CN103667272B - The EST-SSR molecule marker relevant to mitten crab growth traits and application - Google Patents

Abstract

The invention discloses the EST-SSR molecule marker relevant to mitten crab growth traits, described molecule marker called after: SSR1107, by core sequence (TG) _n, upstream primer and downstream primer composition, described upstream primer is shown in SEQ ID NO.1, and downstream primer is by shown in SEQ ID NO.2; N=7-9.Molecule marker SSR1107 of the present invention overcomes in traditional breeding way, and the main phenotypic character that relies on is identified, comparatively large by such environmental effects, and not only difficulty is large to make growth traits breeding, and inefficient deficiency.By applying the EST-SSR molecule marker relevant to mitten crab growth traits of the present invention, quick and precisely can screen growth traits Parents for river crab breeding, select target is clear and definite, time saving and energy saving.

Description

The EST-SSR molecule marker relevant to mitten crab growth traits and application

Technical field

The invention belongs to molecular biology and Biotechnology in Genetic Breeding field, relate to EST-SSR molecule marker that is a kind of and mitten crab growth traits significant correlation particularly, also relate to the application of molecule marker in mitten crab breeding simultaneously.

Background technology

Mitten crab (Eriocheir sinensis) is commonly called as river crab, and belonging to Crustachia (Crustacea), Decapoda (Decapoda), Eriocheir (Eriocheir), is one of China Important Economic crustacean.Culture zone spreads all over maritime provinces and the vast hinterland such as China Liaoning, Hebei, Tianjin, Shandong, Jiangsu, Zhejiang, according to statistical information display in 2009, whole nation culture of Chinese mitten crab relates to 30 provinces (city, autonomous region) in the whole nation, area reaches 66.7 ten thousand hectares, output 530,000 tons, the output value 28,000,000,000 yuan, has become the industry that the fresh water fishery single variety output value is maximum.

Culture of Chinese mitten crab is had very early in China; the seventies in last century in the past mainly caught wild juvenile crab carried out artificial extensive cultivation by adopting; the eighties is based on enhancement releasing; the beginning of the nineties is along with the breakthrough of crab seedling rearing technology; river crab artificial seed large-scale production; the pond of river crab and lake lake Area start to rise, and develop rapidly.In recent years, under the promotion of the market requirement and economic benefit, river crab artificial seed breeds and becomes crab to cultivate scale and constantly expands, culture of Chinese mitten crab industry is flourish, and emerge the best brand of product of each tool local characteristic, and as the Yangcheng Lake steamed crab had won fame both at home and abroad, the Clintonia udensis river crab etc. of unique local characteristic.Culture of Chinese mitten crab becomes mainstay industry that is with the fastest developing speed in China's fish production, most characteristic, most potentiality, has effectively driven the development of the related industries such as feed, fishing medicine, food and drink, tourism, trade.

But river crab industry is fast-developing, Seedling production lacks necessity, specification, the technical measures of science and means, causes that the speed of growth is slow, the appearance of the individual kind matter degradation phenomena such as less than normal of sexual maturity.The Sustainable development of crab farming industry is based on the seed of a large amount of good quality and high output, and this just proposes requirements at the higher level to river crab breeding work.Growth traits is typical quantitative character as breeding objective, its central genetic feature jointly regulates and controls by polygene, due to the impact of the factors such as envrionment conditions, clear and definite corresponding relation is lacked between Phenotype and genotype, and traditional breeding method depends on individual phenotype indirectly selects genotype, efficiency of selection is low, and the prevalent variety cultivation cycle is long, and breeding effect is not good.Eliminate such environmental effects, improve efficiency of selection and the accuracy of breeding, optimal method can directly be selected genotype exactly.

