CN102888462A - Method for fast detecting low-frequency exogenous chromosome fragments of genes with SSR markers - Google Patents

Abstract

The invention discloses a method for detecting whether samples to be detected include target DNA. The method comprises the following steps: performing matrix arrangement to the samples to be detected; respectively mixing by rows and columns; respectively detecting whether the mixed samples include the target DNA or equivalent genetic markers thereof; marking the samples to be detected before mixing, corresponding to the mixed samples with positive results; if there is only one row mixed sample with the positive result and/or there is only one column mixed sample with the positive result, marking that the samples to be detected include the target DAN at two times and the rest of samples to be detected do not include the target DNA; if there are more than two row mixed samples with the positive result and column mixed samples with the positive result, respectively detecting whether the samples to be detected and marked at two times include the target DNA and the equivalent genetic markers thereof, wherein the samples to be detected with the positive result include the target DNA, and the rest of the samples to be detected do not include the target DNA. The method is used for tracking and identifying exogenous chromosome fragments of genes of plant genetic background so as to save the experiment cost, improve the working efficiency and quicken the experiment progress.

Description

Utilize the method for SSR mark rapid detection low frequency exogenous chromosome small segment or gene

Technical field

The present invention relates to a kind of method that whether contains target DNA in the sample to be tested that detects, particularly a kind of method of utilizing SSR mark rapid detection low frequency exogenous chromosome small segment or gene.

Background technology

Genetic modification of plants is by target gene being selected, being realized the process of excellent genes polymerization.If import in the process of plant at target gene, to gene and the carrier thereof that is transferred---exogenous chromosome or chromosome segment are followed the trail of and identified, can improve the accuracy of selection, thereby shorten breeding cycle, improve breeding efficiency.Therefore, to import exogenous chromosome in the plant background or chromosome segment, with and entrained foreign gene effectively identify very important.At present, the evaluation exogenous chromosome of setting up based on genetic marker system or the method for gene mainly comprise: morphological markers, cytological marker, biochemical marker, in situ hybridization and molecule marker etc., wherein molecule marker is one of the most effective authentication method.

Along with the development of Protocols in Molecular Biology, the different kinds of molecules labeling techniques such as restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFIP), simple repeated sequence (SSR) mark and single nucleotide polymorphism (SNP) have appearred in recent years.These methods are accurately reliable, and can remedy banding technique and hybridization in situ technique and can only identify shortcoming than the exogenous chromosome of large fragment, can the smaller exogenous chromosome fragment of identification and detection.By utilization RFLP, RAPD, AFLP, SSR, the methods such as SNP are set up the molecule marker of various exogenous chromosome fragments or gene, then utilize these marks to identify the exogenous genetic material of transferring in the plant background.Traditional method identifies that various resistances and disease-resistant gene and other proterties relatively waste time and energy, and the molecule marker of utilization and these genes and the linkage of characters detects and identifies, and is not only time saving and energy saving, and accuracy is high, has improved efficient.Qi Lili etc. are to coming from the upward research of the molecule marker of mildew-resistance gene of H. villosa chromosome 6V, use OPH17, OPAL01, the primer amplifications such as OPHAN03 and OPHAL03 go out the special band of cluster hair wheat, can be used as the RAPD mark that derives from cluster hair wheat 6VS mildew-resistance gene Pro21.The brave grade of Liu Zhi further is converted into OPH17 the SCAR mark chain with Pm21, i.e. SCAR1400 and SCAR1265.Wang Furong, Zhang Jun, Liu Renchong etc. utilize Pollen Tube Pathway Technique that the genome DNA in sea island cotton kind sea 7124 is imported in the upland cotton Cultivar stone far away 321, obtained the good new germ plasm system of fibrous quality, utilize the SSR mark that mutative material is studied discovery, foreign DNA has been incorporated in the genome of upland cotton, and infers that some dna fragmentation may be that mechanism integration by homologous recombination is to upland cotton karyomit(e).

