CN103194541B

CN103194541B - Purple yam variety molecular identification method based on genome RAPD (random amplified polymorphic DNA) analysis

Info

Publication number: CN103194541B
Application number: CN201310139003.9A
Authority: CN
Inventors: 刘庞源; 宋曙辉; 赵泓; 王慧杰; 何伟明; 王文琪
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2013-04-19
Filing date: 2013-04-19
Publication date: 2014-10-22
Anticipated expiration: 2033-04-19
Also published as: CN103194541A

Abstract

The invention discloses a purple yam variety molecular identification method based on genome RAPD (random amplified polymorphic DNA) analysis, comprising the steps of: 1) sampling and treating a sample; 2) extracting the DNA and detecting the purity and concentration; 3) screening primers and analyzing RAPD; 4) acquiring a standard atlas of eight kinds of purple yam normal strains by utilizing a primer S1255; and 5) analyzing the to-be-identified novel purple yam varieties according to the steps, thereby obtaining an RAPD atlas of the novel purple yam varieties required to be identified. Through comparison and identification of the RARD atlas and the standard atlas, different purple yam varieties can be determined, and the purple yam variety molecular identification method based on genome RAPD analysis has the characteristics of speediness, stability and low expense.

Description

A kind of purple yam variety molecular assay method based on genome RAPD analysis

Technical field

The present invention relates to a kind of purple yam variety molecular assay method of analyzing based on Genome RAPD (randomly amplified polymorphic DNA mark, random amplified polymorphic DNA), belong to plant variety Molecular Identification technical field.

Background technology

Purple Chinese yam Dioscoreaceae yam, the plant of using as dish is mainly Chinese yam and yampi in China, and yampi originates in Tropical Asian area, and China is also one of its country of origin.Just on the books to Chinese yam in the China that records the earliest Chinese yam pharmacology works " Shennong Bencaojing " the earliest, be listed in top grade, material that can dietotherapeutic.In this book, do not distinguish Chinese yam and yampi, they are referred to as to Chinese yam.Because experiencing Tang and Song two, towards the forbidden word that evades emperor, made Chinese yam into afterwards.In " Compendium of Materia Medica " of bright LI Shi-Zhen, still there is no clear and definite two kind of plant are separated, be still referred to as Chinese yam.

At present, in the world the planting site of yampi mainly on West Africa, South East Asia and other places.It is the plant of the main grain dish dual-purpose of the states such as West Africa.At south China, economize more, there is no large-scale planting base, poor because of its profile, market sales volume is low, and with single household, it is main marketing one's own products.In yampi, have plain boiled pork and purple meat kind, purple meat kind is because the effect of anthocyanidin is well known, to increase gradually in recent years.Purple Chinese yam (purple meat yampi) is as a kind of emerging Vegetable Resources, also very limited to its research.Some researchs of carrying out in advance show, purple Chinese yam approaches even higher than RHIIZOMA DIOSCOREAE from Henan of China on nutritive ingredient and partial function composition with RHIIZOMA DIOSCOREAE from Henan of China.Show that purple Chinese yam has potential exploitation to be worth, both can be used as vegetables and eaten, can look like again RHIIZOMA DIOSCOREAE from Henan of China as a kind of medicinal material of gentleness.

Purple yam variety is mostly local variety, although plant on a small quantity southern some areas, cultivation history is long.Area, hillside, hills mostly, plantation area, planting soil soil layer is deep, and quality is loose, and organic content is high, and water conservation, fertilizer-preserving ability are strong, and pH value is 4.5-6.5, is subacidity.

Purple Chinese yam is because of purple the gaining the name of the red middle band of its meat.Purple Chinese yam stem tuber is loose, single stem tuber weighs 500 grams of left and right, maximum reaches more than 2500 gram, meat is soft and smooth, unique flavor, the beautiful deliciousness of color and luster, the commercialization indexs such as nutritious, meat, color and luster, taste, viscosity, smell, mouthfeel, Analysis on Nutritious Ingredient of Fattening are leading in the Chinese yam in the whole province, the whole nation.

