CN104152443A - Molecular marker linked with turnip clubroot resistant gene and method for obtaining molecular marker - Google Patents

Abstract

The invention discloses a molecular marker linked with a turnip clubroot resistant gene and a method for obtaining the molecular marker. The method comprises the following steps: hybridizing a clubroot resistant turnip parent material P1('077-03') and an infected parent material P2('077-04') to obtain a population F1 and constructing corresponding populations BC1, F2 and F3; clearly determining that the pathogeny of the disease is plasmodiophora brassicae woron.; according to a disease-resistant genetic analysis result, indicating that the resistance of the disease is controlled by a dominant single gene; and performing clubroot resistant marker screening on the turnip population F2 by use of the SSR molecular marker technology and the improved BSA method, thereby obtaining the molecular marker BrSSR133 linked with the turnip clubroot resistant gene; the genetic distance between the SSR molecular marker and the resistance gene is 5.02cm. The research result lays a foundation for separating out the new clubroot resistant gene; the molecular marker and the method for obtaining the molecular marker are significant for both breeding practices and disease-resistant theoretical research of compestris crops.

Description

Molecule marker and preparation method with the anti-club root gene linkage of turnip

Technical field

The invention belongs to biological technical field, relate in particular to a kind of molecule marker and the preparation method thereof mutually chain with the anti-club root gene of turnip.

Background technology

Crop in cruciferae club root also claims " crown gall ", is to infect by rape plasmodiophora brassicae (Plasmodiophora brassicae Woron.) the worldwide soil-borne disease causing.Worldwide harm is serious at present, and the crop in cruciferae in China's overwhelming majority area is also being faced with the serious threat of club root.After being infected by plasmodiophora brassicae, crop in cruciferae can cause output significantly to decline, even total crop failure.Produce at present upper general employing Agro-chemicals control, but after Symptoms, re-use chemicals treatment effect not obvious, this has brought very large difficulty to control.Research shows, the life-span of plasmodiophora brassicae statospore in soil reaches 8～10 years, and the sustainability that has a strong impact on crop in cruciferae is produced.Therefore, on producing, cultivating the crop in cruciferae new variety with club root resistance is safe and the most economic method.

For solving the above-mentioned significant problem that affects China's crop in cruciferae Sustainable development, excavate new antigen, the men of breeding in the past carry out assistant breeding by morphology mark and biochemical marker mostly, obtain some good anti-club root self-mating systems through screening, but had long, the shortcoming such as schedule of operation is loaded down with trivial details of seed selection time apply traditional method screening resistant plant in disease-resistant transformation process time; Resistance performance is subject to the impact of envrionment conditions on the other hand, is difficult to adapt to the needs of breeding practice.In recent years, along with molecular biological development, utilize molecule marker ancillary technique to carry out Cruciferae breeding for disease resistance and show huge potentiality.The molecular marking technique of one class based on DNA variation is applied to the disease-resistant research of Cruciferae by domestic and international many scholars.Application molecular marking technique, not only can exempt chosen process heavy in traditional breeding method, and follows the tracks of, detects foreign gene, can also be multiple anti-club root gene pyramidings in same improved seeds, make that it obtains lastingly, resistance widely.

Vegetable Crops of Brassica is an important elementary species during Cruciferae rape belongs to, it includes AA (2n=20) genome, can be divided into three subspecies such as Chinese cabbage subspecies (6 mutation such as Chinese cabbage, Plantula Brassicae chinensis, cabbage heart, purple tsai-tai, a kind of sedge dish and Wuta-tsai), turnip subspecies and Japanese water dish subspecies, be a kind of important vegetables and oil crops.Forefathers' result of study shows, turnip is that in Vegetable Crops of Brassica, unique discovery contains the mutation to club root resistant gene, by hybridization and the technology that backcrosses, resistant gene in turnip can be transferred in the mutation such as Chinese cabbage of the same race, Plantula Brassicae chinensis, cabbage heart and purple tsai-tai, thereby be provided Innovation Germplasm for the breeding of new variety of anti-club root.Therefore, find with the closely linked molecule marker of club root resistant gene and not only may be used in marker assisted selection, improve Vegetable Crops of Brassica crop disease-resistant breeding efficiency, in agricultural, there is important using value, but also can lay the foundation for further cloning relevant disease-resistant gene and analyzing its biological function.

Summary of the invention

The object of the invention is to for the deficiencies in the prior art, a kind of molecule marker and the preparation method thereof mutually chain with the anti-club root gene of turnip are provided.

The object of the invention is to be achieved through the following technical solutions: a kind of molecule marker mutually chain with the anti-club root gene of turnip, the sequence of described molecule marker (BrSSR133) is SEQ ID NO.3, its upstream primer is SEQ ID NO.1, and downstream primer is SEQ ID NO.2.

The present invention is a kind of and the preparation method of the molecule marker BrSSR133 that the anti-club root gene of turnip is mutually chain, comprises the following steps:

1) utilize the anti-club root turnip parent material ' 077-03 ' and the Susceptible parent material ' 077-04 ' that select through inbreeding of more generation, described disease-resistant parent ' 077-03 ' and Susceptible parent ' 077-04 ' are hybridized and obtained F ₁colony, and build corresponding BC ₁, F ₂, F ₃colony;

2) according to the disease symptom of field natural occurrence material, and cause of disease statospore microscopy, the cause of disease that specifies this disease is rape plasmodiophora brassicae;

3) to BC ₁, F ₁, F ₂colony carries out artificial inoculation qualification, finds that the club root resistance of ' 077-03 ' is by a pair of dominant nuclear gene control;

4) utilize SSR molecular marking technique, utilization improved BSA method is carried out the screening with turnip club root resistance molecule marker;

5) filter out a SSR molecule marker BrSSR133, through individual plant check analysis, the genetic distance of this molecule marker and club root resistant gene is 5.02cm.

Compared with prior art, beneficial effect of the present invention is: club root is to affect at present a kind of serious plant disease that Cruciferae is produced.The present invention utilizes SSR molecule marking method, obtains a SSR molecule marker relevant to turnip club root resistance in conjunction with the BSA method of improvement, can be directly used in the assisted selection of the anti-club root molecule marker of Vegetable Crops of Brassica crop.Utilize this molecule marker, not only overcome the shortcomings such as conventional breeding method required time cycle length, the on purpose multiple anti-club root genes of polymerization, cultivate the Vegetable Crops of Brassica crop varieties with stable multiresistance.Also can utilize the anti-club root gene of molecular marker clone of this new club root resistant gene, and it is carried out to structure and function analysis, this molecular genetic mechanism for the anti-club root of further understanding turnip has positive effect simultaneously.Therefore, the present invention is all significant in the practice of Vegetable Crops of Brassica crop breeding and disease-resistant theoretical investigation.

Brief description of the drawings

Fig. 1 be the observations of rape plasmodiophora brassicae statospore under 600 times of opticmicroscopes (scale length be 50 μ m).A figure: from root nodule separate tissue; B figure: separation graph from soil;

Fig. 2 is the result figure of BrSSR133 to two parents;

Fig. 3 is the amplification figure of BrSSR133 antagonism (sense) gene pool.1 and 2: orthogonal F ₂for disease-resistant pond; 3 and 4: orthogonal F ₂for susceptible pond;

Fig. 4 is the F of BrSSR133 ₂for individual plant the result figure;

Fig. 5 is the F of BrSSR133 ₃for individual plant the result figure.