Chromosome segment substitution line (chromosome segment substitution lines, CSSL) be the whole genomic a series of near isogenic lines of nurse crop that utilization is hybridized, backcrossed and molecular marker assisted selection (marker-assisted selection, MAS) makes up.Only from a chromosome segment that isozygotys of donor parents, and genomic rest part is identical with recurrent parent in its genome.It is to carry out genome research, the particularly ideal material of QTL location.The people such as the Wang Peng of Agricultural University Of Nanjing, Zhang Tianzhen are take upland cotton Genetic standard line TM-1 as receptor parent, and sea island cotton sea 7124 is overlapped the chromosome segment substitution line that 330 strains be made of at BC5S1 for cultivated one by molecular marker assisted selection (MAS) for donor parents.

In scientific experiment, often we can run in a large group, and one or a few positive plant or target gene are carried out Screening and Identification.If Screening and Identification not only needs a large amount of manpower and materials, and needs the long period one by one, could obtain qualification result.Therefore, mouse out a shortcut, improve the Screening and Identification efficient of large group low and medium frequency exogenous chromosome fragment or gene, just seem particularly important.

Summary of the invention

The purpose of this invention is to provide a kind of method that whether contains target DNA in the sample to be tested that detects.The method is particularly useful for containing the detection of a large amount of samples to be tested of low frequency goal gene.

The method that whether contains target DNA in the detection sample to be tested provided by the present invention specifically can comprise the steps:

(a) N sample to be tested arranged according to matrix A as the formula (1).

$A=\left(\begin{array}{cccc}{a}_{11}& {\mathrm{<figure-callout\; id="12"\; label="a"\; filenames="HDA00002273799900011.png,HDA00002273799900012.png"\; state="\{\{state\}\}">a</figure-callout>}}_{12}& \&CenterDot;\&CenterDot;\&CenterDot;& a_{1n}\\ a_{21}& a_{22}& \&CenterDot;\&CenterDot;\&CenterDot;& a_{2n}\\ \&CenterDot;\&CenterDot;\&CenterDot;& \&CenterDot;\&CenterDot;\&CenterDot;& \&CenterDot;\&CenterDot;\&CenterDot;& \&CenterDot;\&CenterDot;\&CenterDot;\\ a_{m1}& a_{\mathrm{<figure-callout\; id="2"\; label="m"\; filenames="HDA00002273799900011.png,HDA00002273799900012.png"\; state="\{\{state\}\}">m</figure-callout>}2}& \&CenterDot;\&CenterDot;\&CenterDot;& a_{\mathrm{mm}}\end{array}\right)$

Formula (1)

(b) mix all described samples to be tested that are arranged in the every delegation of described matrix A, obtain altogether m capable mixing sample; Mix all described samples to be tested that are arranged in described each row of matrix A, obtain altogether n row mixing sample.

(c) whether there is respectively the genetic marker of equal value of described target DNA in m capable mixing sample of detecting step (b) gained and n the row mixing sample;

Described genetic marker of equal value is the genetic marker chain with described target DNA, and the existence of described target DNA is indicated in the existence of described genetic marker of equal value.Described genetic marker of equal value can be single nucleotide polymorphism (single nucleotide polymorphism, abbreviation SNP), simple repeated sequence (SSR) mark etc.; In one embodiment of the invention, described genetic marker of equal value is specially the SSR mark.

(d) will before detecting mixing corresponding to all described row mixing samples of the genetic marker of equal value have described target DNA, step (c) all carry out mark by described sample to be tested; To before detecting mixing corresponding to all described row mixing samples of the genetic marker of equal value have described target DNA, step (c) all carry out mark by described sample to be tested;

(e) according to the mark result of step (d), determine as follows whether a described N sample to be tested contains target DNA:

There is and only has 1 if detect the described row mixing sample of the genetic marker of equal value that has described target DNA through step (c), and/or there is and only has 1 through the described row mixing sample that step (c) detects the genetic marker of equal value there is described target DNA, then step (d) acceptance of the bid is recorded a demerit, and twice described sample to be tested contains described target DNA or the candidate is contained described target DNA, and all the other all described samples to be tested do not contain described target DNA or the candidate is not contained described target DNA;