The pharmaceutical use of purple Chinese yam is very high, both can make the delicacies on dining table, can make health-care medicinal again, according to < < Compendium of Materia Medica > >, record, often edible purple Chinese yam, can reduce blood pressure, blood sugar, anti-ageingly lengthen one's life, brain tonic is mended intelligence, is strengthened body immunity and improve sexual function, is the precious dietotherapeutic resource that benefits spleen, lung, renal function.

Along with the growing interest of people to purple Chinese yam, also evoked the plantation enthusiasm of peasant to purple Chinese yam, the kind of the purple Chinese yam of south plantation also has multiple, is all referred to as purple Chinese yam simultaneously, from form, be difficult to accurately differentiate, therefore differentiate that rapidly and accurately purple yam variety is urgent problem.Research shows on molecular level, to exist significant difference between purple yam variety, its difference of direct-detection from DNA level, and the most reliable for the discriminating of Clonal Cultivar.Therefore, purple Chinese yam is carried out to genetic analysis significant.

Summary of the invention

The object of the present invention is to provide a kind of purple Chinese yam conventional variety molecular assay method based on genome RAPD analysis.

For achieving the above object, the present invention takes following technical scheme:

A method for purple yam variety Molecular Identification based on genome RAPD analysis, comprises the following steps:

1) sampling and sample preparation;

Adopt the purple yam variety of normal growth, each kind 10 strain, 10 fresh spires of every strain are put into ice chest, and being placed in-40 ℃ of Ultralow Temperature Freezers, to store 24h postlyophilization powdered, and it is fully mixed;

2) DNA extraction and purity thereof and concentration monitor:

Take spire dry powder as material, get 0.2g, adopt 2 * CTAB to extract total DNA, use 0.8w% agarose to detect the purity of obtained genomic dna, utilize protein nucleic acid quantitative determination instrument to detect DNA concentration;

3) primer screening and RAPD analyze:

Purple Chinese yam is being carried out after the pcr amplification system optimization of RAPD analysis, choose 8 above purple yam varieties that diversity ratio is larger in form and carry out RAPD primer screening, filter out 1 primer S1255 that amplified band is clear, polymorphism is obvious, reproducible, its nucleotides sequence is classified TGACGCACGG as, carry out pcr amplification, on Gene Amp PCR System 9700PCR amplification instrument, carry out DNA cloning;

4) the separated 2-3h of the 1.6w% agarose gel electrophoresis containing 1v%Golden view for amplified production, voltage is 6V/cm; Under UV light, detect amplification and pass through gel imaging system (Gel Doc XR ⁺) take a picture, obtain the randomly amplified polymorphic DNA standard diagram (the peculiar DNA fingerprint that S1255 primer amplification goes out) of 8 above purple yam varieties;

5) purple yam variety to be identified is analyzed according to above-mentioned steps, obtain the randomly amplified polymorphic DNA collection of illustrative plates of purple yam variety to be identified, this randomly amplified polymorphic DNA collection of illustrative plates and standard diagram are compared to judgement, judge whether it is certain purple yam variety.

In aforesaid method, the method for described DNA extraction is:

(1) get 6ml and be preheating to 2 * CTAB solution of 60 ℃ in the centrifuge tube of blade powder is housed, 60 ℃ of water-baths 30 minutes, or jog several times, powder and solution are mixed;

(2) take out centrifuge tube, add after cooling 6ml chloroform-primary isoamyl alcohol (volume ratio 24:1), be placed in shaking table and fully mixed upper 30 minute;

(3) 12000rpm is centrifugal 10 minutes;

(4) supernatant liquor is proceeded in new centrifuge tube, add 2 times of volume precoolings (20 ℃) dehydrated alcohol, mix, in-20 ℃ of refrigerators, put 30 minutes, nucleic acid is precipitated into cotton-shaped;

(5) centrifugal 10 minutes of 12000rpm under room temperature;

(6) abandon supernatant, after DNA precipitation is air-dry, be dissolved in 800 μ l deionized waters, as pcr template DNA ,-20 ℃ save backup.

