CN104004838B - Sudan. grass Varieties verity detection method and primer special - Google Patents

Abstract

The invention discloses a kind of to detect or auxiliary detection arabian cron state examines kind method.The invention provides one group for detect or auxiliary detection arabian cron state examines the primer sets of kind, by primer pair P01-primer pair P18 totally 18 primer pairs form.Experiment of the present invention proves, the present invention filters out 18 pairs of SSR primers, with it, pcr amplification is carried out to herbage, the bands of a spectrum that the electrophoretogram that electrophoretogram and 9 kinds of arabian cron states of the 18 kinds of products obtained examine kind carries out object specific band compare, if in the electrophoretogram of 18 kinds of products at least 16 kinds consistent with the bands of a spectrum of object specific band, then can candidate judge this herbage with as same kind.Identify according to primer of the present invention and method, can simply, fast, whether efficient, precise Identification to go out arabian cron to be measured be that 9 states examine kind true or false.

Description

Sudan. grass Varieties verity detection method and primer special

Technical field

The present invention relates to biological technical field, particularly relate to a kind of Sudan. grass Varieties verity detection method and primer special.

Background technology

Arabian cron (Sorghum, Sudanense) is Gramineae annual herb, originates in African northern the Sudan (plateau) area, all has cultivated area extremely extensive in China from north to south.At northern area as blue or green forage grass and ensiling in winter grass in summer, and define the main seed produces base of China; Southern area, as domestic animal ensiling grass all the year round and excellent fish forage grass, have " king of fishery green fodder " laudatory title.At present there are 9 by the kind of country's authorization, are Ning Nong (improved variety) respectively, No. 2, Wu Late (improved variety), cover agriculture green grass or young crops and raise No. 3 (improved variety), cover agriculture green grass or young crops and raise No. 2 (improved variety), newly to revive No. 2 (improved variety), No. 1, Wu Late (improved variety), Qitai (improved variety), salt pond (improved variety), No. 1, interior agriculture (improved variety).

The Ministry of Agriculture started from 2006 to have carried out continuous selective examination to the grass seeds quality of the production and selling in the whole nation, the phenomenon that result shows the important grass seeds kinds such as arabian cron adulterated is more serious, illegal operators usually occurs utilizes fake and forged seed to cheat the farmers harmful agriculture, not only upset normal Seed Market trade, cause again very large financial loss.Owing to lacking authentication method quickly and accurately, this problem slowly can not get solving.

Traditional cultivar identification many employings Morphological Identification method, but its qualification cycle is long, costly and easily affected by environment.In other cultivar identification methods existing, the application of Isozyme and protein electrophoresis is comparatively wide, but the site detected is few, protein types few, polymorphism level is low, effectively can not distinguish same crop different varieties; RFLP technology is more loaded down with trivial details, requires higher to personnel equipment; RAPD technology is relatively simple, but poor reliability is difficult to widespread use in cultivar identification.

SSR fingerprint pattern technology is widely used in crop breeding and cultivar identification.Utilize the superiority of the SSR finger printing differential variety true and false to be: 1. quantity is large, and polymorphism level is high, can identify that phenotype is difficult to the kind differentiated; 2. not by the impact of any environmental factors, qualification of can drawing materials in any stage of growing; 3. isolated sample DNA can be preserved for a long time, this for carry out retrospective or arbitration property qualification highly beneficial; Not only accurately and reliably, but also be convenient to realize automatization 4..

Summary of the invention

An object of the present invention is to provide for detect or whether auxiliary detection grass seeds to be measured is the primer sets that arabian cron state examines kind.

Primer sets provided by the invention, by primer pair P01-primer pair P18 totally 18 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;

The equal independent packaging of each bar primer in above-mentioned primer sets;

Described arabian cron state examines that kind is Qitai, new No. 2, Soviet Union, No. 1, Ning Nong, Wu Late, salt pond, No. 2, Wu Late, No. 1, interior agriculture, cover agriculture green grass or young crops and raise No. 2 or cover agriculture green grass or young crops and raise No. 3.

Another object of the present invention is to provide a kind of for detect or whether auxiliary detection grass seeds to be measured is the PCR reagent group that arabian cron state examines kind.

PCR reagent group provided by the invention, is made up of following 18 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.

In above-mentioned PCR reagent group, the final concentration of every bar primer in the described PCR reagent of correspondence in each primer pair is 0.25 μm of ol/L; The equal independent packaging of described PCR reagent; Described arabian cron state examines that kind is Qitai, new No. 2, Soviet Union, No. 1, Ning Nong, Wu Late, salt pond, No. 2, Wu Late, No. 1, interior agriculture, cover agriculture green grass or young crops and raise No. 2 or cover agriculture green grass or young crops and raise No. 3.

Test kit containing above-mentioned primer sets or the test kit containing above-mentioned PCR reagent group are also the scope of protection of the invention.

Whether above-mentioned primer sets, above-mentioned PCR reagent group or above-mentioned test kit are the application examined in kind of arabian cron state in detection or auxiliary detection testing sample is also the scope of protection of the invention.

In above-mentioned application: described arabian cron state examines that kind is Qitai, new No. 2, Soviet Union, No. 1, Ning Nong, Wu Late, salt pond, No. 2, Wu Late, No. 1, interior agriculture, cover agriculture green grass or young crops and raise No. 2 or cover agriculture green grass or young crops and raise No. 3.

Another object of the present invention is to provide a kind of to detect or whether auxiliary detection grass seeds to be measured is the method that 9 arabian cron states to examine in kind any one.