If detect the described row mixing sample of the genetic marker of equal value that has described target DNA has more than 2 through step (c), exist the described row mixing sample of the genetic marker of equal value of described target DNA to have more than 2 through step (c) detection simultaneously, twice the described sample to be tested of then step (d) acceptance of the bid being recorded a demerit takes out, whether detect respectively exists the genetic marker of equal value of described target DNA (can carry out pcr amplification, the evaluation of also can checking order), exist the described sample to be tested of the genetic marker of equal value of described target DNA to contain described target DNA or the candidate is contained described target DNA, all the other all described samples to be tested do not contain described target DNA or the candidate is not contained described target DNA;

If all described row mixing samples detect the genetic marker of equal value that does not have described target DNA through step (c), if and/or all described row mixing samples detect the genetic marker of equal value there is not described target DNA through step (c), then all described samples to be tested do not contain described target DNA or the candidate is not contained described target DNA;

Wherein, m and n are the integer more than 3, and N equals m * n.

In aforesaid method step (c), whether exist the method for the genetic marker of equal value of described target DNA specifically to can be in m capable mixing sample of described respectively detecting step (b) gained and n row mixing sample: with m capable mixing sample of described step (b) gained and n row mixing sample respectively as template, adopt the Auele Specific Primer of the genetic marker of equal value of the described target DNA of amplification to carry out pcr amplification, according to the genetic marker of equal value that whether has described target DNA in m capable mixing sample of amplified production determining step (b) gained and n the row mixing sample.

Aforesaid method is specially adapted to detect the described target DNA of low frequency occurrence in some described samples to be tested, as in the new variety that cultivate plants, and after the structure BC2F2 colony, the exogenous chromosome small segment or the gene that utilize SSR marker detection low frequency to occur.Described low frequency is that the frequency of occurrences is lower than 5%.

In one embodiment of the invention, described sample to be tested is specially take the new land of cotton variety early No. 48 for maternal, take sea island cotton chromosome segment substitution line CSSL-120 as male parent, obtain F1 for after, take the new land of cotton variety early No. 48 as recurrent parent, the genomic dna of the BC2F2 colony of acquisition; Described target DNA is that segment number is the chromosome segment of IL024-D2-6 among the described sea island cotton chromosome segment substitution line CSSL-120; The Auele Specific Primer of the genetic marker of equal value of the described target DNA of described amplification is the SSR mark of the karyomit(e) small segment of IL024-D2-6---the Auele Specific Primer of NAU2987-320 or BNL3145-290 for the described segment number of amplification, more concrete, the Auele Specific Primer of described NAU2987-320 of increasing is two single stranded DNAs shown in sequence 1 and the sequence 2 in the sequence table, and the Auele Specific Primer of the described BNL3145-290 that increases is two single stranded DNAs of sequence 3 and sequence 4 in the sequence table.

In one embodiment of the invention, described m and n are 8, and described N is 64.

In one embodiment of the invention, detected result shows that described segment number is that the frequency that the karyomit(e) small segment of IL024-D2-6 occurs is 2/64 in 64 parts of described samples to be tested.

A kind of method that whether contains target DNA in the sample to be tested that detects provided by the present invention also can comprise the steps:

(a) N sample to be tested arranged according to matrix A as the formula (1);

$A=\left(\begin{array}{cccc}{a}_{11}& {\mathrm{<figure-callout\; id="12"\; label="a"\; filenames="HDA00002273799900011.png,HDA00002273799900012.png"\; state="\{\{state\}\}">a</figure-callout>}}_{12}& \&CenterDot;\&CenterDot;\&CenterDot;& a_{1n}\\ a_{21}& a_{22}& \&CenterDot;\&CenterDot;\&CenterDot;& a_{2n}\\ \&CenterDot;\&CenterDot;\&CenterDot;& \&CenterDot;\&CenterDot;\&CenterDot;& \&CenterDot;\&CenterDot;\&CenterDot;& \&CenterDot;\&CenterDot;\&CenterDot;\\ a_{m1}& a_{\mathrm{<figure-callout\; id="2"\; label="m"\; filenames="HDA00002273799900011.png,HDA00002273799900012.png"\; state="\{\{state\}\}">m</figure-callout>}2}& \&CenterDot;\&CenterDot;\&CenterDot;& a_{\mathrm{mm}}\end{array}\right)$

Formula (1)