The compound method of 2 * CTAB:

Tris-HCl(pH8.0) 100mmol/L, EDTA-Na ₂(EDETATE SODIUM) be 20mmol/L (pH8.0), NaCl 1.4mol/L, CTAB(cetyl trimethylammonium bromide) 2w%(20g/L); Before DNA extraction, add 0.2v% beta-mercaptoethanol.

Use 0.8w% agarose to detect the purity of obtained genomic dna, utilize protein nucleic acid quantitative determination instrument to detect DNA concentration.Record DNA concentration and be greater than 10mg/ml, the ratio of OD260/280 is between 1.8-2.0, and the ratio of OD260/230 is between 2.0-2.5.

In step 3), the pcr amplification system optimization that purple Chinese yam is carried out to RAPD analysis refers to DNA profiling amount is optimized; As to DNA profiling amount from 5ng, 10ng, 20ng, 40ng, 60ng, 80ng, 100ng is optimized, and finally selects 20ng.

The method of described pcr amplification is: adopt 20 μ L reaction systems to carry out pcr amplification, reaction system cumulative volume is 20 μ L, containing 10 μ L2 * EasyTag PCR SuperMix (+dye) (Beijing Quanshijin Biotechnology Co., Ltd)+2 μ L 10mM primer+20ng DNA.

The response procedures of described pcr amplification is: 94 ℃ of denaturation 5min 30s; Follow 94 ℃ of sex change 1min 30s, 40 ℃ of renaturation 1min, 72 ℃ are extended 2min, 40 circulations; After last 72 ℃, extend 10min.

The present invention is based on 8 kinds of above purple Chinese yam conventional variety molecular assay method of genome RAPD analysis, by 1) sampling and sample preparation; 2) DNA extraction and purity thereof and concentration detect; 3) primer screening and RAPD analyze; 4) obtain the standard diagram of 8 kinds of conventional strains of purple Chinese yam use primer S1255(Shanghai Sheng Gong biotechnology company limited); 5) purple Chinese yam new variety to be identified are analyzed according to above-mentioned steps, obtain the randomly amplified polymorphic DNA collection of illustrative plates of the purple Chinese yam new variety of required evaluation, by this randomly amplified polymorphic DNA collection of illustrative plates and standard diagram Identification, can judge different purple yam varieties, the feature have fast, stable, expense is low.

The present invention is to purple yam cultivation kind, particularly the purple yam variety of new seed selection carries out DNA fingerprint analysis, set up kind DNA fingerprinting, find specific DNA fragment sequence, to explore, utilize molecular marking technique to carry out the method for kind Rapid identification, for purple yam cultivation kind quick and precisely identify and the preliminary election of purple Chinese yam hybridization Parents provides theoretical foundation.

Accompanying drawing explanation

Fig. 1 is that 8 purple yam varieties adopt primer S1255(TGACGCACGG) the RAPD standard diagram of amplification.

Fig. 2 is that 8 purple yam varieties and purple Chinese yam to be identified adopt primer S1255(TGACGCACGG) the RAPD collection of illustrative plates of amplification.

Embodiment

Below in conjunction with the drawings and specific embodiments, the present invention is described in detail.

One, materials and methods

1, material

For totally 8 of materials of examination, within 2010, introduce a fine variety respectively in Zhejiang, the ground such as Jiangxi, Fujian, Guangdong, Yunnan, Taiwan, refer to table 1.

Random primer is purchased from Shanghai Sheng Gong bio-engineering corporation, and 2 * EasyTaq PCR SuperMix and DNA molecular amount standard Trans5K DNA Marker are purchased from Beijing Quanshijin Biotechnology Co., Ltd.