Method provided by the invention, comprises the steps:

1) with the primer pair of 18 in above-mentioned primer sets, above-mentioned PCR reagent group or test kit according to claim 5, kind is examined to grass seeds to be measured and 9 arabian cron states and carry out SSR amplification, electrophoresis detection amplified production, obtain 18 kinds of electrophoretograms of grass seeds to be measured and 9 arabian cron states examine each in kind 18 kinds containing specific amplified band electrophoretogram;

Each arabian cron state that electrophoretogram and the same primer pair amplifies of the grass seeds to be measured that described same primer pair amplifies obtains obtain examines the corresponding containing specific amplified band electrophoretogram of kind;

2) 9 arabian cron states that the electrophoretogram of the grass seeds to be measured obtained by each primer pair amplifies is corresponding with it examine comparing respectively containing specific amplified band electrophoretogram in kind, if the collection of illustrative plates of the grass seeds to be measured that at least 16 primer pair amplifies obtain is consistent respectively with the collection of illustrative plates that these 16 pairs of primer amplifications that certain arabian cron state examines kind obtain, then grass seeds to be measured is or candidate examines kind for this arabian cron state; If the electrophoretic band being less than the electrophoretogram of the grass seeds to be measured that 16 primer pair amplifies obtain and certain arabian cron state examine and form consistent containing specific amplified band in specific amplified band electrophoretogram in kind, then grass seeds to be measured is not or candidate does not examine kind for this arabian cron state;

Described 9 arabian cron states examine kind and are specially Qitai, new No. 2, Soviet Union, No. 1, Ning Nong, Wu Late, salt pond, No. 2, Wu Late, No. 1, interior agriculture, cover agriculture green grass or young crops and raise No. 2 and cover agriculture green grass or young crops and raise No. 3.

3rd object of the present invention be to provide a kind of detect or auxiliary detection grass seeds to be measured with contrast the method whether Sudan. grass Varieties is same kind.

Method provided by the invention, comprises the steps:

1) with the primer pair of 18 in above-mentioned primer sets, above-mentioned PCR reagent group or above-mentioned test kit, SSR amplification is carried out to grass seeds to be measured and contrast Sudan. grass Varieties, electrophoresis detection amplified production, obtains 18 kinds of electrophoretograms of grass seeds to be measured and 18 kinds of contrast Sudan. grass Varieties contain specific amplified band electrophoretogram;

The electrophoretogram of the grass seeds to be measured that described same primer pair amplifies obtains and the corresponding containing specific amplified band electrophoretogram of the contrast Sudan. grass Varieties that same primer pair amplifies obtains;

2) electrophoretogram of the grass seeds to be measured obtained by each primer pair amplifies being compared respectively with the specific amplified band electrophoretogram that contains contrasted in Sudan. grass Varieties, if what the electrophoretic band of the electrophoretogram of the grass seeds to be measured that at least 16 primer pair amplifies obtain all had a contrast Sudan. grass Varieties corresponding with it forms consistent band containing specific amplified band in specific amplified band electrophoretogram, then grass seeds to be measured with contrast Sudan. grass Varieties and is or candidate is same kind; If the electrophoretic band being less than the electrophoretogram of the grass seeds to be measured that 16 primer pair amplifies obtain there is the contrast Sudan. grass Varieties corresponding with it form consistent band containing specific amplified band in specific amplified band electrophoretogram, then grass seeds to be measured with contrast Sudan. grass Varieties and is not or candidate is same kind.

In aforesaid method, described electrophoresis adopts denaturing polyacrylamide gel; The template of described PCR is genomic dna.

Testing sample is arabian cron or doubtful Sudan. grass Varieties.

Each arabian cron state examines kind also can examine kind for the arabian cron state determined for the arabian cron state of correspondence examine kind.

The composition that above-mentioned each arabian cron state examines specific amplified band in the electrophoretogram of 18 pairs of primer amplifications of kind is as shown in table 4.

Experiment of the present invention proves, the present invention filters out 18 pairs of SSR primers, with it, pcr amplification is carried out to herbage, the bands of a spectrum that the electrophoretogram that electrophoretogram and 9 kinds of arabian cron states of the 18 kinds of products obtained examine kind carries out object specific band compare, if in the electrophoretogram of 18 kinds of products at least 16 kinds consistent with the bands of a spectrum of object specific band, then candidate can judge this herbage with it as same kind, if be less than in the electrophoretogram of 18 kinds of products 16 kinds consistent with the bands of a spectrum of object specific band, then candidate can judge this herbage with it as different varieties.Identify according to primer of the present invention and method, can simply, fast, whether efficient, precise Identification to go out arabian cron to be measured be that 9 states examine kind true or false.

Accompanying drawing explanation

Fig. 1 is the denaturing polyacrylamide gel electrophoresis result of P01 primer amplification sample 1-10

Fig. 2 is the denaturing polyacrylamide gel electrophoresis result of P02 primer amplification sample 1-10

Fig. 3 is the denaturing polyacrylamide gel electrophoresis result of P03 primer amplification sample 1-10

Fig. 4 is the denaturing polyacrylamide gel electrophoresis result of P04 primer amplification sample 1-10

Fig. 5 is the denaturing polyacrylamide gel electrophoresis result of P05 primer amplification sample 1-10

Fig. 6 is the denaturing polyacrylamide gel electrophoresis result of P06 primer amplification sample 1-10

Fig. 7 is the denaturing polyacrylamide gel electrophoresis result of P07 primer amplification sample 1-10

Fig. 8 is the denaturing polyacrylamide gel electrophoresis result of P08 primer amplification sample 1-10

Fig. 9 is the denaturing polyacrylamide gel electrophoresis result of P09 primer amplification sample 1-10

Figure 10 is the denaturing polyacrylamide gel electrophoresis result of P10 primer amplification sample 1-10

Figure 11 is the denaturing polyacrylamide gel electrophoresis result of P11 primer amplification sample 1-10