(b) mix all described samples to be tested that are arranged in the every delegation of described matrix A, obtain altogether m capable mixing sample; Mix all described samples to be tested that are arranged in described each row of matrix A, obtain altogether n row mixing sample;

(c) whether there is described target DNA in m capable mixing sample of detecting step (b) gained and n row mixing sample respectively;

(d) will before detecting mixing corresponding to all the described row mixing samples have described target DNA, step (c) all carry out mark by described sample to be tested; To before detecting mixing corresponding to all the described row mixing samples have described target DNA, step (c) all carry out mark by described sample to be tested;

(e) according to the mark result of step (d), determine as follows whether a described N sample to be tested contains target DNA:

If exist the described row mixing sample of described target DNA to have and only have 1 through step (c) detection, and/or detect through step (c) and to exist the described row mixing sample of described target DNA to have and only have 1, then step (d) acceptance of the bid is recorded a demerit, and twice described sample to be tested contains described target DNA or the candidate is contained described target DNA, and all the other all described samples to be tested do not contain described target DNA or the candidate is not contained described target DNA;

If exist the described row mixing sample of described target DNA to have more than 2 through step (c) detection, detecting through step (c) simultaneously exists the described row mixing sample of described target DNA to have more than 2, twice the described sample to be tested of then step (d) acceptance of the bid being recorded a demerit takes out, whether detect respectively exists described target DNA (can carry out pcr amplification, the evaluation of also can checking order), exist the described sample to be tested of described target DNA to contain described target DNA or the candidate is contained described target DNA, all the other all described samples to be tested do not contain described target DNA or the candidate is not contained described target DNA;

If detecting through step (c), all described row mixing samples all do not have described target DNA, if and/or all described row mixing samples detect through step (c) and all do not have described target DNA, then all described samples to be tested do not contain described target DNA or the candidate is not contained described target DNA;

Wherein, m and n are the integer more than 3, and N equals m * n.

In aforesaid method step (c), whether exist the method for described target DNA specifically to can be in m capable mixing sample of described respectively detecting step (b) gained and n row mixing sample: with m capable mixing sample of described step (b) gained and n row mixing sample respectively as template, adopt the Auele Specific Primer of the described target DNA of amplification to carry out pcr amplification, according to whether having described target DNA in m capable mixing sample of amplified production determining step (b) gained and n row mixing sample.

Aforesaid method is specially adapted to detect the described target DNA of low frequency occurrence in some described samples to be tested.Described low frequency is that the frequency of occurrences is lower than 5%.

In one embodiment of the invention, described sample to be tested is specially take the new land of cotton variety early No. 48 for maternal, take sea island cotton chromosome segment substitution line CSSL-120 as male parent, obtain F1 for after, take the new land of cotton variety early No. 48 as recurrent parent, the genomic dna of the BC2F2 colony of acquisition; Described target DNA is that segment number is the karyomit(e) small segment of IL024-D2-6 among the described sea island cotton chromosome segment substitution line CSSL-120.

A kind of method that whether contains target DNA in the sample to be tested that detects provided by the present invention, adopted sample to be tested has been carried out the mode that arranged is mixed, be used for tracking and the evaluation of plant genetic background exogenous chromosome fragment or gene, can save experimental cost, increase work efficiency, accelerate the experiment process.In the present invention, 64 individual plant DNA are mixed into 16 samples, and direct-detection goes out to contain the plant of foreign gene.Only directly to carry out the evaluation of exogenous chromosome fragment or gene in 1000 individual plant colonies, needs 1000 individual plant DNA samples * 60 yuan/DNA sample=6.0 ten thousand yuan, and the method for employing arranged hybrid dna sample, the testing cost of 1000 individual plants only is: ten thousand yuan of 1000 DNA sample * 60 yuan/DNA sample * 16/64=1.5, therefore 4.5 ten thousand yuan can be saved, the experiment process can be accelerated simultaneously.

Description of drawings

Fig. 1 is arrangement and the combination treatment situation of 64 samples to be tested (individual plant genomic dna).