The purple yam variety of table 1 and source thereof

Numbering	Affiliated kind	Kind	Source
				1	Dioscoreaceae Wild yam Dioscorea alata L.	Purple Chinese yam	The Jiangxi ten thousand years
2	Dioscoreaceae Wild yam Dioscorea alata L.	Purple Chinese yam	Wenzhou District of Zhejiang Province
				3	Dioscoreaceae Wild yam Dioscorea alata L.	Purple Chinese yam	Wuyi Mountain, fujian
4	Dioscoreaceae Wild yam Dioscorea alata L.	Purple Chinese yam	Guangdong Huizhou
				5	Dioscoreaceae Wild yam Dioscorea alata L.	Purple Chinese yam	Guangzhou Guangdong
6	Dioscoreaceae Wild yam Dioscorea alata L.	Purple Chinese yam	Yunnan mountain of papers
				7	Dioscoreaceae Wild yam Dioscorea alata L.	Purple Chinese yam	Honghe, Yunnan
8	Dioscoreaceae Wild yam Dioscorea alata L.	Purple Chinese yam	Taiwan

2, sampling and sample preparation;

Adopt the purple yam variety of normal growth, each kind 10 strain, 10 fresh spires of every strain are put into ice chest, are placed in-40 ℃ of Ultralow Temperature Freezers and store the powdered (CoolSafe being produced by SCANVAC company of 24h postlyophilization ^tMafter the freeze-drying of-55-4 vacuum-freeze-dry machine, with have gentle hands micro-rub broken), it is fully mixed;

3, DNA extraction and purity thereof and concentration monitor:

Take spire dry powder as material, get 0.2g, adopt improvement 2 * CTAB(Extraction buffer) extract total DNA, and revise slightly: use 0.8w% agarose to detect the purity of obtained genomic dna, utilize protein nucleic acid quantitative determination instrument to detect DNA concentration.

The compound method of 2 * CTAB:

Composition final concentration

Tris-HCl damping fluid (pH8.0) 100mmol/L,

EDTA-Na ₂(EDETATE SODIUM) be 20mmol/L (pH8.0),

NaCl 1.4mol/L，

CTAB 2w%（20g/L），

Before DNA extraction, add beta-mercaptoethanol 2ml/L(0.2w%).

DNA extraction method: (1) is got 6ml and is preheating to 2 * CTAB solution of 60 ℃ in the centrifuge tube of 0.2g blade powder is housed, 60 ℃ of water-baths 30 minutes, or jog several times, powder and solution are mixed; (2) take out centrifuge tube, add after cooling 6ml chloroform-primary isoamyl alcohol (volume ratio 24:1), be placed in shaking table and fully mixed upper 30 minute; (3) 12000rpm is centrifugal 10 minutes; (4) supernatant liquor is proceeded in new centrifuge tube, add 2 times of volume precoolings (20 ℃) dehydrated alcohol, mix, in-20 ℃ of refrigerators, put 30 minutes, nucleic acid is precipitated into cotton-shaped; (5) centrifugal 10 minutes of 12000rpm under room temperature; (6) abandon supernatant, after DNA precipitation is air-dry, be dissolved in 800 μ l deionized waters, as pcr template DNA ,-20 ℃ save backup;

Use 0.8w% agarose to detect the purity of obtained genomic dna, utilize protein nucleic acid quantitative determination instrument to detect DNA concentration.Use the NANODROP2000 of Thermo company micro-spectrophotometer directly to record DNA concentration, the ratio of OD260/280 is between 1.8-2.0, and the ratio of OD260/230 is between 2.0-2.5, and DNA concentration is greater than 10mg/ml.

4, primer screening and RAPD analyze

Purple Chinese yam is being carried out to the pcr amplification system optimization of RAPD analysis---to DNA profiling amount from 5ng, 10ng, 20ng, 40ng, 60ng, 80ng, 100ng is optimized, and the most always selects 20ng.Then, choose the purple yam variety that eight (table 1) diversity ratio in form is larger and carry out 130 primers of RAPD (in Table 2) screening.

From the DNA fingerprinting obtaining, filter out 1 primer S1255(TQACGCACGG that amplified band is clear, polymorphism is obvious, reproducible), carry out pcr amplification.