Molecule marker provides possibility to genotypic direct selection, because the genotype of molecule marker can identify for realizing.If target gene and certain molecule marker close linkage, so by the detection to molecular marker gene type, the genotype of target gene just can be known.Therefore, we can select by the genotype of molecule marker to objective trait, are applied in the middle of breeding work by molecule marker.Micro-satellite (Microsatellites) molecule marker has rich polymorphism, follows the feature such as Mendel's law of segregation, codominant inheritance, has been widely used at present in molecular marker breeding research.The EST-SSR obtained by expressed sequence tag (EST) exploitation marks, and is more better than genome microsatellite marker, is more suitable for Biotechnology in Genetic Breeding field in conservative property.Wu Yan etc. have carried out correlation analysis to 30 full-length genome microsatellite locus and growth traits in a river crab colony, and wherein 4 sites and typical growth proterties are significant correlation in result display.So far, there is not yet the report of mitten crab EST-SSR molecule marker and growth traits correlation analysis.

Summary of the invention

The object of the invention is to fill up mitten crab molecular breeding technology field growth traits to be correlated with the blank of EST-SSR molecule marker, provide a kind of EST-SSR molecule marker relevant to mitten crab growth traits.

Second object of the present invention is to provide the EST-SSR molecule marker application in growth traits breeding relevant to mitten crab growth traits.

Technical scheme of the present invention is summarized as follows:

An EST-SSR molecule marker relevant to mitten crab growth traits, described molecule marker called after: SSR1107, by core sequence (TG) _n, upstream primer and downstream primer composition, described upstream primer is shown in SEQ ID NO.1, and downstream primer is by shown in SEQ ID NO.2; N=7-9,

An application for the EST-SSR molecule marker relevant to mitten crab growth traits, comprises the steps:

(1) individual 60 of random choose male and female in river crab sexual maturity colony, number record one by one;

(2) every river crab clip step, extracts muscle DNA with phenol-chloroform method;

(3) utilize molecule marker SSR1107 to carry out gene type to selected river crab, retain the individuality containing genotype A, the close crab as river crab breeding carries out breeding production; Described genotype A refers to molecule marker core sequence (TG) _nin n=7.

Advantage of the present invention:

The present invention obtains EST-SSR molecule marker SSR1107 that is a kind of and mitten crab growth traits significant correlation first.Overcome in traditional breeding way, the main phenotypic character that relies on is selected, comparatively large by such environmental effects, and not only difficulty is large to make growth traits breeding, and inefficient deficiency.By applying the EST-SSR molecule marker relevant to mitten crab growth traits of the present invention, quick and precisely can screen growth traits Parents for river crab breeding, select target is clear and definite, time saving and energy saving.

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.

Embodiment 1

F6 is for the measurement of Clintonia udensis river crab population growth trait phenotypes value and record

Individual 60 of random choose male and female in river crab sexual maturity colony, number record one by one;

Measurement primary growth proterties body is long, body is wide, (unit: g) index, recording individual sex, data are as shown in table 1 for height, IV step meropodium long (unit: mm) and body weight.