Embodiment

Mainly comprise the following steps with the preparation method of the anti-club root molecule marker of turnip BrSSR133:

1. utilize the anti-club root turnip parent material P selecting through inbreeding of more generation ₁(' 077-03 ') and Susceptible parent material P ₂(' 077-04 '), hybridizes described disease-resistant parent ' 077-03 ' and Susceptible parent ' 077-04 ' to obtain F ₁colony, and build corresponding BC by test cross and selfing approach ₁, F ₂and F ₃colony.

2. according to the disease symptom of field natural occurrence material, and cause of disease statospore microscopy, the cause of disease that specifies this disease is rape plasmodiophora brassicae.

Specific practice is: observe the disease symptom of field natural occurrence material, the statospore of bacterial isolate from old complaint and sick soil, in the morphological specificity of 600 times of optical microphotograph Microscopic observation statospores.Shown in Fig. 1: consistent with Cruciferae plasmodiophora brassicae from size, the morphological specificity of old complaint and the statospore separating sick soil.

The separation of plasmodiophora brassicae statospore in knee tissue:

(1) get knee and organize 10g, in mortar, grind, add 50mL aqua sterilisa, by 4 layers of filtered through gauze.

(2) filtrate moves into 50mL centrifuge tube, the centrifugal 15min of 3100rpm.

(3) abandon supernatant liquor, precipitation is dissolved in 50mL aqua sterilisa, moves into 50mL centrifuge tube, the centrifugal 15min of 3100rpm.

(4) repeating step (3) 2～3 times.

(5) abandon supernatant liquor, in the precipitation obtaining in step (4), adding 5mL mass percent is 50% sucrose solution, after mixing, moves into 50mL centrifuge tube, the centrifugal 10min of 3100rpm.

(6) with liquid-transfering gun, the supernatant liquor after centrifugal is carefully moved into 50mL centrifuge tube, add 30mL aqua sterilisa, the centrifugal 10min of 3100rpm.

(7) abandon the supernatant liquor of step (6) after centrifugal, the precipitation obtaining is heavily dissolved in to 30mL aqua sterilisa, move in 50mL centrifuge tube, the centrifugal 10min of 3100rpm, this step operation repeats 2～3 times.

(8) finally precipitation is dissolved in 5mL aqua sterilisa for subsequent use.

The separation of plasmodiophora brassicae statospore in soil:

(1) get 10g sick soil, being dissolved in 20mL mass percent is in 0.05%tween-80 (polysorbate) aqueous solution, is placed in and on shaking table, shakes 2～3h.

(2) 4 layers of filtered through gauze for mixed solution step (1) being obtained.

(3) filtrate moves into 50mL centrifuge tube, the centrifugal 15min of 3100rpm.

(4) abandon supernatant liquor, precipitation is heavily dissolved in 50mL aqua sterilisa, moves into 50mL centrifuge tube, the centrifugal 15min of 3100rpm.

(5) repeating step (4) 2～3 times.

(6) abandon supernatant liquor, in the precipitation obtaining in step (5), adding 5mL massfraction is 50% sucrose solution, after mixing, moves into 50mL centrifuge tube, the centrifugal 10min of 3100rpm.

(7) with liquid-transfering gun, supernatant liquor is carefully moved into 50mL centrifuge tube, add 30mL aqua sterilisa, the centrifugal 10min of 3100rpm.

(8) abandon supernatant liquor, precipitation is heavily dissolved in 30mL aqua sterilisa, the centrifugal 10min of 3100rpm, and this step operation can repeat 2～3 times.

(9) finally precipitation is dissolved in 5mL aqua sterilisa for subsequent use.

3. couple BC ₁, F ₂, F ₃colony carries out artificial inoculation qualification, finds P ₁the club root resistance of (' 077-03 ') is by a pair of dominant nuclear gene control.

Specific practice is: to P ₁, P ₂, orthogonal F _lwith reciprocal cross F _l, orthogonal F ₂with reciprocal cross F ₂, orthogonal F ₃with reciprocal cross F ₃, BC ₁the seed of these six generations carries out club root resistant proof, under adapt circumstance, cultivate, sow the susceptible situation that records each generation after 6 weeks, be divided into four standards according to severity, and calculate sickness rate, disease index, disease index (table 1), data are carried out to chi square test, and the assay of table 2 shows P ₁the club root resistance of (' 077-03 ') is by a pair of dominant nuclear gene control.

Table 1 club root resistant proof result

Table 2 club root genetics of resistance test card side assay

From generation to generation	Disease-resistant plant number	Disease plant number	Theoretical segregation ratio	X 2Value	P value
						P 1	41	2	1:0	0.093	0.70<P<0.80
P 2	3	46	0:1	0.184	0.50<P<0.70
						Orthogonal F 1	67	2	1:0	0.058	0.80<P<0.90
Reciprocal cross F 1	35	1	1:0	0.028	0.80<P<0.90
						Orthogonal F 2	55	20	3:1	0.111	0.70<P<0.80
Reciprocal cross F 2	37	13	3:1	0.027	0.80<P<0.90
						BC 1	32	35	1:1	0.134	0.70<P<0.80

The Resistance Identification key step of club root is as follows:

(1) hot water treatment of seeds: get P ₁, P ₂, orthogonal F _lwith reciprocal cross F _l, orthogonal F ₂with reciprocal cross F ₂, orthogonal F ₃with reciprocal cross F ₃, BC ₁the seed of these six generations, is placed on the 7～8min that soaks seed in the thermostat(t)ed water of 55 DEG C～60 DEG C, notes during this time constantly stirring, guarantee is heated evenly, and then warm water is outwelled, with twice of normal temperature tap water flushing, flat grain and impurity are rejected, finally change clean normal temperature tap water into, soak seed 1～3h.

(2) vernalization: in the culture dish of diameter 15cm, spread the filter paper of two-layer cleaning, on filter paper, spray a small amount of tap water, make filter paper completely moistening but there is no mobile water, then above-mentioned seed of hoting water treatment of seeds is evenly placed on moistening filter paper, finally on seed, covers the moistening filter paper moisturizing of one deck cleaning.Cover culture dish lid, be placed in indoor, room temperature vernalization.During vernalization, note water spray moisturizing, until the long 0.5～1.2cm of radicle.

(3) sowing and the inoculation of rape plasmodiophora brassicae: its disease resistance is inoculated and identified to application interpolation to above-mentioned materials, sowing is carried out with inoculation simultaneously.The peat composed of rotten mosses, vermiculite and perlite are mixed according to the ratio of 6:3:1, as the matrix (pH5.5～6.5) of material normal growth.At the bottom in each cave of 15 hole seedling culture hole plates (length × wide × height=8cm × 6cm × 6cm), spread the above-mentioned matrix of about 4cm thickness, spread the bacterium soil with rape plasmodiophora brassicae (physiological strain is ECD16/0/0) of about 2cm thickness on matrix upper strata, then above-mentioned seed of having urged bud is evenly placed on bacterium soil, the seed of every cave 5～6 grain germinations, finally on seed, cover one deck peat composed of rotten mosses, thickness is as the criterion with the seed that just covers germination.Substrate culture is all used in contrast, does not add any matrix or soil with pathogenic bacteria.

(4) daily administration: day temperature is controlled at more than 25 DEG C, and nocturnal temperature is controlled at more than 20 DEG C.During launching to true leaf after emerging, ensure sufficient illumination, control moisture, prevent excessive growth, manage afterwards according to the required illumination of conventional cress and moisture, water and nutritive medium are adjusted to pH5.5 left and right with nitric acid.