Table 2,130 random primers are numbered and sequence

Sequence number	Primer numbering	Series	Sequence number	Primer numbering	Series
						1	S1001	TCCGCAACCA	66	S1266	TCTCTAGGGG
2	S1002	CACTTCCGCT	67	S1267	AGACCCTTGG
						3	S1003	GGTTACTGCC	68	S1268	CACCGATCCA
4	S1004	CTCCCCAGAC	69	S1269	ACGGCCAATC
						5	S1005	TTGCAGGCAG	70	S1270	GGCGTATGGT
6	S1006	GTAAGCCCCT	71	S1271	CTTCTCGGTC
						7	S1007	CCCTACGGAG	72	S1272	CCACTCGTGT
8	S1008	TTCCCGTGCC	73	S1273	CCAAGCACAC
						9	S1009	AGAACCGAGG	74	S1274	CACCTCGACC
10	S1010	GGGATGACCA	75	S1275	TGCTGACGAC
						11	S1011	TCCGCTGAGA	76	S1276	TCTTAGGCGG
12	S1012	TCCAACGGCT	77	S1277	TTGGCATCCC
						13	S1013	TGAGTCCGCA	78	S1278	CACCACTAGG
14	S1014	TGTGGCCGAA	79	S1279	AGCCTGGGGA
						15	S1015	CTACAGCGAG	80	S1280	GTCGAAACCC
16	S1016	CAAGGTGGGT	81	S1321	GTGTGCCGTT
						17	S1017	CAGTGGGGAG	82	S1322	GGGAGGCAAA
18	S1018	GGGCTAGTCA	83	S1323	CCAAGAGGCT
						19	S1019	GGCAGTTCTC	84	S1324	TCCCCAGGAG
20	S1020	GGAAGGTGAG	85	S1325	AGTGCACACC
						21	S1021	GGCATCGGCT	86	S1326	AGGCATCGTG
22	S1022	AGCCGTTCAG	87	S1327	ACGCGACAGA
						23	S1023	GGGTCCAAAG	88	S1328	AGTATGGCGG
24	S1024	CTATCCTGCC	89	S1329	GGAAGTCCTG
						25	S1025	GTCGTAGCGG	90	S1330	CCAGGCTGAC
26	S1026	TGCCGCACTT	91	S1331	TGATTGCGGG
						27	S1027	ACGAGCATGG	92	S1332	GGTCGGGTCA
28	S1028	AAGCCCCCCA	93	S1333	GAGCACTGCT

29	S1029	TCGCTGGTGT	94	S1334	CACGGGCTTG
						30	S1030	TCGGGGCATC	95	S1335	CAGCAATCCC
31	S1031	ACGGCGATGA	96	S1336	GTCTGTGCGG
						32	S1032	GACGCGAACC	97	S1337	TGGGCTCTGG
33	S1033	ACGCTGCGAC	98	S1338	GTGTGCAGTG
						34	S1034	TGGTGCACTC	99	S1339	CCCTGTCGCA
35	S1035	GACACAGCCC	100	S1340	ACACTCGGCA
						36	S1036	AAGGCACGAG	101	S1341	GTCCACCTCT
37	S1037	CCTCACGTCC	102	S1342	TGCGAAGGCT
						38	S1038	TCGCGGAACC	103	S1343	TTTCCGGGAG
39	S1039	GGCAAAGCTG	104	S1344	AAGGCTCGAC
						40	S1040	CCTGTTCCCT	105	S1345	TCGCTGCGTT
41	S1241	CAGTGGTTCC	106	S1346	GGCTTCGCAA
						42	S1242	CAGGTCTAGG	107	S1347	GACCGTCTGT
43	S1243	GACTGGGAGG	108	S1348	AGGCTTCCCT
						44	S1244	TTGCCTCGCC	109	S1349	CCGATCCAAC
45	S1245	ACACCTGCCA	110	S1350	CAAGGCCCCT
						46	S1246	CCGTCCCTGA	111	S1351	ACGCGCCTTC
47	S1247	ACTGCGACCA	112	S1352	CTGTCGGCGT
						48	S1248	TCCTCGTGGG	113	S1353	CCGCTCGTAA
49	S1249	CCGTTAGCGT	114	S1354	GGTGGGTAGA
						50	S1250	ACCTCCGGTC	115	S1355	CCAAGAGGCA
51	S1251	CCAGATCTCC	116	S1356	GGTGTGGTTC
						52	S1252	CTGCCTAGCC	117	S1357	GGTGATTCGG
53	S1253	CTGGTGGAAG	118	S1358	ACCCCAACCA
						54	S1254	GTGCCGCACT	119	S1359	AACTTGGCCC
55	S1255	TGACGCACGG	120	S1360	TCATTCGCCC
						56	S1256	CTCTCCGTAG	121	S1361	TCGGATCCGT
57	S1257	AGCGACTGCT	122	S1362	CCTGAACGGA
						58	S1258	CCAGCTGTGA	123	S1363	GGCTGTGTGG