Figure 12 is the denaturing polyacrylamide gel electrophoresis result of P12 primer amplification sample 1-10

Figure 13 is the denaturing polyacrylamide gel electrophoresis result of P13 primer amplification sample 1-10

Figure 14 is the denaturing polyacrylamide gel electrophoresis result of P14 primer amplification sample 1-10

Figure 15 is the denaturing polyacrylamide gel electrophoresis result of P15 primer amplification sample 1-10

Figure 16 is the denaturing polyacrylamide gel electrophoresis result of P16 primer amplification sample 1-10

Figure 17 is the denaturing polyacrylamide gel electrophoresis result of P17 primer amplification sample 1-10

Figure 18 is the denaturing polyacrylamide gel electrophoresis result of P18 primer amplification sample 1-10

Embodiment

The experimental technique used in following embodiment if no special instructions, is ordinary method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

In following embodiment, 9 arabian cron states used examine kind: No. 2, Ning Nong, Wu Late, cover agriculture green grass or young crops raise No. 3, cover agriculture green grass or young crops raise No. 2,

No. 2, new Soviet Union, Qitai, salt pond, No. 1, Wu Late, No. 1, interior agriculture, all can buy from market;

Table 1 examines kind reference numeral for state

Embodiment 1, examine the screening of the SSR primer of kind for detecting arabian cron state

Filter out from 108 pairs of sorghum primers 18 to have polymorphism, amplification stablize, be easy to add up primer for cultivar identification, the sequence of 18 pairs of SSR primers is in table 2.

Table 2 is the SSR primer for detecting

Embodiment 2, examine the application of the SSR primer of kind for detecting arabian cron state

One, SSR primer detects the specificity research that arabian cron state examines kind

Respectively 9 of table 1 arabian cron states are examined the seed distilled water immersion that kind and 1 Ge Fei arabian cron state examine the grass seeds sample of kind, be placed under 4 DEG C of storage cabinets plant for 7 days afterwards.The seed trays of having planted is placed in incubator, and set temperature is 23 DEG C.Get 40 strain blades after 7 days and mix sample, Liquid nitrogen storage or directly carry out next step analysis.

1, the extraction of STb gene

(1) 2 × CTAB solution and β-thin base ethanol are joined solution with the ratio of 85:1, in 65 DEG C of water-baths, are added hot reserve;

(2) collect in the 2ml centrifuge tube of blade material and add little steel ball, then put into liquid nitrogen 5 minutes.Take out, forward to rapidly on beveller, ground material is powdered;

(3) solution got in 700 μ l (1) adds in each centrifuge tube, then puts into 65 DEG C of water-baths, and 85rpm shakes incubation 2 hours;

(4) often pipe adds isopyknic CI (chloroform: primary isoamyl alcohol=24:1), and shake mixing: 4 DEG C, centrifugal 10 minutes of 12000rpm, takes out aqueous phase, proceed in new centrifuge tube;

(5) repeating step (4);

(6) Virahol of equal-volume precooling is added ,-20 DEG C, 1 hour;

(7) 4 DEG C, the centrifugal 10min of 12000rpm, abandons supernatant liquor;

(8) precipitate with the ethanol purge of 70%, 4 DEG C, the centrifugal 10min of 12000rpm, abandons supernatant liquor;

(9) air-dry, dissolve with 0.1 appropriate × TE, measure concentration, in 4 DEG C of short-term preservations, preserve for a long time, obtain genomic dna for-20 DEG C.

2, SSR amplification

The mixed sample genomic dna of each sample obtained with 1 is template, uses 18 pairs of SSR primer pairs (P01-P18) in table 2 to carry out pcr amplification according to the condition of table 3 and table 4 respectively.

(1) reaction system

Test reaction volume 20 μ l, each component configuration is in table 3.

Table 3PCR amplification reaction system

(2) reaction conditions

94 DEG C of denaturation 5min, a circulation; 94 DEG C of sex change 40s; 60 DEG C of annealing (annealing temperature is determined with material) 35s, 72 DEG C extend 45s, circulation (cycle number is determined with material); 72 DEG C extend 5min, 4 DEG C of preservations.

The sex change of 2.4PCR product

In PCR primer, add 4 μ l denaturation buffer, fully in PCR instrument, run denaturation program, 95 DEG C of sex change 5min after mixing, 4 DEG C of cooling more than 10min.

Obtain the SSR amplified production that each sample 40 strain mixes 18 pairs of primers of sample.

3, denaturing polyacrylamide gel electrophoresis

Each sample 40 strain obtained above-mentioned 2 mixes SSR amplified production 4.5% denaturing polyacrylamide gel electrophoresis of 18 pairs of primers of sample, DNA molecular amount standard used is pUC18DNA/MspI (TIANGEN Biotech (Beijing) Co., Ltd., catalog number MD203-02).

The numbering of 10 samples is as follows respectively: sample 1 is peaceful agriculture, sample 2 for non-state examine kind arabian cron, sample 3 is No. 2, Wu Late, in order to cover, agriculture green grass or young crops raises No. 3 to sample 4, in order to cover, agriculture green grass or young crops raises No. 2 to sample 5, sample 6 is new No. 2, Soviet Union, sample 7 is Qitai, sample 8 is salt pond, sample 9 is No. 1, Wu Late, sample 10 is No. 1, interior agriculture.