(5) anti-/ susceptible qualification: sow after 6 weeks (42 days), the careful area of the plant root being grown in the dish of cave is dug out, the matrix of then root being adhered to and the soil with pathogenic bacteria are rinsed well with tap water, observe the root growth situation of each plant, be divided into 4 standards according to coincident with severity degree of condition:

0 grade: root growth is grown normal, not susceptible

1 grade: main root grows normally, and minority lateral root has small tumour

2 grades: main root has and slightly expands symptom or lateral root and have larger tumour

3 grades: main root expand obviously even check surface or part rot, do not have lateral root or lateral root similar to main root situation

Conventionally because 0 grade and 1 grade of plant have no significant effect final product production and quality in production, thus be categorized as resistant, and the plant of 2 grades and the 3 grades state of an illness is larger on the impact of yield and quality, is categorized as susceptible type.

Illness analysis and account form:

Sickness rate=morbidity strain number/total strain number × 100%

Disease index (Disease Index, DI)=100 × ∑ (diseased plant numbers at different levels × typical values at different levels)/(investigating total strain number × highest typical value).Wherein, DI<10, is decided to be disease-resistantly, and DI >=10, are decided to be susceptible.

4. utilize SSR molecular marking technique, utilization improved BSA method is carried out the screening with turnip club root resistance molecule marker.

(1) extraction of the total DNA of blade:

Specific practice is: adopt anti-club root turnip parent material P ₁(' 077-03 ') and Susceptible parent material P ₂the combination of (' 077-04 ') preparing hybrid, builds F ₂and F ₃for segregating population.Determine anti-(sense) property of each individual plant by club root Resistance Identification, by F ₃the individual plant that is 0 grade and 3 grades for incidence in colony samples respectively, adopts CTAB method to extract genomic dna.Also extracted parent P simultaneously ₁and P ₂genomic dna.

(2) structure of anti-sense club root gene pool

Specific practice is: the present invention adopts the BSA method of improvement to build respectively disease-resistant gene DNA pond and susceptible gene DNA pond: get orthogonal F ₂mix respectively sample for 0 grade of individual plant and 3 grades of individual plant 30 pnca gene group DNA in colony, build orthogonal F ₂for disease-resistant pond, orthogonal F ₂for susceptible pond.

(3) with the screening of the anti-sex-linked SSR mark of club root

With 233 pairs of SSR primers (sequence is in table 3) to 2 gene pool (orthogonal F ₂for disease-resistant pond and orthogonal F ₂for susceptible pond) carry out pcr amplification, every pipe PCR repeats once.The reaction system (20 μ L) that PCR detects: 75ng genomic dna template, 0.5U Taq archaeal dna polymerase (Fermentas ^tM), 0.4 μ L (10 μ molL ^-1) dNTPs, 1 μ L (10 μ molL ^-1) upstream and downstream primer, 2 μ L10 × PCR Buffer (200mmolL ^-1trispH8.0,200mmolL ^-1kCl, 100mmolL ^-1(NH ₄) ₂sO ₄and 20mmolL ^-1mgSO ₄), add ddH ₂o to 20 μ L.PCR detect amplification program: 94 DEG C of denaturation 3min, enter 35 circulations (94 DEG C of sex change 30s, 53 DEG C annealing 30s, 72 DEG C extend 30s), finally again 72 DEG C extension 7min.PCR reaction process is at BIO-RADS1000 ^tMon Thermal Cycler instrument, carry out, sample is preserved under 4 DEG C of conditions.

Table 3 is for screening 233 pairs of primer sequences of SSR mark

Primer title	Upstream primer (5 '-3 ')	Downstream primer (5 '-3 ')
			BrSSR-OPC11-1	TTACAGCTGGACCAAGAACATAG	ATCGATGTTTGTGAGTCTCTACT
BrSSR-OPC11-2	GTAACTTGGTACAGAACAGCATAG	ACTTGTCTAATGAATGATGATGG
			BrSSR-TCR05	AGAATCATGACCGGGGAAAT	GCAGCTAAGTCATCGACCAA
BrSSR-TCR09	GCAGCAACCGATAATATAAGGA	AACCAGAAGAAGAAAAACAAAAA
			BrSSR-TCR10	AACTCTTGAAGAAAGCAAAGAAGC	GCAGGAATAAGAAGGAACACCA
BrSSR-Crr3-1	CTTCAGAACATCAGAAAGGGTCTT	TTGTTAATCTTGGTTGGGATGTTA
			BrSSR-Crr3-2	GGAAAGACACTTGTTTCCAGAACT	AAACATTCTGAAGAGGGTAGATGC
BrSSR-Crr3-3	CGTATAGACATAGAAGACATGGAAGC	GTGTTTATGCTGATTCCTTCACAG
			BrSSR-HC352b-SCAR	CTTTATAATGGCTACTATTTA	TGCTCATGAGTGTATAACTA
BrSSR-TCR08	GCAGAATTATAACCTGAGCGTGT	ATTACCGGAGTATGCGATCC
			BrSSR-BRMS-297	AAACTCAAAAACCTCCACTTTCTCG	ATGTGGAGGTGGGACCCATTA
BrSSR-BRMS-088	TATCGGTACTGATTCGCTCTTCAAC	ATCGGTTGTTATTTGAGAGCAGATT
			BrSSR-BRMS-100	CTCTTGAGAATCAGAGAGAGATTAC	GATCTTCATTATATTCATCTCTCTC
BrSSR-BRMS-096	AGTCGAGATCTCGTTCGTGTCTCCC	TGAAGAAGGATTGAAGCTGTTGTTG
			BrSSR-DBC16	AAAGTCGTGGGAAGTATCGT	AGGTGTAAGGATGGTGGTAGT
BrSSR-BRMS-056	GATCAAGGCTACGGAGAGAGAG	CGTGACGCTAGAGTAATCGAGT
			BrSSR-BRMS-026	CCTATCCTCGGACTAATCAGAA	GTGCTTGATGAGTTTCACATTG
BrSSR-BRMS-026-2	CCTATCCTCGGACTAATCAG	CTTGATGAGTTTCACATTGC
			BrSSR-BRMS-042-2	AGCTCCCGACAGCAACAAAAGA	TTCGCTTCCTTTTCTGGGAATG
BrSSR-BRMS-043	GCGATGTTTTTTCTTCAGTGTC	TTAATCCCTACCCACAATTTCC
			BrSSR-BRMS-034	GATCAAATAACGAACGGAGAGA	GAGCCAAGAAAGGACCTAAGAT
BRMS-027	GCAGGCGTTGCCTTTATGTA	TCGTTGGTCGGTCACTCCTT
			BRMS-005	ACCTCCTGCAGATTCGTGTC	GCTGACCTTTCTTACCGCTC
BRMS-040	TCGGATTTGCATGTTCCTGACT	CCGATACACAACCAGCCAACTC