59	S1259	ACCAAGGCAC	124	S1364	CCAGCCTCAG
						60	S1260	ACATCAGCCC	125	S1365	TCCGCATACC
61	S1261	GGGATGGAAC	126	S1366	CCTTCGGAGG
						62	S1262	CCAACCCGCA	127	S1367	CACGAGTCTC
63	S1263	ACGAAACGGG	128	S1368	TCGCTCGTAG
						64	S1264	GGCTTCTGTC	129	S1369	CCTTGACCCC
65	S1265	GAGCTACCGT	130	S1370	ACTCTGGGGA

8 purple yam varieties adopt S1255 primer, on Gene Amp PCR System9700PCR amplification instrument, carry out DNA cloning.Adopt 20 μ L reaction systems to carry out pcr amplification, reaction system cumulative volume is 20 μ L, containing 10 μ L2 * EasyTag PCR SuperMix (+dye) (Beijing Quanshijin Biotechnology Co., Ltd)+2 μ L5mM primer+20ng DNA.

PCR response procedures is: 94 ℃ of denaturation 5min30s; Follow 94 ℃ of sex change 1min30s, 40 ℃ of renaturation 1min, 72 ℃ are extended 2min, 40 circulations; After last 72 ℃, extend 10min.

1.6w% for amplified production, containing the separated 2-3h of agarose gel electrophoresis of 1v%Golden view, voltage is 6V/cm; Under UV light, detect amplification and pass through gel imaging system (Gel Doc XR ⁺) take a picture, obtain respectively the randomly amplified polymorphic DNA standard diagram (Fig. 1) of 8 kinds of purple Chinese yams.

Two, results and analysis

1, the RAPD finger printing of different purple yam varieties

Each primer amplification has produced a kind of peculiar RAPD finger printing, if Fig. 1 is the RAPD finger printing with S1255 primer amplification, in figure, collection of illustrative plates from left to right numbering is respectively M and 1-8, M:DNA molecular weight standard wherein, Trans5K DNA Marker Go149 (Beijing Quanshijin Biotechnology Co., Ltd); 1-8: purple yam variety numbering, in Table 1.For each 5 repetition for the purple yam variety of examination is individual, primer S1255 increases respectively and produces consistent DNA fingerprinting, does not occur polymorphism, from molecular level, illustrates, vegetative propagation does not affect the genetic construction for examination plant.

By the randomly amplified polymorphic DNA standard diagram of 8 kinds of purple Chinese yams, can see the peculiar DNA fingerprint that S1255 primer amplification goes out, 8 kinds of purple yam variety amplification collection of illustrative plates have obvious difference each other, can distinguish 8 kinds of purple Chinese yams simultaneously.

2, purple yam variety to be identified is analyzed according to above-mentioned steps, obtain the randomly amplified polymorphic DNA collection of illustrative plates of the purple yam variety of required evaluation, this randomly amplified polymorphic DNA collection of illustrative plates and standard diagram are compared to determine, can determine whether certain purple yam variety.

