amplified band forms consistent band if the electrophoretic band of the electrophoretogram of the grass seeds to be measured that the amplification of at least 18 primer pairs obtains all has with its any corresponding high red careless state examines kind, grass seeds to be measured be or the candidate is the careful kind of corresponding high red careless state; Have to examine with its any corresponding high red careless state and contain that in specific amplified band electrophoretogram, the specific amplified band forms consistent band in kind if be less than the electrophoretic band of the electrophoretogram of the grass seeds to be measured that the amplification of 18 primer pairs obtains, grass seeds to be measured be or the candidate is not the careful kind of corresponding high red careless state;Described 7 high pellet careless states examine kinds and are specially day agriculture green grass or young crops and raise No. 2, No. 1, day agriculture, No. 3, Anhui grass, happy food, No. 2, Anhui grass, No. 2, Ji grass and Shanxi and herd No. 1.
- 9. one kind is detected or whether auxiliary detection grass seeds to be measured is the method for same kind with the contrast Pacetter varieties, comprises the steps:1) with the described PCR reagent set of 20 primer pairs, claim 3 or 4 in the described primer sets of claim 1 or 2 or test kit claimed in claim 5, grass seeds to be measured and contrast Pacetter varieties are carried out the SSR amplification, the 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;The electrophoretogram of the grass seeds to be measured that described same primer pair amplification obtains and same primer pair increase the contrast Pacetter varieties that obtains to contain specific amplified band electrophoretogram corresponding;The electrophoretogram of the grass seeds to be measured that 2) each primer pair amplification is obtained is compared respectively with the specific amplified band electrophoretogram that contains in the contrast Pacetter varieties, if the electrophoretic band of the electrophoretogram of the grass seeds to be measured that the amplification of at least 18 primer pairs obtains all have with its corresponding contrast Pacetter varieties contain that in specific amplified band electrophoretogram, the specific amplified band forms consistent band, grass seeds to be measured with the contrast Pacetter varieties is or the candidate is same kind; If be less than the electrophoretic band of the electrophoretogram of the grass seeds to be measured that the amplification of 18 primer pairs obtains have with its corresponding contrast Pacetter varieties contain that in specific amplified band electrophoretogram, the specific amplified band forms consistent band, grass seeds to be measured with the contrast Pacetter varieties be not or not the candidate be same kind.
- 10. according to claim 8 or 9 described methods, is characterized in that: described electrophoresis employing denaturing polyacrylamide gel; The template of described PCR is genomic dna.
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| CN103525928A (en) * | 2013-10-13 | 2014-01-22 | 兰州大学 | Kit for identifying the authenticity of Gaodan grass seeds and detection method thereof |
| CN105296482A (en) * | 2015-12-02 | 2016-02-03 | 全国畜牧总站 | Method for authenticating national examination and approval variety of oat for feeding and special primer group thereof |
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