Table 1 mitten crab population growth trait phenotypes value

Numbering	Body is long	Body is wide	Height	IV step meropodium is long	Body weight	Male and female
							1	61.76	71.90	32.88	49.12	184.06	♂
2	56.68	63.16	31.56	40.38	108.96	♀
							3	54.34	60.30	29.32	42.46	114.22	♂
4	49.56	55.34	28.44	35.10	77.46	♀
							5	62.18	69.12	33.98	49.62	184.92	♂
6	60.90	68.52	33.32	47.26	160.32	♂
							7	54.54	60.82	29.00	44.62	117.96	♂
8	53.00	60.86	28.86	42.44	110.38	♂
							9	53.52	59.42	29.78	42.24	109.92	♂
10	51.74	57.26	27.42	39.32	97.65	♂
							11	46.68	51.06	26.26	36.28	60.08	♀
12	56.78	62.40	30.62	44.38	128.82	♂
							13	51.12	57.36	29.00	38.22	81.38	♀
14	55.90	62.00	30.00	43.00	121.42	♂
							15	55.00	60.88	31.18	38.58	100.87	♀
16	56.28	63.68	30.72	41.42	105.84	♀
							17	54.52	59.42	29.06	42.32	103.93	♂
18	53.12	57.42	31.34	43.40	87.07	♀
							19	65.60	67.48	35.64	45.80	155.20	♂
20	46.24	50.40	27.86	28.32	57.30	♀
							21	44.54	49.92	26.42	23.98	54.03	♀
22	54.52	62.38	31.66	41.00	97.79	♀
							23	50.02	56.18	29.12	36.20	76.49	♀
24	54.52	60.06	34.08	39.54	100.24	♀
							25	50.44	55.78	29.14	38.10	76.19	♀
26	56.18	62.16	32.54	40.58	108.20	♀
							27	47.78	52.58	27.92	36.20	63.98	♀
28	50.72	56.22	28.00	40.32	91.20	♂
							29	60.80	67.52	34.72	47.66	155.31	♂
30	51.00	58.44	29.56	37.56	81.66	♀
							31	57.78	64.56	32.88	48.52	146.38	♂
32	56.80	64.84	33.18	42.72	132.62	♂
							33	47.66	52.56	27.18	29.14	63.21	♀
34	50.42	55.18	29.32	34.82	76.15	♀
							35	52.60	56.10	29.50	36.90	78.10	♀
36	54.30	58.30	31.70	37.90	85.06	♀
							37	62.30	66.50	35.50	50.80	158.40	♂
38	56.10	60.80	39.20	41.60	115.90	♂
							39	49.10	54.20	27.20	38.50	66.90	♂
40	56.70	62.90	31.80	44.20	116.90	♂
							41	46.00	51.70	25.56	36.22	61.22	♂
42	47.20	51.24	28.74	34.86	57.14	♀
							43	43.94	47.44	26.22	31.62	43.96	♀
44	56.04	61.26	34.06	39.76	94.15	♀
							45	58.06	63.24	32.54	39.76	119.29	♂
46	52.34	56.18	30.54	35.64	76.18	♀
							47	57.46	61.92	32.26	46.46	125.24	♂
48	58.94	64.60	34.38	44.72	125.80	♂
							49	56.60	66.60	34.20	39.36	96.42	♀
50	50.98	54.94	28.00	40.12	82.22	♂
							51	53.50	58.88	30.24	42.66	95.60	♂
52	53.76	59.02	30.44	42.66	98.04	♂
							53	55.54	61.00	30.66	44.22	106.95	♂
54	52.76	57.64	30.34	38.34	79.80	♀
							55	50.80	52.62	31.46	35.68	71.40	♀
56	62.20	69.02	38.90	41.98	138.85	♀
							57	65.36	72.36	37.06	50.00	188.00	♂
58	50.74	55.86	27.32	33.42	83.15	♀
							59	50.66	53.90	27.22	32.42	80.64	♀
60	54.62	59.88	30.04	35.24	103.72	♀

Embodiment 2. mitten crab F6 is for the extraction (step of every river crab clip) of breeding population muscle STb gene

Adopt phenol-chloroform method to extract muscle STb gene, concrete steps are as follows:

1) cut the muscle samples 100mg of freezen protective, 1.5ml sterile centrifugation tube is put in chopping;

2) add extraction buffer (6M urea, 10mM Tris-HCl, 125mM NaCl, 1%SDS, 10mM EDTA, pH7.5) 500 μ l, Proteinase K (20mg/ml) 8 μ l, 37 DEG C of digestion are spent the night;

3), after sample complete digestion, isopyknic PCI(phenol is added: chloroform: primary isoamyl alcohol=25:24:1), the centrifugal 15min of Stirring 20min, 5000rpm room temperature, gets supernatant liquor in the sterile centrifugation tube that another is new.Repeat PCI extracting 3 times;

4) supernatant liquor is carefully proceeded in new centrifuge tube, adds isopyknic CI(chloroform: primary isoamyl alcohol=24:1), the centrifugal 15min of Stirring 10min, 5000rpm room temperature, gets supernatant liquor.Repeat CI extracting 3 times;

5) by the mixing of the sodium-acetate (3M, pH5.2) of supernatant liquor and 1/10 volume, then add 100% dehydrated alcohol of 2.5 times of volume precoolings ,-20 DEG C of preservation of spending the night, precipitate DNA;

6) mixed solution is at 4 DEG C, the centrifugal 15min of 12000rpm.Carefully pour out all liquid, note not allowing the white precipitate particle of bottom pour out;

7) by 70% washing with alcohol 2 times of precooling, then 1 time is replaced with the absolute ethanol washing of precooling;

8) after DNA complete drying, 50 μ l1 × TE(10mM Tris-HCl are dissolved in, 1mM EDTA, pH8.0) in, 4 DEG C of dissolvings are spent the night;

9) with ultraviolet spectrophotometer, DNA concentration is adjusted to 100ng/ μ l, be placed in 4 DEG C for subsequent use.