Electrophoresis result as shown in Fig. 1-18,

Fig. 1 is the denaturing polyacrylamide gel electrophoresis result of P01 primer amplification sample 1-10; Remove the Marker on both sides in figure, 10 middle swimming lanes are from left to right followed successively by: No. 1, interior agriculture, No. 1, Wu Late, salt pond, Qitai, new No. 2, Soviet Union, cover agriculture green grass or young crops and raise No. 2, cover that agriculture green grass or young crops raises No. 3, No. 2, Wu Late, non-state examine kind arabian cron, Ning Nong;

Fig. 2 is the denaturing polyacrylamide gel electrophoresis result of P02 primer amplification sample 1-10; Remove the Marker on both sides in figure, 10 middle swimming lanes are from left to right followed successively by: No. 1, interior agriculture, No. 1, Wu Late, salt pond, Qitai, new No. 2, Soviet Union, cover agriculture green grass or young crops and raise No. 2, cover that agriculture green grass or young crops raises No. 3, No. 2, Wu Late, non-state examine kind arabian cron, Ning Nong;

Fig. 3 is the denaturing polyacrylamide gel electrophoresis result of P03 primer amplification sample 1-10; Remove the Marker on both sides in figure, 10 middle swimming lanes are from left to right followed successively by: No. 1, interior agriculture, No. 1, Wu Late, salt pond, Qitai, new No. 2, Soviet Union, cover agriculture green grass or young crops and raise No. 2, cover that agriculture green grass or young crops raises No. 3, No. 2, Wu Late, non-state examine kind arabian cron, Ning Nong;

Fig. 4 is the denaturing polyacrylamide gel electrophoresis result of P04 primer amplification sample 1-10; Remove the Marker on both sides in figure, 10 middle swimming lanes are from left to right followed successively by: No. 1, interior agriculture, No. 1, Wu Late, salt pond, Qitai, new No. 2, Soviet Union, cover agriculture green grass or young crops and raise No. 2, cover that agriculture green grass or young crops raises No. 3, No. 2, Wu Late, non-state examine kind arabian cron, Ning Nong;

Fig. 5 is the denaturing polyacrylamide gel electrophoresis result of P05 primer amplification sample 1-10; Remove the Marker on both sides in figure, 10 middle swimming lanes are from left to right followed successively by: No. 1, interior agriculture, No. 1, Wu Late, salt pond, Qitai, new No. 2, Soviet Union, cover agriculture green grass or young crops and raise No. 2, cover that agriculture green grass or young crops raises No. 3, No. 2, Wu Late, non-state examine kind arabian cron, Ning Nong;

Fig. 6 is the denaturing polyacrylamide gel electrophoresis result of P06 primer amplification sample 1-10; Remove the Marker on both sides in figure, 10 middle swimming lanes are from left to right followed successively by: No. 1, interior agriculture, No. 1, Wu Late, salt pond, Qitai, new No. 2, Soviet Union, cover agriculture green grass or young crops and raise No. 2, cover that agriculture green grass or young crops raises No. 3, No. 2, Wu Late, non-state examine kind arabian cron, Ning Nong;

Fig. 7 is the denaturing polyacrylamide gel electrophoresis result of P07 primer amplification sample 1-10; Remove the Marker on both sides in figure, 10 middle swimming lanes are from left to right followed successively by: No. 1, interior agriculture, No. 1, Wu Late, salt pond, Qitai, new No. 2, Soviet Union, cover agriculture green grass or young crops and raise No. 2, cover that agriculture green grass or young crops raises No. 3, No. 2, Wu Late, non-state examine kind arabian cron, Ning Nong;

Fig. 8 is the denaturing polyacrylamide gel electrophoresis result of P08 primer amplification sample 1-10; Remove the Marker on both sides in figure, 10 middle swimming lanes are from left to right followed successively by: No. 1, interior agriculture, No. 1, Wu Late, salt pond, Qitai, new No. 2, Soviet Union, cover agriculture green grass or young crops and raise No. 2, cover that agriculture green grass or young crops raises No. 3, No. 2, Wu Late, non-state examine kind arabian cron, Ning Nong;

Fig. 9 is the denaturing polyacrylamide gel electrophoresis result of P09 primer amplification sample 1-10; Remove the Marker on both sides in figure, 10 middle swimming lanes are from left to right followed successively by: No. 1, interior agriculture, No. 1, Wu Late, salt pond, Qitai, new No. 2, Soviet Union, cover agriculture green grass or young crops and raise No. 2, cover that agriculture green grass or young crops raises No. 3, No. 2, Wu Late, non-state examine kind arabian cron, Ning Nong;

Figure 10 is the denaturing polyacrylamide gel electrophoresis result of P10 primer amplification sample 1-10; Remove the Marker on both sides in figure, 10 middle swimming lanes are from left to right followed successively by: No. 1, interior agriculture, No. 1, Wu Late, salt pond, Qitai, new No. 2, Soviet Union, cover agriculture green grass or young crops and raise No. 2, cover that agriculture green grass or young crops raises No. 3, No. 2, Wu Late, non-state examine kind arabian cron, Ning Nong;

Figure 11 is the denaturing polyacrylamide gel electrophoresis result of P11 primer amplification sample 1-10; Remove the Marker on both sides in figure, 10 middle swimming lanes are from left to right followed successively by: No. 1, interior agriculture, No. 1, Wu Late, salt pond, Qitai, new No. 2, Soviet Union, cover agriculture green grass or young crops and raise No. 2, cover that agriculture green grass or young crops raises No. 3, No. 2, Wu Late, non-state examine kind arabian cron, Ning Nong;

Figure 12 is the denaturing polyacrylamide gel electrophoresis result of P12 primer amplification sample 1-10; Remove the Marker on both sides in figure, 10 middle swimming lanes are from left to right followed successively by: No. 1, interior agriculture, No. 1, Wu Late, salt pond, Qitai, new No. 2, Soviet Union, cover agriculture green grass or young crops and raise No. 2, cover that agriculture green grass or young crops raises No. 3, No. 2, Wu Late, non-state examine kind arabian cron, Ning Nong;