BRMS-036	GGTCCATTCCTTTTTGCATCTG	CATGGCAAGGGGTAACAAACAT
			BRMS-006	TGGTGGCTTGAGATTAGTTC	ACTCGAAGCCTAATGAAAAG
BRMS-029	AACAAATGACACACACCACACT	ATTGAAAATCTTAACCGTGAAG
			BRMS-051	GGCCAAGCCACTACTGCTCAGA	GCGGAGAGTGAGGGAGTTATGG
BRMS-058	GCAGACAAGAAATTCTCGCCATGTC	GACATTGGCGAAAGTCTTGAACTGG
			BRMS-303	ACTCAACAACCGAACAAGAAAAACA	CGGTAGAGAACAGAGGAAGCCTAAG
BRMS-124	GAGACGAGTGTTTTGTTGGCAGTTG	CGACGAGAACCAACACATAACAACC
			BRMS-296	CATCCTAATGTTGCTGAGAAAGAGG	TATATGAAACCGATGAAGCTCCTTT
BRMS-144	CCATCTGTTGAGAGCTTCTTCTTC	AAGTTCATTTGCTCCGATGC
			BRMS-324	AACTTAACCGAAACCGAGATAGGTG	AATCTCGAAATTCATCGACTTCCTC
BrSSR-Na12-E02	TTGAAGTAGTTGGAGTAATTGGAGG	CAGCAGCCACAACCTTACG
			BrSSR-Ol11-B05	TCGCGACGTTGTTTTGTTC	ACCATCTTCCTCGACCCTG
BrSSR-Ol11-H02	TCTTCAGGGTTTCCAACGAC	AGGCTCCTTCATTTGATCCC
			BrSSR-Na12-A08	AACACTTGCAACTTCATTTTCC	CATTGGTTGGTGAATTGACAG
BrSSR-KBRH139B23	ATCTCATGGTTGGTTCACCG	ATTTCCAAAACACACACGCA
			BrSSR-ENA28	GGAGTCCGAGCGTTATGAAT	CTTCATCGACCCACCTTGTT
BrSSR-ENA23	GCTGTGCCAGTTCCTCTTTC	TCATTCCAAATGGCCTTACC
			BrSSR-E129	AGATGGTAAAAGAGCACAAGCC	TTCAAGCTACCGATCCAACTG
BrSSR-KBRH048O11	GCCTCTACCTGGCTTCAGCA	TCATTTGGCGCATACTTCCA
			BrSSR-ENA2	GATGGTGATGGTGATAGGTC	GAAGAGAAGGAGTCAGAGATG
BrSSR-KBRH138G23	TTTGACATCGTGCAATGCTA	TTGGGCTGGTCCTGAAGATA
			BrSSR-E138	TGCTATCACAGTAGGGATTGCTT	CACTCCCACTCCTCCTAGTCC
BrSSR-ENA17	CAGTTATTTCGCCTCGTCT	TATTTGTGGTCTGTTATTGGA
			BrSSR-ENA4	ACTTCTCTTTATTCACTTCCCA	GAGGGTGGTTGGTTCATT
BrSSR-ENA25	ACACCCTCCTTCTCCTCTC	GCTTTGTTGAGTATCTTCGTC
			BrSSR-EJU5	GGCACGTACATGGAGGATTC	TGTTGGTCGAGCTGTTTCAG
BrSSR-KBRH043E02	ATGCAAGCTTCATGGTGTCA	CATCAGCAAAATTTCATTTGTGT
			BrSSR-EJU4	CACCTTATCATCTCTCTATCCC	CCTCTGTTTCTCTCCTTGTG
BrSSR-ENA6	CTCGTCTTCTTCACCTACAAC	CTGACATCTTTCTCACCCAC
			BrSSR-KBRH143H15	TCTGCATCAAAATGCTAAAATGA	TGATCTTTTAGAAACAAAGATCGAG
BrSSR-EJU3	CCTCTTTTAATTCAAACAAGAAATCA	TTCGGACAATGGCAGTGATA
			BrSSR-KBRH143D22	GATGTGATACTTTGGCGACGG	TGAAGGATAATATGGTCTTGGCC
BrSSR-E120	ATCATAACCCTCAGGTTTGACATC	ACATCAAGCTCCTCTCTGGGTA
			BrSSR-KBRH143K20	CAAATGTCTCAAGACACATAAACCA	CTAAAGCAGCAATTGGGTGTTC
BrSSR-KBRH143F19	GCATGCAAGCTTGGAACTGAT	CAGTCACGCTTTCTGACGAAAA
			BrSSR-ENA18	TTAAAATGAAACCCACCCGA	TGTTGGGCAACATCCATTTA
BrSSR-E039	CTTGAGTGCTCAGGTCAAAGC	GAACCCTTACCCCCAAGACTAC
			BrSSR-BC7	TCGGCGATGGCTCTTTTCACC	AAGGATCGTTCAGCGGAGAGTAT
BrSSR-BC46	CTTTTGCTGCCCGACGAGA	AAGGAAGCAGGAAAGAGATAAAAG
			BrSSR-BC48	CTGGTGATGGAGACGCTATTA	ACTGTCCCAAAACCGCCTCTC
BrSSR-BC38	TCGTTGCGTTTGAATGTT	CTCGTGGAAGCGGTTAC
			BrSSR-BC63	GATGGCTCCGGCAAGGTC	TTAACAAAAGATCCAAAAAGAAAC
BrSSR-BC65	CTTCCAGTAGCCATTGTTGA	ATCGGGTTTTATACTCCTGAA
			BrSSR-BC113	ATACAATCTTCGTGACTCTACAG	AGCATCAACGCCAACTTTATCC
BrSSR-BC105	CGTCCGTAGCGCTATTTTTCAGA	ACGTTGTCGATCGCCCAGTTC
			BrSSR-BC51	GGTGGTGGGCTGGGGAGTA	CGTCGATCGATTCATAACCGTAGA
BrSSR-BC89	CACGCGTCCGCACAAATAAAC	TGAGCCGGCGAAGACAAAGAT
			BrSSR-BC107	GACGCCTCAATTGCTTACTT	AGGGAATGAGGATGGGTCTG
BrSSR-FITO045	ATGGCTGTAGAAACACATTGA	CTGACAACACGAGCATCTTAC