The exploitation of embodiment 3.EST-SSR molecule marker

1) log in ncbi database (http://www.ncbi.nlm.nih.gov/dbEST) and search for mitten crab est sequence, (http://www.gramene.org/db/searches/ssrtool) searches the cDNA fragment containing microsatellite sequence online, and standard is that multiplicity (comprises 5 times) more than 5 times;

2) according to sequencing result and est database the selection result, choose the suitable DNA segment containing microsatellite sequence, utilize Primer Premier5.0 (http://www.premierbiosoft.com/) software design primer, and send the raw work with Shanghai to synthesize.The theoretical parameter that design primer is considered comprises: GC content, annealing temperature, mispairing, primer dimer, hairpin structure, 3 ' hold last bit base and stability thereof etc.;

3) grads PCR amplification is carried out to synthesized primer, agarose gel electrophoresis EB dyes preliminary screening, and determine the suitableeest annealing temperature of often pair of primer, choose the primer that can amplify single clear band;

The screening of embodiment 4.EST-SSR molecule marker polymorphism

1) from F6 for Stochastic choice 10 samples Clintonia udensis river crab colony, the template as screening primer polymorphism carries out pcr amplification reaction;

2) 10 μ l PCR reaction systems comprise 100ng template DNA, 2mM dNTP, 1.5mM MgCl ₂, 0.25U Taq enzyme, each 10 μMs of forward and reverse primers and 1 × Buffer;

3) PCR response procedures is 94 DEG C of 3min; 94 DEG C of 1min, annealing 1min, 72 DEG C of 1min, circulate 35 times; 72 DEG C extend 5min.

4) preparation of denaturing polyacrylamide gel plate:

A. the sheet glass of silver dye order-checking must be very clean, generally first with stain remover washing, then uses deionized water rinsing sheet glass, finally clean with ethanol.

B. the cleaned square glass of strict process and ear glass respectively is all needed before each encapsulating.The stripping silane (Repel Silane) of ear sheet glass wiping 1ml2%, square sheet glass affine silane (Binding Silane) (1.5ml dehydrated alcohol, 7.5 μ l glacial acetic acids, the affine silane of 2 μ l0.5%) carries out silication.Side, ear sheet glass, in turn in treating processes, first will be changed gloves, prevent two pieces of silane crossed contaminations.At least dry 10min after silanization.

C. sheet glass assembling is carried out.With the sheet glass left and right sides, the edge strip side of being placed in that 0.4mm is thick, by ear sheet glass pressure thereon, both sides clip is fixed.When using clip fastening glass panels, the strength of best clip is slightly larger, prevents because strength deficiency makes to occur the phenomenon of leakage in the process of encapsulating.

D. by 60ml6% denaturing polyacrylamide gel storage liquid (420g urea, 57g acrylamide, 3g methylene diacrylamide, 100ml10 × TBE, is dissolved in sterilizing distilled water, be settled to 1L, 4 DEG C save backup), after adding 0.024g ammonium persulphate and 24 μ l TEMED, enter along encapsulating light irrigation fond of food that is not salty, treat that glue flow to bottom sheet glass, at encapsulating light insertion shark tooth comb fond of food that is not salty.Note strictly to prevent bubble in encapsulating process, otherwise the result of impact order-checking.After encapsulating terminates, static placement makes it to be polymerized at least 2.5h, if allow glue spend the night, spreads preservative film in case dry glue at two of glue.

5) denaturing polyacrylamide gel electrophoresis:

A. after gel polymerisation is complete, extract shark tooth comb, be assembled into by sheet glass on electrophoresis chamber, dilution 10 × tbe buffer liquid to 1 × TBE, adds in upper and lower two electrophoresis chambers, 60W invariable power prerunning 30min by this damping fluid.The process of prerunning removes the foreign ion of gel, makes the temperature that gel slab reaches required simultaneously.High temperature electrophoresis can prevent GC from enriching the hairpin like fold of district's formation, the result of impact order-checking.