Figure 13 is the denaturing polyacrylamide gel electrophoresis result of P13 primer amplification sample 1-10; Remove the Marker on both sides in figure, 10 middle swimming lanes are from left to right followed successively by: No. 1, interior agriculture, No. 1, Wu Late, salt pond, Qitai, new No. 2, Soviet Union, cover agriculture green grass or young crops and raise No. 2, cover that agriculture green grass or young crops raises No. 3, No. 2, Wu Late, non-state examine kind arabian cron, Ning Nong;

Figure 14 is the denaturing polyacrylamide gel electrophoresis result of P14 primer amplification sample 1-10; Remove the Marker on both sides in figure, 10 middle swimming lanes are from left to right followed successively by: No. 1, interior agriculture, No. 1, Wu Late, salt pond, Qitai, new No. 2, Soviet Union, cover agriculture green grass or young crops and raise No. 2, cover that agriculture green grass or young crops raises No. 3, No. 2, Wu Late, non-state examine kind arabian cron, Ning Nong;

Figure 15 is the denaturing polyacrylamide gel electrophoresis result of P15 primer amplification sample 1-10; Remove the Marker on both sides in figure, 10 middle swimming lanes are from left to right followed successively by: No. 1, interior agriculture, No. 1, Wu Late, salt pond, Qitai, new No. 2, Soviet Union, cover agriculture green grass or young crops and raise No. 2, cover that agriculture green grass or young crops raises No. 3, No. 2, Wu Late, non-state examine kind arabian cron, Ning Nong;

Figure 16 is the denaturing polyacrylamide gel electrophoresis result of P16 primer amplification sample 1-10; Remove the Marker on both sides in figure, 10 middle swimming lanes are from left to right followed successively by: No. 1, interior agriculture, No. 1, Wu Late, salt pond, Qitai, new No. 2, Soviet Union, cover agriculture green grass or young crops and raise No. 2, cover that agriculture green grass or young crops raises No. 3, No. 2, Wu Late, non-state examine kind arabian cron, Ning Nong;

Figure 17 is the denaturing polyacrylamide gel electrophoresis result of P17 primer amplification sample 1-10; Remove the Marker on both sides in figure, 10 middle swimming lanes are from left to right followed successively by: No. 1, interior agriculture, No. 1, Wu Late, salt pond, Qitai, new No. 2, Soviet Union, cover agriculture green grass or young crops and raise No. 2, cover that agriculture green grass or young crops raises No. 3, No. 2, Wu Late, non-state examine kind arabian cron, Ning Nong;

Figure 18 is the denaturing polyacrylamide gel electrophoresis result of P18 primer amplification sample 1-10; Remove the Marker on both sides in figure, 10 middle swimming lanes are from left to right followed successively by: No. 1, interior agriculture, No. 1, Wu Late, salt pond, Qitai, new No. 2, Soviet Union, cover agriculture green grass or young crops and raise No. 2, cover that agriculture green grass or young crops raises No. 3, No. 2, Wu Late, non-state examine kind arabian cron, Ning Nong;

After above-mentioned experiment is repeated 5 times, 9 states being examined kind and 1 Ge Fei state, to examine the bands of a spectrum that the object specific band sequencing result in 18 pairs of primer SSR amplified production collection of illustrative plates of kind adds up as shown in table 4.

Table 4 is the bands of a spectrum (unit: bp) that 9 states examine that kind and 1 Ge Fei state examine the SSR object specific band of kind arabian cron

Represent there is no object specific band.

Therefore, aforesaid method can have following purposes:

1) be used for detect or auxiliary detection grass seeds to be measured whether be that arabian cron state examines kind, concrete grammar is as follows:

A, respectively kind is examined to grass seeds to be measured and 9 arabian cron states with 18 pairs of SSR primers and carry out SSR amplification, electrophoresis detection amplified production, obtain 18 kinds of electrophoretograms of grass seeds to be measured and 9 arabian cron states examine each in kind 18 kinds containing specific amplified band electrophoretogram; Each arabian cron state that electrophoretogram and the same primer pair amplifies of the grass seeds to be measured that described same primer pair amplifies obtains obtain examines the corresponding containing specific amplified band electrophoretogram of kind;

B, 9 arabian cron states that the electrophoretogram of grass seeds to be measured obtained by each primer pair amplifies is corresponding with it examine comparing respectively containing specific amplified band electrophoretogram in kind, if the collection of illustrative plates of the grass seeds to be measured that at least 16 primer pair amplifies obtain is consistent respectively with the collection of illustrative plates that these 16 pairs of primer amplifications that certain arabian cron state examines kind obtain, then grass seeds to be measured is or candidate examines kind for this arabian cron state; If the electrophoretic band being less than the electrophoretogram of the grass seeds to be measured that 16 primer pair amplifies obtain has to examine with certain arabian cron state form consistent band containing specific amplified band in specific amplified band electrophoretogram in kind, then grass seeds to be measured not for or candidate do not examine kind for this arabian cron state;

9 arabian cron states examine kind: No. 2, Ning Nong, Wu Late, cover agriculture green grass or young crops raise No. 3, cover agriculture green grass or young crops raise No. 2, new No. 2, Soviet Union, Qitai, salt pond, No. 1, Wu Late, No. 1, interior agriculture.