BrSSR-FITO063	GTTCAGTTCCCAGATTCCTAA	TTTCCTCTTCCTTCTCTCTTC
			BrSSR-FITO035	AAAGTCGTGGGAAGTATCGT	AGGTGTAAGGATGGTGGTAGT
BrSSR-FITO036	GGATTGCCTGAGTTTATTCTT	TCTGGAGTAGATGCTTTGGT
			BrSSR001	TTTGTCCCTATTGCTCAGGG	CCGAGAACGTCTTCTCCTTG
BrSSR002	GGCAGTCTTCAGGTGATCGT	CGGAACTCAGAGTGTCGTGA
			BrSSR003	CTCTTTTGGTTGCTCTTCCG	ACAAAACAAGATCGGGATGC
BrSSR005	TCTTTTGCACGCCATTAACA	CTCCTCCAGCGTAAGGACTG
			BrSSR008	CCGACGAAGCTGTAGAAAGG	CGGATCATCACCTGGAAACT
BrSSR009	CCAACCAACCACAGACACAG	TTGTTCGTCTCCATTCCTCC
			BrSSR011	ATGCTGCTCTTAAGGACGGA	TGGACGGTGGGTATTGATCT
BrSSR013	TCTCCGCAAAGTCCATCTCT	CCTCACCACTACCACCGTCT
			BrSSR014	TTCTGAATCTCCGCCGTATC	CTGCTTTTTAGCCAAATCGG
BrSSR015	TTCTGAATCTCCGCCGTATC	CTGCTTTTTAGCCAAATCGG
			BrSSR016	GATCCGATTGTGCGAGAGAG	TAGATCTCAGCCTCGGGAGA
BrSSR018	ATCACCCTCCCTCTTCGTCT	CGCTTTCACCCATTTTTGTT
			BrSSR019	ATCACCCTCCCTCTTCGTCT	CGCTTTCACCCATTTTTGTT
BrSSR020	TTGAGGAAGAAGAATGTGGTGA	GCTTCACCTCTGGGCTTATG
			BrSSR024	CATTCTTCATCATTAGCATCCAA	ACCATTCCAGACCTTTCACG
BrSSR026	AGCATTGCCTCCTTCTGGTA	GGGCGATGGAGAAGTGTTAC
			BrSSR027	GCTTGAGACCCGTTATGGAA	TCTCAGATCGTTTCTCCGGT
BrSSR028	TTGGGGAAGTTTGATTTTTGA	CTCCAAGGTGAGGTTTCCAA
			BrSSR030	GAGACGAGGACGACGAGAAC	TGACAACAATGGAGTTGGTGA
BrSSR031	TCTTCCATGACCTTCGTCGT	AAGGTGTCTGGACGCTGAGT
			BrSSR032	TTCGCCGTTGTTTTCTCTCT	TTGTTAACGCAGAGACGGTG
BrSSR033	CACCGTCTCTGCGTTAACAA	AGGATTCGACGGTTGATTTG
			BrSSR035	AACATTCGAGTTTTCAGGCG	TCCATAGTCCAGGAACCAGC
BrSSR036	GAAAACCAGAGTGACGGAGC	TGCAACAATACCGAACTTTGA
			BrSSR071	CGACAGCAACAAACCAAAGA	ATATCCACAGCCGTCCTCAG
BrSSR073	AAGGCTTCTTCCTCCTCTGG	GAAAGCCCTTTGGATCATCA
			BrSSR074	GGGTTGGACAGGCATGATAC	GAACCAAACCAACCCCTCTA
BrSSR076	GGTGATGTTTCTGGAGCGTT	ACAAATCTGCCCAAGCAATC
			BrSSR077	TTTTCGTCAAAGTCTTCTTCAGC	CATCATGCCATAAAAGCCCT
BrSSR078	ATCGTGCGTGGATCTTCTCT	AAATCAAGTTCTGGATCCGC
			BrSSR079	ACGCAGAGATGGCGTAGTTT	AGCTTCTTCAGCCACTCGTC
BrSSR080	TAAGGTTCCTCTGTGCAGCC	TGCCGGTTCAGAGGTCTACT
			BrSSR081	GCAAGAGGAGAAGCGAGAGA	CAGCGGATTGATCTTTGGAT
BrSSR083	GTCAGCCCTACTGGCTTGTC	AGGTCTCGTGGTCTCTTCCA
			BrSSR084	TGGTTGAATCGCATAACGAG	AGCAGTAGGGAAGAGGCACA
BrSSR085	TACCTGTCGTGGTCAGCAAG	GTGGTGGGTCCATTTGGTAG
			BrSSR086	TCTTGTGTCGTCTACGCTGG	CCAATTCTGAACCAAAACCG
BrSSR088	TCACGTGCGTATCAAAGCTC	GCTTCGTGTGGATTCTTGGT
			BrSSR089	GAATCCTTTGGAAACGACGA	GGGGACACAATTCTCCTGAA
BrSSR090	ACCGGCTCCAAATCCTACTT	CGTGGACCATGAAGATGATG
			BrSSR092	CCAAAGAATCCTCATCCGAA	GTCGAAGCTCGTTCACCATT
BrSSR093	CATCATCAAGAGCGAACGAA	GGGGTGAACAGGAGAAATCA
			BrSSR094	GGTTTGTGAGATTCATGGGG	GAATCACTCCTCTTCCGTCG
BrSSR096	CGTCCAACAAGCAGACTCAA	ATGCTTCACTGCTGATGCTG
			BrSSR097	AAACCATATGGCTGCCTCAG	TTGCAAGCTCCTTTTCGTTT
BrSSR100	CCCCTTCACTCCAATTTCAA	GAGGACGTTGTGAGCTTGGT

BrSSR104	CTTCCCACTGTCAACGGTTT	TCATACTCGTCGATGGGACA
			BrSSR105	CAAGGGTGAACCTTCTGAGC	GAAACGCCTGCTGTTCTTCT
BrSSR106	TGCTATGAGTGTGGTGAGCC	CTACTGTAGCTGCGACCACG
			BrSSR107	AAGAAGAAAGAAGGCGTCCC	CACGAGATTCCGAAGTAGGG
BrSSR109	GTTGCATCCTTAGCCCTCAC	CACGCCTCTCCTCCTACTTG
			BrSSR112	GCCTCTCTACCATCAGCAGC	TTGGAGGAGGAGAGTGAGGA
BrSSR113	GCCTCTCTACCATCAGCAGC	TTGGAGGAGGAGAGTGAGGA
			BrSSR114	GTTCTCCTCACTGGAGCGAC	TTGCGTACGACATCTTCAGC
BrSSR115	GCTCCAAGGTCGCTACAAGT	CCACAAAACCAAAACCCAAA
			BrSSR116	CCTTTGATCCGTTTGCTGAT	GACCAGCCTGAACAAGGAAG
BrSSR117	CTTGCAAATGACCTGCTTGA	CCATGTCGGAAAGCAGAGTT
			BrSSR119	TGAGAATGCGGAGTTCATTG	AACATCGTCCTCATTCCCTG
BrSSR120	GACCAAGCCAGACATCGAAT	GCGACAGGCTTCCATATTCT
			BrSSR121	CGACACGTGGCTTAGACTCA	CGATGATCAAATTAGTGACCAAA
BrSSR122	CTTGTGGTTCGTGTTTGTGG	AGGCAAAGGAAGTGTCGCTA
			BrSSR123	CACCAGAGTCTGCAAATCCA	CTCCTTTCCTTTTCTCGGCT
BrSSR128	TGATGATGAACCACAAGGGA	GATCTCCCGGTATTGCTGAA
			BrSSR130	TGTTCCTGGGAAAGAAGCTG	CCTCCTTGAGGACCAGTCAA
BrSSR131	AATGTTCGTTTCCGACAAGC	CGTTTCTTGGGAGCTTTCAC
			BrSSR132	CGACAAACCTACAACCACAAA	ATGCCAATTGTTGGAGGAAG
BrSSR133	GAATCTGCCGGATGTTTGTT	GGCTAATAGCTTAATCCCCAGAA
			BrSSR134	CGCGTTAAAGAGCATTCACA	TTGCAGCAACAATAAACGGT
BrSSR136	ACCAGCTTGATTTTGTTGCC	CCAAGGATTTGGTGCAAACT
			BrSSR137	GGAGTTCAACGAAACACCGT	TGCTTCCTTGGACAGAACAG
BrSSR138	CCTCCCTAAGCAAGCTGGTA	CTGCAATCGCTGACCATCTA
			BrSSR139	TGTTCTAAAGGTGGCGGTTC	TTGGGCTTCTCTGCTTGTTT
BrSSR140	CCACCACCTCCCTACGTCTA	GGTGGTGGAGACTTGTAGGC
			BrSSR141	CCACCACCTCCCTACGTCTA	GGTGGTGGAGACTTGTAGGC
BrSSR142	CACTTTTATGATTTTCCATTTCG	AAGCTAGAGCCTCCGATTCC
			BrSSR143	CTTTGCATGCATGTTGTTCC	ACAGAGGGCAGATCAACACC
BrSSR144	GCTGAAGAACAACGGAGGAG	TGAAGAATCGTCGTCAACCA
			BrSSR145	TTGAAACTTGGTTGACGACG	TTCTTCTTTGCGGCTGATTT
BrSSR146	CAGAGAAGAAACCAGCCGAG	ACCTGCTTGAGGACCTTGAA
			BrSSR147	CCATCTTGATTTTGTTGCCC	CTGTCAGGGTGAAATTGCCT
BrSSR149	CTCCTTTCTATTCGGTTGCG	TCACCGAGCTTCTTCGTCTT
			BrSSR150	GCCAGTCTTCAAATCCCAAA	CTTCTCAAACAAGTTTTGGTCG
BrSSR151	CTCATGAGAAAGTCGCCCTC	GCCAATAGCCTGCACAAAAT
			BrSSR152	GTAGCATCACCACTTCCGGT	CACATCTTCATCGTCCAATCA
BrSSR153	CGCAGCTCAACAACAACACT	ATCCCAATCGTTCTGTCTGC
			BrSSR154	AACTTTACGGCGAGACCAAA	CTGATCCAGTCGAGGGAGAG
BrSSR156	GATTCGGTTTGATTCCCATC	TTCAATGTCGTTGTACGGGA
			BrSSR158	TTCCTCTTCACCTTTGGTGG	GGAAGAAACGAAGAGCAACG
BrSSR159	CCACCATCGCTTCTCAGATT	GTCTCGTCAGGCCTCTTTTG
			BrSSR160	GCTGGAGAAGACCAAAGCTG	CGGATACCTCTCAGGGATGA
BrSSR161	TCATCCCTGAGAGGTATCCG	AGGAGGGGTTAAAGAGCTGC
			BrSSR162	TCGTGTTCTCTCGTGATTCC	TTGGTGGCACAAAGTGTGTT
BrSSR163	GGTTATACACACTTTGATGGGGA	ATCTTTGGGACTTGCCACTG
			BrSSR164	GTCTCCTCCACCACCGAATA	ACTGGTAGACCGTCTGGTGG
BrSSR165	GATTAGCGACGAGGTTGCTC	CTGAAACCAACATGCTTCAA