B. prerunning simultaneously, carries out the preparation of sample.PCR primer mixes with formamide denaturation agent 1:1,95 DEG C of sex change 5min, then ice bath immediately.

C., after prerunning terminates, powered-down, inhales buffer solution for cleaning sample well with needle tubing, removes the urea diffused out when prerunning.By shark tooth comb conversely, having in the insertion gel of tooth.Each loading wells clicks and enters the sample of 6 μ l through sex change.After application of sample, 60W invariable power electrophoresis immediately.

6) cma staining and development:

A., after electrophoresis, careful separately two pieces of sheet glass, gel can be close on the square sheet glass of the affine silane of painting.

B. fix: gel slab is placed in 10% glacial acetic acid solution (fixing/stop solution), shakes dyestuff completely dissolve in 15-30min to sample gently.

C. rinse: wash glue 3 times, each 5-10min with tri-distilled water vibration.

D. dye: in 2L tri-distilled water, add 2g AgNO ₃be made into staining fluid with 3ml37% formaldehyde, gel slab be placed in staining fluid and fully shake 40min.

E. rinse: from staining fluid, take out offset plate put into tri-distilled water and embathe 5-10s.Noting that gel is transferred to time of developing solution from tri-distilled water can not be long, otherwise causes weak output signal, even loses signal.

F. develop: moved on to rapidly by gel slab in the developing solution of 2L cooling (2L water adds 60g anhydrous Na CO3, is cooled to 4 DEG C, adds 37% formaldehyde 3.5ml before using, 10mg/ml Sulfothiorine 400 μ l), fully vibration is until band line occurs.

G. fixing: fixing 3-5min in fixing/stop solution.

H. rinse: embathe gel 3-5min at tri-distilled water.

I. kept dry: be scanned into image file after waiting offset plate drying and preserve.

The gene type of embodiment 5.EST-SSR polymorphism primer in mitten crab colony and with growth traits correlation analysis

1) using river crab Meta-genomic DNA as template, pcr amplification reaction, denaturing polyacrylamide gel electrophoresis somatotype are carried out to primary dcreening operation primer, manually am-plified fragments is read according to 10bp DNA Marker, use analysis software GENEPOP3.4(Raymond et al, 1995) and MICROSATELLITE ANALYSER(MSA) (Dieringer et al, 2003) analyze microsatellite locus characteristic;

2) SPSS11.5 software is applied, adopt general linear model (General Linear Model, GLM), with marker gene somatotype and river crab sex for independent variable(s), grow that typical proterties body is long, body is wide, the long and body weight of height, IV step meropodium is dependent variable, carry out multivariate analysis of variance, and the multiple comparisons between interior different genotype is marked to the mark of having of detecting and growth traits significant correlation, find the genotype with proterties significant correlation.

Result shows: mark SSR1107(amplification is as shown in table 2, locus trait is as shown in table 3) long with primary growth proterties body, body is wide, IV step meropodium length and body weight are significant correlation (P<0.05) (table 4 and 5), multiple comparisons between different genotype shows, the average body of genotype A is long, body is wide, height, IV step meropodium length and body weight are all higher than genotype B and C(table 6 and 7), and genotype A and genotype B significant difference (P<0.05), genotype C belongs to rare genotype, probability of occurrence is only 3%, therefore can reach a conclusion: genotype A is the preponderant genotype of growth traits.Described genotype A refers to molecule marker core sequence (TG) _nin n=7, clip size is 167bp; Genotype B refers to n=7 and 8, and clip size is 167bp and 169bp; Genotype C refers to n=7 and 9, and clip size is 167bp and 171bp.