2) can be used for detect or auxiliary detection grass seeds to be measured whether be same kind with contrasting Sudan. grass Varieties, concrete grammar is as follows:

A) respectively SSR amplification is carried out to grass seeds to be measured and contrast Sudan. grass Varieties with the primer pair of 18 in above-mentioned primer sets, above-mentioned PCR reagent group or above-mentioned test kit, electrophoresis detection amplified production, obtains 18 kinds of electrophoretograms of grass seeds to be measured and 18 kinds of electrophoretograms of contrast Sudan. grass Varieties;

The electrophoretogram of the grass seeds to be measured that described same primer pair amplifies obtains is corresponding with the electrophoretogram of the contrast Sudan. grass Varieties that same primer pair amplifies obtains;

The electrophoretogram of the grass seeds to be measured B) obtained by each primer pair amplifies is compared respectively with the electrophoretogram contrasting Sudan. grass Varieties, if the electrophoretogram that the electrophoretic band of the electrophoretogram of the grass seeds to be measured that at least 16 primer pair amplifies obtain all has the contrast Sudan. grass Varieties corresponding with it forms consistent band, then grass seeds to be measured with contrast Sudan. grass Varieties for or candidate be same kind; If the electrophoretogram that the electrophoretic band being less than the electrophoretogram of the grass seeds to be measured that 16 primer pair amplifies obtain has the contrast Sudan. grass Varieties corresponding with it forms consistent band, then grass seeds to be measured with contrast Sudan. grass Varieties not for or candidate for same kind;

Above-mentioned contrast Sudan. grass Varieties is that the arabian cron state that arabian cron state examines kind or phenotypic evaluation examines kind.

The test kit (primer pair independent packaging) of all 18 pairs of SSR primers adopted containing above-mentioned SSR amplification or the test kit of PCR reagent group (the equal independent packaging of each PCR reagent in PCR reagent group) adopted containing above-mentioned SSR amplification all can be used to detect or whether auxiliary detection is that arabian cron state examines kind or object arabian cron state examines kind;

Above-mentioned PCR reagent group is made up of 18 kinds of PCR reagent, and each PCR reagent is become to be grouped into by following: the 1 pair of SSR primer pair (in table 2 in 20 pairs of SSR primer pairs any one), 10 × PCR buffer, dNTP, Taq archaeal dna polymerase and water form; Wherein, the final concentration of every bar SSR primer in PCR reagent is 0.25 μ lmol/L; The final concentration of each dNTP in PCR reagent is 0.15mmol/L; The final concentration of archaeal dna polymerase in PCR reagent is 1.0U.

Whether embodiment 3, detection sample to be tested are that arabian cron state examines kind

10 samples to be tested are that phenotypic evaluation has been the grass seeds that following 9 arabian cron states examine kind and unknown arabian cron:

Ning Nong: plant height 250-320cm; The wide bar shaped of blade, long 40-80cm; Red or the red-purple of grain husk shell, above close raw grey pubescence, seed falls oval, thousand seed weight 18-22g.

No. 2, Wu Late: plant height 290cm; The long 50-60cm of leaf, Glabrous; Caryopsis obovate, seed grain husk shell color is red, thousand seed weight 12.6g.

Cover agriculture green grass or young crops and raise No. 3: plant height 380cm; Blade is long lanceolar; Seed oblong, slightly flat, clever shell is black, red-brown, oyster white, thousand seed weight about 22g.

Cover agriculture green grass or young crops and raise No. 2: plant height 350cm; The long 75-110cm of leaf, the long lanceolar of blade; Seed oblong, slightly flat, tawny, grain husk white or milk yellow, slightly brown speckle, thousand seed weight 21g.No. 2, new Soviet Union: plant height 225-270cm; Blade is slightly wide, light green; The many black of seed grain husk shell, thousand seed weight 12.5-14g.

Qitai: plant height 213cm; Fruit oval, variegated, tool is yellow, red, black, thousand seed weight about 12.4g.

Salt pond: plant height 160-190cm; Stem leaf band shape is smooth open and flat; Caryopsis black bright, oval, thousand seed weight is about 15g.

No. 1, Wu Late: plant height is about 295cm; Grain husk shell is black.

No. 1, interior agriculture: plant height 315-350cm; The long 70-78cm of leaf, two sides without hair, the sharp-pointed seta of edge tool; Caryopsis is oval, and planting shell color is black (yellow, red) look, thousand seed weight 24g.

Unknown arabian cron (negative findings): stem stalk is elongated, and plant height is about 262cm, has 9 stipes, diameter 1cm, saves long 23cm; Blade is long and narrow, the long 20cm of leaf sheath, the long 26cm of leaf, the wide 5.1cm of leaf.Panicle, every strain fringe 9 fringe, spike length 37.5cm, seed oval, filbert to black, thousand grain weigth 12.58g.

The 40 strain blades collecting each sample after the plantation of the seed of each sample to be tested above-mentioned are mixed sample test as follows.

1, the extraction of STb gene:

According to embodiment 2 one 1 40 strains that method extracts sample to be tested respectively and 9 kinds of arabian cron states examine kind mix the STb gene of sample blade.

2, SSR amplification: with according to embodiment 2 above-mentioned one 2 identical;

3, denaturing polyacrylamide gel electrophoresis: method is identical with above-mentioned one 3;

Obtain 18 kinds of electrophoretograms of each sample to be tested and each arabian cron state examine kind containing specific amplified band electrophoretogram.Each arabian cron state that electrophoretogram and the same primer pair amplifies of the grass seeds to be measured that same primer pair amplifies obtains obtain examines the corresponding containing specific amplified band electrophoretogram of kind;

9 arabian cron states that the electrophoretogram of the grass seeds to be measured obtained by each primer pair amplifies is corresponding with it examine comparing respectively containing specific amplified band electrophoretogram in kind, if the collection of illustrative plates of the grass seeds to be measured that at least 16 primer pair amplifies obtain is consistent respectively with the collection of illustrative plates that these 16 pairs of primer amplifications that certain arabian cron state examines kind obtain, then grass seeds to be measured is or candidate examines kind for this arabian cron state; If the electrophoretic band being less than the electrophoretogram of the grass seeds to be measured that 16 primer pair amplifies obtain and certain arabian cron state examine and form consistent containing specific amplified band in specific amplified band electrophoretogram in kind, then grass seeds to be measured is not or candidate does not examine kind for this arabian cron state.