BrSSR166	TCGGAGATTATCGGAAGTGG	TCAGAAACCCTGAGAATCCG
			BrSSR169	ATTTGCACTGAGGCTGGTCT	TCCTACGCAGAAGCAAAATG
BrSSR170	ACCGTTACGATGTTGAAGCC	TAAAGAGCAGCTGCGTCAAA
			BrSSR171	ACTCTTGGGATAATTGGGGG	TTTCTTATCCCCTGCACCAC
BrSSR172	AATGGGAAACCAGACACTGC	CATGCAATGGCTTACAATGG
			BrSSR173	GCTCTCTCCATCTCGTGCTC	TCGGAGAAGAAAGCTTCGAG
BrSSR174	TCTCTAGACTCTTTGAAAGGACG	GATGTTGTGCTTGCACTGCT
			BrSSR175	AAGCTCTGCTAGATCATTTTGG	AGGCACAACCTCCACAATTC
BrSSR176	ACAACGCATCAACCAAAACA	TGCTCTTGAACCAGCTTCCT
			BrSSR177	TCTGCATGAAACAAGAGACGA	GAAGTTGGTCCGCTGAGAAG
BrSSR178	TAACTTCTCCCCCGTCCTCT	GAGATGGCTACAGTGGCTCC
			BrSSR179	TGCTATGAGTGTGGTGAGCC	CTACTGTAGCTGCGACCACG
BrSSR180	GCGGTTGTCTCTCACCAACT	AGCTCCGATTGCCAGAGTAA
			BrSSR181	CTCGCTGGTCTCAGGGTAAT	CACGACACTGCAACCAAAAG
BrSSR182	GCCGAACAATTGGAATCATC	GATGGTTGGTTAGCTGCGAT
			BrSSR183	TCAACGACCGTAGAGACGTG	TCGTTTAACAACCAGTACGCA
BrSSR184	TTCATCAGTGCATTCGCTTC	TGGCCCAATAGATTAACCAAA
			BrSSR185	ATGCTCAGCTTCCTGGAGAT	GAAGATGAAAACCGCCAAAA
BrSSR186	GCTCTATCTCGTACGTCGCC	TGGATCCATCAAATTCCGTT
			BrSSR187	CGCTAGTCTCCTTCTCCCCT	ATCACCCAACCAGGAGTGAG
BrSSR188	TGTTCTTACATGCCGTCGAA	AAGACCCATTTGCACAAACC
			BrSSR189	CATCAGGGGTTGCTTCAAAT	TTATCTTTGCCCAAACCTCG
BrSSR190	TCATTCTCTTTCCCTCTGCG	ATTGGAGCAGAGGTTCATGG
			BrSSR191	CCATGAACCTCTGCTCCAAT	CTGAATCAGCGGGATTTGTT
BrSSR192	ACACTACATTCACCGGCTCC	GCATGTTGCATCATACCCAG
			BrSSR193	CTTTCTATTCGGTTGCGCTC	CACCGAGCTTCTTCGTCTTC
BrSSR194	TTCTATTCGGTTGCGCTCTT	CACCGAGCTTCTTCGTCTTC
			BrSSR195	AGGCCAACCCTTACTCTCGT	CCACCAGGAAACAACAAAAA
BrSSR196	AGGCAAAGGACAAAGAAGCA	AATCGTTTGGCTCACGAATC
			BrSSR197	ATGTGCGTTGTGCGTTATGT	CCTGGGACAAGGTTGACAGT
BrSSR198	GTCCTCCTCCTCCTCTGCTT	GAATCCAATGGTGGGAACAG
			BrSSR199	TTTGGCCCTATCGTCATAGC	TCAATGTCGTCGTCGTCTTC
BrSSR200	TCTGATCCCATCAATGCTGA	GCCGGAGGTCATTAGGTTCT
			BrSSR201	GTTGACAAGGAAGCAGAGGC	ATGGTTGCACACCAAGACAA
BrSSR202	GGCTTACTCTCTGTCGTCGG	TGGGTGTCCTTTTCTTCAGG
			BrSSR203	GTTTCTGGCCACTTGTCGTT	ATCGTAAGTGAGGCTGGCAC
BrSSR204	ACTCCATCTCCATGGCTGAA	AGACGAGAATAGACGGCGAA
			BrSSR205	CACCTCCACCCATGAAATCT	ACTGGAGGAGGTGGTGAATG
BrSSR206	AATCTTTGCGTCACCGTTTC	TAAGCGTGGTTTTCAGGACC
			BrSSR207	TGATCGTTCATGGAAAGATTGT	CTGCTGCTGCTCTCTTCCTT
BrSSR208	AGCTAAGGTCAAGCAAGCCA	ACAATCTGTTTCAAAGGCGG
			BrSSR209	GAAAGGTGGATCTAGCTGCG	TATTCCCTTCTTCTCCCGCT
BrSSR210	ACGTAGAGTGCGTTGACACG	ACGACTCACGAAATCGAACC
			BrSSR211	TGGCGAAGAAGGAAGAAAGA	GTTCACACGACTCATTCGGA
BrSSR282	GAGATGAAGCATCAGGGAGC	TTTGCCATAACCTGAAGAAGA
			BrSSR283	ACGGTGGTGTCTTGCCTAAC	CTGAGGAGGAATCAAACCCA
BrSSR284	CTACAAGTCCGACCCATCGT	TCTCCTTCTCCTCCTCCTCC
			BrSSR286	CACTGTCCCTCTGTGTGACG	GCCATACGGAGGAGGGTATT
BrSSR291	TCCTCCGACTACGCTCCTTA	CTGTTTTCAAGTGCTGCCAA