Table 2 molecule marker SSR1107 amplification

Numbering	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24	25	26	27	28	29	30
																															Genotype	A	A	A	B	A	A	A	A	A	A	A	A	A	A	A	A	B	A	A	A	B	A	A	A	A	A	B	B	A	A
Numbering	31	32	33	34	35	36	37	38	39	40	41	42	43	44	45	46	47	48	49	50	51	52	53	54	55	56	57	58	59	60
																															Genotype	A	A	B	A	A	C	A	A	A	A	A	B	A	A	A	A	A	A	A	C	A	A	A	A	A	A	A	A	A	A

Table 3 microsatellite locus SSR1107 characteristic

Note: primer sequence F is SEQ ID NO.1, R is SEQ ID NO.2; Repeating unit (TG) _nmiddle n=7-9

The factor between table 4 main body

The inspection of effect between table 5 main body

A R side=.350 (adjustment R side=.290)

B R side=.338 (adjustment R side=.276)

C R side=.177 (adjustment R side=.101)

D R side=.566 (adjustment R side=.526)

E R side=.454 (adjustment R side=.404)

Table 6 is estimated

Table 7 paired comparison

Based on the marginal average of estimation

* average difference is remarkable at 0.05 level difference.

A is to the adjustment of multiple comparison: least marked difference (being equivalent to not adjust).

Embodiment 6. 1 kinds and the application of EST-SSR molecule marker SSR1107 in Parent Mitten Crab seed selection of mitten crab growth traits significant correlation, the steps include:

1) individual 60 of random choose male and female in river crab sexual maturity colony, number record one by one, measurement primary growth proterties body is long, body is wide, (unit: g) index, recording individual sex, data are as shown in table 8 for height, IV step meropodium long (unit: mm) and body weight.

Table 8 mitten crab population growth trait phenotypes value

Numbering	Body is long	Body is wide	Height	IV step meropodium is long	Body weight	Sex
							1	48.43	52.68	28.57	36.00	66.47	♀
2	46.26	50.98	23.75	37.91	60.72	♂
							3	53.35	58.00	28.70	43.17	102.94	♂
4	46.70	46.68	22.76	33.58	48.94	♂
							5	47.22	50.40	27.78	34.70	65.92	♂
6	44.04	49.92	24.62	32.18	51.66	♀
							7	48.76	55.08	28.00	36.76	69.22	♀
8	47.56	50.50	26.58	34.60	54.29	♀
							9	49.64	55.12	25.98	38.72	82.04	♂
10	44.32	47.26	24.00	33.84	51.42	♂
							11	43.54	48.20	22.56	34.92	56.55	♂
13	48.28	55.56	26.12	40.96	81.10	♂
							14	44.00	47.38	23.44	34.12	52.31	♂
15	42.32	46.72	24.44	30.72	45.35	♀
							16	41.58	45.50	22.40	32.68	45.63	♂
17	43.74	48.12	22.52	34.64	55.11	♂
							18	41.62	46.48	23.32	31.12	43.34	♀
20	45.70	50.20	23.46	36.78	60.57	♂
							21	44.82	48.14	25.40	31.88	56.11	♂
22	47.96	55.28	26.06	30.80	70.22	♂
							23	44.60	48.34	24.42	34.92	52.37	♂
24	44.74	49.18	24.54	31.62	53.01	♀
							25	51.58	54.60	31.38	35.86	74.38	♀
26	48.12	52.88	26.62	33.14	63.61	♀
							27	44.84	49.76	25.00	33.84	55.42	♀
28	42.56	44.72	23.84	30.00	39.29	♀
							29	43.72	48.70	24.32	32.98	50.39	♀
30	47.56	51.58	29.04	32.78	62.63	♀
							31	46.98	51.78	25.80	34.12	61.53	♀
32	41.04	45.78	22.12	28.40	39.99	♀
							33	45.26	48.58	24.92	36.00	58.34	♂
34	42.64	47.46	22.60	34.48	52.94	♂
							35	44.72	47.96	25.48	31.82	49.12	♀
36	45.44	49.26	24.30	32.82	52.04	♀
							37	39.82	42.58	22.10	30.78	38.25	♂
38	43.44	49.00	23.14	36.72	55.16	♂
							39	40.38	43.14	21.08	32.32	42.57	♂
40	44.32	47.92	26.40	31.22	47.23	♀
							41	43.12	46.74	23.56	29.88	46.33	♀
42	43.34	48.12	24.48	32.56	49.91	♂
							43	43.88	48.52	23.90	32.12	50.28	♀
44	43.52	45.84	24.44	29.92	42.70	♀
							45	42.76	46.48	24.32	30.40	45.82	♀
46	42.62	46.00	24.38	30.00	42.91	♀
							47	42.16	46.00	23.94	28.88	42.25	♀
48	38.84	42.36	32.62	28.84	32.48	♀
							49	39.82	43.52	20.54	25.86	39.26	♂
50	39.92	43.54	20.88	30.62	40.68	♂
							51	41.76	44.72	23.64	29.92	39.60	♀
53	39.52	44.42	21.62	29.58	37.97	♀
							54	40.84	46.02	22.96	30.78	42.38	♀
56	44.32	49.64	23.34	35.52	58.04	♂
							57	41.12	44.38	23.84	30.82	38.60	♀
58	46.00	49.38	24.92	33.24	59.36	♂
							59	39.18	42.96	21.18	33.44	36.53	♂
60	43.24	48.14	23.82	34.72	53.62	♂