18 of each sample to be tested kinds of electrophoretograms and each arabian cron state are examined comparing containing specific amplified band electrophoretogram (bands of a spectrum of specific amplified band are shown in the table 4 obtained of embodiment 2) of kind, result is as follows:

All have in electrophoretic band in the electrophoretogram of SSR primer P01, P02, P03, P04, P05, P07, P08, P09, P10, P11, P12, P13, P14, P15, P16, P17, P18 acquisition of sample to be tested 1 and form consistent band (electrophoretogram that the sample to be tested electrophoretogram that same primer obtains and arabian cron state examine kind compares) with specific amplified band in the electrophoretogram of the peaceful agriculture shown in the table 4 of corresponding embodiment 2, sample to be tested 1 is described really for state examines the peaceful agriculture of kind;

All have in electrophoretic band in the electrophoretogram of SSR primer P01, P03, P04, P05, P07, P08, P09, P10, P11, P12, P13, P14, P15, P16, P17, P18 acquisition of sample to be tested 2 and form consistent band (electrophoretogram that the sample to be tested electrophoretogram that same primer obtains and arabian cron state examine kind compares) with specific amplified band in the electrophoretogram of No. 2, the Wu Late shown in the table 4 of corresponding embodiment 2, sample to be tested 2 is described really for state examines No. 2, kind Wu Late;

All have 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 of sample to be tested 3 obtain and raise specific amplified band in the electrophoretogram of No. 3 with the illiteracy agriculture green grass or young crops shown in the table 4 of corresponding embodiment 2 and form consistent band (electrophoretogram that the sample to be tested electrophoretogram that same primer obtains and arabian cron state examine kind compares), illustrate that sample to be tested 3 raises No. 3 for state examines kind illiteracy agriculture green grass or young crops really;

All have 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 of sample to be tested 4 obtain and raise specific amplified band in the electrophoretogram of No. 2 with the illiteracy agriculture green grass or young crops shown in the table 4 of corresponding embodiment 2 and form consistent band (electrophoretogram that the sample to be tested electrophoretogram that same primer obtains and arabian cron state examine kind compares), illustrate that sample to be tested 4 raises No. 2 for state examines kind illiteracy agriculture green grass or young crops really;

All have in electrophoretic band in the electrophoretogram of SSR primer P01, P02, P03, P04, P05, P07, P08, P09, P10, P11, P12, P13, P14, P15, P16, P17, P18 acquisition of sample to be tested 5 and form consistent band (electrophoretogram that the sample to be tested electrophoretogram that same primer obtains and arabian cron state examine kind compares) with specific amplified band in the electrophoretogram of No. 2, the new Soviet Union shown in the table 4 of corresponding embodiment 2, illustrate that sample to be tested 5 newly revives No. 2 for state examines kind really;

All have in electrophoretic band in the electrophoretogram of SSR primer P01, P02, P03, P04, P05, P07, P08, P09, P10, P11, P12, P13, P14, P15, P16, P17, P18 acquisition of sample to be tested 6 and form consistent band (electrophoretogram that the sample to be tested electrophoretogram that same primer obtains and arabian cron state examine kind compares) with specific amplified band in the electrophoretogram of the Qitai shown in the table 4 of corresponding embodiment 2, sample to be tested 6 is described really for state examines kind Qitai;

All have in electrophoretic band in the electrophoretogram of SSR primer P01, P02, P03, P04, P05, P07, P08, P09, P10, P11, P12, P13, P14, P15, P16, P17, P18 acquisition of sample to be tested 7 and form consistent band (electrophoretogram that the sample to be tested electrophoretogram that same primer obtains and arabian cron state examine kind compares) with specific amplified band in the electrophoretogram in the salt pond shown in the table 4 of corresponding embodiment 2, sample to be tested 7 is described really for state examines kind salt pond;

All have in electrophoretic band in the electrophoretogram of SSR primer P01, P02, P03, P04, P05, P07, P08, P09, P10, P11, P12, P13, P14, P15, P16, P17, P18 acquisition of sample to be tested 8 and form consistent band (electrophoretogram that the sample to be tested electrophoretogram that same primer obtains and arabian cron state examine kind compares) with specific amplified band in the electrophoretogram of No. 1, the Wu Late shown in the table 4 of corresponding embodiment 2, sample to be tested 8 is described really for state examines No. 1, kind Wu Late;

All have 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 of sample to be tested 9 obtain with shown in the table 4 of corresponding embodiment 2 in No. 1, agriculture electrophoretogram in specific amplified band form consistent band (electrophoretogram that the sample to be tested electrophoretogram that same primer obtains and arabian cron state examine kind compares), sample to be tested 9 is described really for state examines No. 1, agriculture in kind;

The SSR primer P01 of sample to be tested 10, P02, P03, P04, P05, P07, P08, P09, P10, P11, P12, P13, P14, P15, P16, P17, compared with examining with 9 states shown in the table 4 of corresponding embodiment 2 band (electrophoretogram that the sample to be tested electrophoretogram that same primer obtains and arabian cron state examine kind compares) that in the electrophoretogram of kind, specific amplified band forms in electrophoretic band in the electrophoretogram that P18 obtains, all be not equal to or greater than 16 consistent sites, illustrate sample to be tested 10 be not really 9 kinds of states examine in kind any one: Ning Nong, No. 2, Wu Late, cover agriculture green grass or young crops and raise No. 3, cover agriculture green grass or young crops and raise No. 2, No. 2, new Soviet Union, Qitai, salt pond, No. 1, Wu Late, No. 1, interior agriculture.