BrSSR292	AATCGGTTTTAAGGGAGGGA	GCTTTGAAACAAAGAGATCAAACA
			BrSSR300	CCCCAGATTGTCGATTCAGT	AAAACGGTGGGCACATAGTC
BrSSR301	CAGAAGCTTTCCCTCCCTCT	CTCTTGAACCATAGGAGGCG
			BrSSR304	TCAACTTCAACAAGAGAGAGAGAGA	TACCAAGAGCTCCAACCCAC
BrSSR306	TTAGGGGTGTGAACCAAAGC	AATTGTTCCTCGAACTCCCC
			BrSSR307	GAGCCTCGCCTCTATTTGTG	CCGATTTGGTAGATCTCGGA
BrSSR308	CCAAGGAGATCTGTGGTCGT	TCAGTCTCCAGATCCATCCC
			BrSSR310	TGCCATTCAGATCCTTACCC	TCAGGCTCTCTTCCACCTTC
BrSSR311	TGGCTAGCAGTGAGATGGAA	GTCCAGCACTCACAGACGAA
			BrSSR315	AGCAATTCCAAAACCAACCA	ATGGATACTCCTCCGTTCCA
BrSSR316	CAACACCAATCGCATAATCG	CTTGACGCCATGTTGAGCTA
			BrSSR324	TCGAAAAGGAAGTGGGATTG	GGTTGGTTTGGCACAGAACT
BrSSR327	GAGAGGGAATCGTTGATGGA	TCCGAACAAGCGGTAGACTT

The polyacrylamide gel electrophoresis that PCR product is 12% with mass percent detects.PAGE electrophoresis and dyeing agents useful for same and instrument are as follows:

(1) the acrylamide mixed solution (100mL) of 0.3g/mL, compound method is: 29g acrylamide (Acrylamide), 1gN, N '-first bisacrylamide (Bisacrylamide), adds ddH ₂o to 100mL, 37 DEG C of stirring and dissolving, 4 DEG C keep in Dark Place.

(2) Ammonium Persulfate 98.5 of 0.1g/mL (10mL), compound method is: 1g Ammonium Persulfate 98.5 (Ammonium persulfate, APS) is dissolved in 10mL ddH ₂in O, 4 DEG C of preservations.

(3) 5 × tbe buffer liquid (pH8.3,500mL), compound method is: 27g Tris alkali, 13.75g boric acid, 1.86gNa ₂eDTAH ₂o, adds ddH ₂o constant volume is to 500mL, 4 DEG C of preservations.

(4) TEMED (N ', N ', N ', N '-Tetramethyl Ethylene Diamine).

(5) sample-loading buffer: wherein contain 0.25% tetrabromophenol sulfonphthalein (W/V), 40% aqueous sucrose solution (W/V), 4 DEG C of storages.

(6) back cover glue: massfraction is 1% agarose solution, configures with 1 × TBE.

(7) staining fluid: dehydrated alcohol 50ml, Silver Nitrate 0.5g, is dissolved in ddH ₂o is also settled to 500ml.Be stored in brown bottle room temperature preservation.

(8) nitrite ion: sodium hydroxide 10g, is dissolved in ddH ₂o is also settled to 500ml, room temperature preservation.It is 37% formaldehyde that laboratory adds 2ml mass percent again.

(9) instrument: DYY-6C type voltage stabilization and current stabilization electrophoresis apparatus (Liuyi Instruments Plant, Beijing), DYCZ-30C type double plate Vertial electrophorestic tank (Liuyi Instruments Plant, Beijing), Tanon2500 fully automatic digital gel image analysis system etc.

The key step of PAGE electrophoresis:

(1) will prepare the sheet glass of encapsulating, comb, gum cover, electrophoresis chambers etc., with the flushing of stain remover washing tap water, dry alcohol wipe.

(2) two sheet glass are inserted after rubber coating, put into electrophoresis chamber, make lower sheet glass towards negative pole (black electrodes).Keep electrophoresis chamber level, the screw rod of tightening on electrophoresis chamber makes sheet glass and rubber coating firmly and sealing.

(3) in microwave oven, back cover glue is boiled, the outer side seams of three of sheet glass and rubber coatings is sealed, and the space of a sheet glass bottom is wherein sealed up.

(4) glue (polyacrylamide gel of 12% mass percent) 50mL:19.65mL ddH ₂o+20mL30% collagen solution+10mL5 × TBE+35 μ L TEMED+350 μ L APS.

(5) encapsulating: draw gelating soln with 100ml syringe, turn syringe, emptying enters the air of needle tubing.The syringe needle of syringe is inserted to the space between two sheet glass, inject acrylamide soln, almost fill space.

(6) immediately suitable comb is inserted in gel gently, does not carefully make to leave bubble under broach.The top of comb should be a little more than the upper edge of sheet glass.

(7) acrylamide, in polymerized at room temperature 30～60min, is put into 30 DEG C of baking ovens winter.If gel retraction obviously, should add solution.Two refractive power lines under visible broach when the complete polymerization of gel.

(8) pull out comb: infiltrate the top 2-4 minute of gel with 1 × TBE, carefully extract comb, use immediately 1 × TBE to rinse well, to prevent that a small amount of acrylamide soln that comb retained from, in well polymerization, producing irregular surface.

(9) glued membrane is clipped on electrophoresis chamber, in every 20 μ L systems, adds 2 μ L sample-loading buffers, mix point sample 1 μ L.

(10) electrophoresis: 120V, 60mA, apart from stop (about 3h) to 2/3 place.

The silver of PAGE glue dyes step and adopts fast staining, and concrete steps are as follows:

(1) dyeing: after polyacrylamide gel electrophoresis finishes, unload lower glass plate, gel be transferred in staining fluid, under room temperature on shaking table with 40～80rpm vibration, 8～10min.

(2) clean: use up staining fluid, use ddH ₂o cleans gel 2 times, and 20～30s, cleans after 20～30s for the second time, adds micro-nitrite ion, slightly after vibration, outwells the nitrite ion of blackening.

(3) colour developing: most above-mentioned scavenging solution, then add appropriate nitrite ion, and vibrate to clear band line appearance (about 5min) with 40rpm, use up nitrite ion, and use ddH ₂o rinses 2～3 times.

(4) wash glue: use ddH ₂o cleans, and by careful PAGE glue transferring on white base plate, takes pictures with gel imaging system.

Analyze pcr amplification product electrophoresis result, find that BrSSR133 there are differences (Fig. 2 and Fig. 3) between between two parents and anti-(sense) sick pond, thinks that it likely exists linkage relationship with objective trait (club root resistance).

5. filter out a SSR molecule marker BrSSR133, its sequence is SEQ ID NO.3, and its upstream primer is SEQ ID NO.1, and downstream primer is SEQ ID NO.2; Through individual plant check analysis, the genetic distance of this molecule marker BrSSR133 and club root resistant gene is 5.02cm.

Specific practice is: the BrSSR133 molecule marker that screening is obtained carries out individual plant checking, and statistics resists, feels the frequency of occurrences and the switch type of individual plant indicia band and calculate crossover value, uses Kosambi function to calculate the genetic distance between mark and anti-club root gene.Result shows (Fig. 4), in 41 disease-resistant individual plants, has 37 strains to have special band, and 4 strains do not have special band; In the susceptible individual plant of 39 strain, all there is no special band.This experimental result shows that BrSSR133 molecule marker is is closely linked with the club root resistance of turnip, but there is certain exchange in disease-resistant and susceptible individual plant, exchange rate is 5%, and the genetic distance of SSR mark BrSSR133 and disease-resistant gene is 5.02cm as calculated.

6. the molecule marker BrSSR133 that application the present invention screens acquisition is to F ₃0 grade of generation and 3 grades of materials carry out respectively PCR checking, result demonstration, and the sequence length of BrSSR133 is 261bp, BrSSR133 mark can be directly used in the assisted selection practice of the anti-club root of Vegetable Crops of Brassica crop.