2) every river crab clip step, alleviates the injury caused river crab as far as possible in operation, extract muscle DNA (method is with embodiment 2) with phenol-chloroform method;

3) utilize molecule marker SSR1107 to carry out gene type (method is with embodiment 5) to selected river crab, result is as shown in table 9, and wherein 12,19,52, No. 55 do not have amplified band.

Table 9 molecule marker SSR1107 amplification

Numbering	1	2	3	4	5	6	7	8	9	10	11	13	14	15	16	17	18	20	21	22	23	24	25	26	27	28	29	30
																													Genotype	A	A	A	A	A	A	B	B	B	B	A	A	A	A	A	A	A	A	A	C	A	A	A	A	A	A	A	A
Numbering	31	32	33	34	35	36	37	38	39	40	41	42	43	44	45	46	47	48	49	50	51	53	54	56	57	58	59	60
																													Genotype	A	B	A	A	A	A	B	B	A	A	A	A	A	A	A	B	A	A	A	A	B	B	C	A	A	B	B	A

4) SPSS11.5 software is applied, adopt general linear model (General Linear Model, GLM), to mark the gene type of SSR1107 in river crab colony and river crab sex for independent variable(s), grow that typical proterties body is long, body is wide, the long and body weight of height, IV step meropodium is dependent variable, carry out the multiple comparisons between multivariate analysis of variance and mark different genotype.Result is as shown in Table 10 and Table 11: the gene type of mark SSR1107 in river crab colony has A, B and C tri-kinds, and wherein genotype C belongs to rare genotype, and probability of occurrence is only 3.6%, and in actual breeding work, this genotype can not considered; The average body of genotype A is long, body is wide, height, IV step meropodium are long and body weight all higher than genotype B, genotype A is the preponderant genotype of growth traits.Described genotype A refers to molecule marker core sequence (TG) _nin n=7, clip size is 167bp; Genotype B refers to n=7 and 8, and clip size is 167bp and 169bp; Genotype C refers to n=7 and 9, and clip size is 167bp and 171bp.

5) carry out Marker-assisted selection breeding, retain the individuality containing genotype A, the close crab as river crab breeding carries out breeding production.

The factor between table 10 main body

Table 11 is estimated

Claims (2)

1. an EST-SSR molecule marker relevant to mitten crab growth traits, is characterized in that described molecule marker called after: SSR1107, by core sequence (TG) _n, upstream primer and downstream primer composition, described upstream primer is shown in SEQ ID NO.1, and downstream primer is by shown in SEQ ID NO.2; N=7-9.

2. an application for the EST-SSR molecule marker relevant to mitten crab growth traits, is characterized in that comprising the steps:

(1) individual 60 of random choose male and female in river crab sexual maturity colony, number record one by one;

(2) every river crab clip step, extracts muscle DNA with phenol-chloroform method;

(3) utilize the molecule marker SSR1107 described in claim 1 to carry out gene type to selected river crab, retain the individuality containing genotype A, the close crab as river crab breeding carries out breeding production; Described genotype A refers to molecule marker core sequence (TG) _nin n=7.