The above results illustrates, method of the present invention is correct, can verify whether sample to be tested is that 9 kinds of states examine kind, thus whether checking sample to be tested is the verity of arabian cron.

Claims (9)

1. for detect or whether auxiliary detection grass seeds to be measured is the primer sets that arabian cron state examines kind, by primer pair P01-primer pair P18 totally 18 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 arabian cron state examines that kind is Qitai, new No. 2, Soviet Union, No. 1, Ning Nong, Wu Late, salt pond, No. 2, Wu Late, No. 1, interior agriculture, cover agriculture green grass or young crops and raise No. 2 or cover agriculture green grass or young crops and raise No. 3.

2. primer sets according to claim 1, is characterized in that: the equal independent packaging of each bar primer in described primer sets.

3., for detect or whether auxiliary detection grass seeds to be measured is the PCR reagent group that arabian cron state examines kind, be made up of following 18 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; Described arabian cron state examines that kind is Qitai, new No. 2, Soviet Union, No. 1, Ning Nong, Wu Late, salt pond, No. 2, Wu Late, No. 1, interior agriculture, cover agriculture green grass or young crops and raise No. 2 or cover agriculture green grass or young crops and raise No. 3.

4. PCR reagent group according to claim 3, is characterized in that: the final concentration of every bar primer in the described PCR reagent of correspondence in each primer pair is 0.25 μm of ol/L;

The equal independent packaging of described PCR reagent.

5. the test kit containing primer sets described in claim 1 or 2 or the test kit containing PCR reagent group described in claim 3 or 4.

6. whether PCR reagent group described in primer sets, claim 3 or 4 described in claim 1 or 2 or test kit according to claim 5 are the application that arabian cron state examines in kind in detection or auxiliary detection testing sample; Described arabian cron state examines that kind is Qitai, new No. 2, Soviet Union, No. 1, Ning Nong, Wu Late, salt pond, No. 2, Wu Late, No. 1, interior agriculture, cover agriculture green grass or young crops and raise No. 2 or cover agriculture green grass or young crops and raise No. 3.

7. to detect or whether auxiliary detection grass seeds to be measured is the method that 9 arabian cron states to examine in kind any one, comprise the steps:

1) with PCR reagent group described in the primer pair of 18 in primer sets described in claim 1 or 2, claim 3 or 4 or test kit according to claim 5, kind is examined to grass seeds to be measured and 9 arabian cron states and carry out SSR amplification, electrophoresis detection amplified production, obtain 18 kinds of electrophoretograms of grass seeds to be measured and 9 arabian cron states examine each in kind 18 kinds containing specific amplified band electrophoretogram;

Each arabian cron state that electrophoretogram and the same primer pair amplifies of the grass seeds to be measured that described same primer pair amplifies obtains obtain examines the corresponding containing specific amplified band electrophoretogram of kind;

2) 9 arabian cron states that the electrophoretogram of the grass seeds to be measured obtained by each primer pair amplifies is corresponding with it examine comparing respectively containing specific amplified band electrophoretogram in kind, if the collection of illustrative plates of the grass seeds to be measured that at least 16 primer pair amplifies obtain is consistent respectively with the collection of illustrative plates that these 16 pairs of primer amplifications that certain arabian cron state examines kind obtain, then grass seeds to be measured is or candidate examines kind for this arabian cron state; If the electrophoretic band being less than the electrophoretogram of the grass seeds to be measured that 16 primer pair amplifies obtain and certain arabian cron state examine and form consistent containing specific amplified band in specific amplified band electrophoretogram in kind, then grass seeds to be measured is not or candidate does not examine kind for this arabian cron state;

Described 9 arabian cron states examine that kind is Qitai, new No. 2, Soviet Union, No. 1, Ning Nong, Wu Late, salt pond, No. 2, Wu Late, No. 1, interior agriculture, cover agriculture green grass or young crops and raise No. 2 and cover agriculture green grass or young crops and raise No. 3.

8. detect or auxiliary detection grass seeds to be measured with contrast the method whether Sudan. grass Varieties is same kind, comprise the steps:

1) with PCR reagent group described in the primer pair of 18 in primer sets described in claim 1 or 2, claim 3 or 4 or test kit according to claim 5, SSR amplification is carried out to grass seeds to be measured and contrast Sudan. grass Varieties, electrophoresis detection amplified production, obtains 18 kinds of electrophoretograms of grass seeds to be measured and 18 kinds of contrast Sudan. grass Varieties contain specific amplified band electrophoretogram;

The electrophoretogram of the grass seeds to be measured that described same primer pair amplifies obtains and the corresponding containing specific amplified band electrophoretogram of the contrast Sudan. grass Varieties that same primer pair amplifies obtains;

2) electrophoretogram of the grass seeds to be measured obtained by each primer pair amplifies being compared respectively with the specific amplified band electrophoretogram that contains contrasted in Sudan. grass Varieties, if what the electrophoretic band of the electrophoretogram of the grass seeds to be measured that at least 16 primer pair amplifies obtain all had a contrast Sudan. grass Varieties corresponding with it forms consistent band containing specific amplified band in specific amplified band electrophoretogram, then grass seeds to be measured with contrast Sudan. grass Varieties and is or candidate is same kind; If the electrophoretic band being less than the electrophoretogram of the grass seeds to be measured that 16 primer pair amplifies obtain there is the contrast Sudan. grass Varieties corresponding with it form consistent band containing specific amplified band in specific amplified band electrophoretogram, then grass seeds to be measured with contrast Sudan. grass Varieties and is not or candidate is same kind.

9. the method according to claim 7 or 8, is characterized in that: described electrophoresis adopts denaturing polyacrylamide gel; The template of described PCR is genomic dna.