The concrete practice is:

(6.1) reclaim adhesive tape DNA: cut the non-sex change PAGE glue that contains target dna fragment with aseptic operation cutter, the wide about 2mm of adhesive tape, then adhesive tape is proceeded to 1.5mL centrifuge tube with clean tweezers.Adhesive tape is ground with liquid nitrogen.Add 200 μ L to disperse damping fluid (0.5M CH ₃cOONH ₄, 0.01MMg (CH ₃cOO) ₂, 1mM EDTA, SDS, pH8.0 that massfraction is 0.1%), broken glue and damping fluid are mixed with aseptic rifle head; 60 DEG C of temperature are bathed 60min, and every 15min vibration during this time 1 time, guarantees that damping fluid fully contacts with broken glue, improves the burst size of object fragment.Add 200 μ L chloroform extraction liquid (chloroform: primary isoamyl alcohol: dehydrated alcohol volume ratio is 19:1:5) to mix, with the centrifugal 3min of 13000rpm, suct clear liquid 150 μ L, forward in the aseptic centrifuge tube of new 1.5mL.The dehydrated alcohol that adds 300 μ L precoolings, mixes, and places 30min for-20 DEG C; With the centrifugal 4min of 13000rpm; Abandon supernatant liquor, be inverted dry 15～20min.Finally, add 10 μ L ddH ₂o dissolves ,-20 DEG C of preservations.

(6.2) DNA profiling that the reaction system (20 μ L) of pcr amplification: PCR: 75ng reclaims, 0.5UTaqDNA polysaccharase (Fermentas ^tM), 0.4 μ L (10 μ molL ^-1) dNTPs, 1 μ L (10 μ molL ^-1) upstream and downstream primer, 2 μ L10 × PCR Buffer (200mmolL ^-1tris pH8.0,200mmolL ^-1kCl, 100mmolL ^-1(NH ₄) ₂sO ₄and 20mmolL ^-1mgSO ₄), add ddH ₂o to 20 μ L.PCR detect amplification program: 94 DEG C of denaturation 3min, enter 35 circulations (94 DEG C of sex change 30s, 53 DEG C annealing 30s, 72 DEG C extend 30s), finally again 72 DEG C extension 7min.PCR reaction process is at BIO-RADS1000 ^tMon Thermal Cycler instrument, carry out, sample is preserved under 4 DEG C of conditions.

(6.3) PCR product reclaims: the reclaimer operation step of PCR product is with reference to Axyen ^tMgel reclaims test kit specification sheets to carry out.

(6.4) carrier of recovery product connects: linked system (10 μ L): 5 μ L2 × Rapid Ligation Buffer, 4 μ L reclaim products, 0.5 μ LT-easy carrier (50ng μ L ^-1), 1.5U T4DNA ligase enzyme (Promega ^tM), add ddH ₂o to 10 μ L.4 DEG C connect 10～12h.

(6.5) conversion of connection carrier: the competent escherichia coli cell 100 μ L that take out-75 DEG C of preservations, slightly thaw, add the connection product of 10 μ L.After vortex mixes, put into ice, ice bath 30min, then puts into 42 DEG C of water-bath thermal shock 100s, then ice bath 5min.On Bechtop, every pipe adds the liquid LB substratum of 1mL.On constant-temperature table 37 DEG C, the about 90min of 200rpm jolting.LB solid medium is put into microwave oven, heating and melting.After the LB substratum melting is slightly cooling, every 30mL adds sodium ampicillin (Amp, the 200mgmL of 7.5 μ L ^-1), after mixing, change into two flat boards, cooled and solidified.After bacterium liquid in shaking table is taken out, 12000rpm, centrifugal 1min, abandons supernatant.Residual cleer and peaceful precipitation is mixed with liquid-transfering gun, drip to and be frozen on dull and stereotyped LB substratum, smear evenly with paint daubs.The flat board 30min that faces up, after bacterium liquid is dry, be inverted dull and stereotyped in the substratum of 37 DEG C 10～12h.

(6.6) choose bacterial plaque order-checking: in 30mL liquid LB substratum, add 7.5 μ L Amp (200mgmL-1), mixing rear absorption 1mL substratum divides and is filled in 1.5mL centrifuge tube, the white bacterial plaque of making even on plate with the choicest of liquid-transfering gun rifle, in LB substratum in centrifuge tube inhale beat several under, guarantee that the bacterium colony of picking has been inoculated in the substratum of this centrifuge tube.The above-mentioned LB substratum with target bacterium colony is placed in to 37 DEG C of constant-temperature tables, the about 12h of the horizontal jolting of 200rpm.Taking the above-mentioned bacterium liquid shaking as template, detect with SP6/T7 primer pair bacterium liquid.The reaction system of bacterium liquid PCR and the same step of amplification program (6.2).According to the detected result of bacterium liquid PCR agarose gel electrophoresis, select positive bacteria liquid, draw 500 μ L, and be sent to Shanghai Ying Jun Bioisystech Co., Ltd and check order.Sequencing result is as shown in SEQ ID NO.3.

Above-mentioned enforcement is used for the present invention that explains, instead of limits the invention, and in the protection domain of spirit of the present invention and claim, any amendment and change that the present invention is made, all fall into protection scope of the present invention.

SEQUENCE LISTING

<110> Zhejiang University

Molecule marker and the preparation method of the anti-club root gene linkage of <120> and turnip

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> DNA

<213> synthetic

<400> 1

gaatctgccg gatgtttgtt 20

<210> 2

<211> 23

<212> DNA

<213> synthetic

<400> 2

ggctaatagc ttaatcccca gaa 23

<210> 3

<211> 249

<212> DNA

<213> synthetic

<400> 3

gaatctgccg gatgtttgtt ttagaacaaa gcaatgttct cttgtatatc atcatcaatc 60

acaaagaaaa catatacata tagagattta catataaaaa atctcgtcca tatttgtttt 120

ctttcaggac ttggggtgca tttttctgca ttgcaaagaa agaaagaaag cagaatcatt 180

atgtttcctc tttattcttt ctggcactag tttataaaaa tattacttct ggggattaag 240

ctattagcc 249

Claims (3)

1. a molecule marker mutually chain with the anti-club root gene of turnip, is characterized in that, described molecule marker is brSSR133,sequence is SEQ ID NO.3.

2. a kind of molecule marker mutually chain with the anti-club root gene of turnip according to claim 1, is characterized in that, the upstream primer of described molecule marker is SEQ ID NO.1, and downstream primer is SEQ ID NO.2.

3. a preparation method for the mutually chain molecule marker of claimed in claim 1 and the anti-club root gene of turnip, is characterized in that, mainly comprises the following steps:

(1) utilize the anti-club root turnip parent material ' 077-03 ' and the Susceptible parent material ' 077-04 ' that select through inbreeding of more generation, described disease-resistant parent ' 077-03 ' and Susceptible parent ' 077-04 ' are hybridized and obtained F ₁colony, and build corresponding BC ₁, F ₂, F ₃colony;

(2) according to the disease symptom of field natural occurrence material, and cause of disease statospore microscopy, the cause of disease that specifies this disease is rape plasmodiophora brassicae;

(3) to BC ₁, F ₁, F ₂colony carries out artificial inoculation qualification, finds that the club root resistance of ' 077-03 ' is by a pair of dominant nuclear gene control;

(4) utilize SSR molecular marking technique, utilization improved BSA method is carried out the screening with turnip club root resistance molecule marker;

(5) filter out a SSR molecule marker brSSR133, through individual plant check analysis, the genetic distance of this molecule marker and club root resistant gene is 5.02 cm.