Example: purple Chinese yam to be identified is for buying purple Chinese yam from the market of farm produce, urban district, Yunnan, be numbered No. 9, RAPD finger printing (concrete steps are with noted earlier) with S1255 primer amplification, as shown in Figure 2, in figure, collection of illustrative plates from left to right numbering is respectively M, 1-8 and 9, M:DNA molecular weight standard wherein, Trans5K DNA Marker Go149 (Beijing Quanshijin Biotechnology Co., Ltd); 1-8: purple yam variety numbering, in Table 1; 9: purple Chinese yam to be identified.Through the comparison of RAPD finger print identification, judge that purple Chinese yam to be identified (No. 9) is consistent with the DNA collection of illustrative plates of Yunnan mountain of papers kind (No. 6), determine that this purple yam variety to be identified is Yunnan mountain of papers kind.

Claims

1. a method for the purple yam variety Molecular Identification based on genome RAPD analysis, comprises the following steps:

1) sampling and sample preparation;

2) DNA extraction and purity thereof and concentration monitor:

3) primer screening and RAPD analyze:

Purple Chinese yam is being carried out after the pcr amplification system optimization of RAPD analysis, choose 8 above purple yam varieties that diversity ratio is larger in form and carry out RAPD primer screening, filter out primer S1255, its nucleotides sequence is classified TGACGCACGG as, on Gene Amp PCR System9700PCR amplification instrument, carries out DNA cloning;

4) the separated 2-3h of the 1.6w% agarose gel electrophoresis containing 1v%Golden view for amplified production, voltage is 6V/cm; Under UV light, detect amplification and take a picture by gel imaging system, obtaining the randomly amplified polymorphic DNA standard diagram of 8 kinds of above purple Chinese yams;

2. the method for the purple yam variety Molecular Identification based on genome RAPD analysis claimed in claim 1, is characterized in that: the method for described DNA extraction comprises:

(2) take out centrifuge tube, add after cooling 6ml chloroform-primary isoamyl alcohol, volume ratio 24:1, is placed in shaking table and fully mixed upper 30 minute;

(3) 12000rpm is centrifugal 10 minutes;

(4) supernatant liquor is proceeded in new centrifuge tube, add 2 times of volume precooling dehydrated alcohols, mix, in-20 ℃ of refrigerators, put 30 minutes, nucleic acid is precipitated into cotton-shaped;

(5) centrifugal 10 minutes of 12000rpm under room temperature;

3. the method for the purple yam variety Molecular Identification based on genome RAPD analysis claimed in claim 1, is characterized in that: described DNA concentration is greater than 10mg/ml, and the ratio of OD260/280 is between 1.8-2.0, and the ratio of OD260/230 is between 2.0-2.5.

4. the method for the purple yam variety Molecular Identification based on genome RAPD analysis claimed in claim 1, is characterized in that: the pcr amplification system optimization that purple Chinese yam is carried out to RAPD analysis refers to DNA profiling amount is optimized.

5. the method for the purple yam variety Molecular Identification based on genome RAPD analysis claimed in claim 4, is characterized in that: described DNA profiling amount is 20ng.

6. the method for the purple yam variety Molecular Identification based on genome RAPD analysis claimed in claim 1, it is characterized in that: the method for described pcr amplification is for adopting 20 μ L reaction systems to carry out pcr amplification, reaction system cumulative volume is 20 μ L, containing 10 μ L2 * EasyTag PCR SuperMix (+dye)+2 μ L 5mM primer+20ngDNA.

7. the method for the purple yam variety Molecular Identification based on genome RAPD analysis claimed in claim 6, is characterized in that: the response procedures of described pcr amplification is: 94 ℃ of denaturation 5min 30s; Follow 94 ℃ of sex change 1min 30s, 40 ℃ of renaturation 1min, 72 ℃ are extended 2min, 40 circulations; After last 72 ℃, extend 10min.