Fig. 2 is that primer NAU2987 is in the amplification situation of all DNA samples.Wherein, swimming lane M is dna molecular amount standard, is respectively from top to bottom 750bp, 500bp, 250bp; Swimming lane 1-64 is the genomic dna of numbering 1-64 individual plant; Swimming lane P1 is early No. 48 genomic dna of maternal new land; Swimming lane P2 is the genomic dna of male parent chromosome segment substitution line CSSL-120.

Fig. 3 is that primer BNL3145 is in the amplification situation of all DNA samples.Wherein, swimming lane M is dna molecular amount standard, is respectively from top to bottom 1000bp, 750bp, 500bp, 250bp; Swimming lane 1-64 is the genomic dna of numbering 1-64 individual plant; Swimming lane P1 is early No. 48 genomic dna of maternal new land; Swimming lane P2 is the genomic dna of male parent chromosome segment substitution line CSSL-120.

Fig. 4 is for determining the diagram of exogenous chromosome fragment or Gene Detecting method.

Embodiment

Employed experimental technique is ordinary method if no special instructions among the following embodiment.

Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.

The new land of cotton (Gossypium hirsutum L.) kind early No. 48: company limited provides by the development in agricultural science and technology far away of Xinjiang favour;

Sea island cotton chromosome segment substitution line CSSL-120: be so kind as to give by Agricultural University Of Nanjing's crop genetic and professor Zhang Tianzhen of germplasm innovation National Key Laboratory.Reference: Wang Peng, fourth is already slapped, Lu Qiongxian, Guo Wangzhen, Zhang Tianzhen. the cultivation of the sea island cotton chromosome segment substitution line of upland cotton Genetic standard line TM-1 background. Science Bulletin, 2008,53(9): this sea island cotton chromosome segment substitution line of 1065-1069. is high staple length after testing.

Embodiment 1, utilize SSR mark rapid detection low frequency exogenous chromosome small segment or gene

Present embodiment utilizes the method for SSR mark rapid detection low frequency exogenous chromosome small segment or gene specifically to comprise the steps:

(a) N sample to be tested arranged according to matrix A as the formula (1);

$A=\left(\begin{array}{cccc}{a}_{11}& {\mathrm{<figure-callout\; id="12"\; label="a"\; filenames="HDA00002273799900011.png,HDA00002273799900012.png"\; state="\{\{state\}\}">a</figure-callout>}}_{12}& \&CenterDot;\&CenterDot;\&CenterDot;& a_{1n}\\ a_{21}& a_{22}& \&CenterDot;\&CenterDot;\&CenterDot;& a_{2n}\\ \&CenterDot;\&CenterDot;\&CenterDot;& \&CenterDot;\&CenterDot;\&CenterDot;& \&CenterDot;\&CenterDot;\&CenterDot;& \&CenterDot;\&CenterDot;\&CenterDot;\\ a_{m1}& a_{\mathrm{<figure-callout\; id="2"\; label="m"\; filenames="HDA00002273799900011.png,HDA00002273799900012.png"\; state="\{\{state\}\}">m</figure-callout>}2}& \&CenterDot;\&CenterDot;\&CenterDot;& a_{\mathrm{mm}}\end{array}\right)$

Formula (1)

(b) mix all described samples to be tested that are arranged in the every delegation of described matrix A, obtain altogether m capable mixing sample; Mix all described samples to be tested that are arranged in described each row of matrix A, obtain altogether n row mixing sample;

(c) whether there is respectively the genetic marker of equal value (such as the SSR mark) of described target DNA in m capable mixing sample of detecting step (b) gained and n the row mixing sample;

(d) will before detecting mixing corresponding to all described row mixing samples of the genetic marker of equal value have described target DNA, step (c) all carry out mark by described sample to be tested; To before detecting mixing corresponding to all described row mixing samples of the genetic marker of equal value have described target DNA, step (c) all carry out mark by described sample to be tested;

(e) according to the mark result of step (d), determine as follows whether a described N sample to be tested contains target DNA:

There is and only has 1 if detect the described row mixing sample of the genetic marker of equal value that has described target DNA through step (c), and/or there is and only has 1 through the described row mixing sample that step (c) detects the genetic marker of equal value there is described target DNA, then step (d) acceptance of the bid is recorded a demerit, and twice described sample to be tested contains described target DNA or the candidate is contained described target DNA, and all the other all described samples to be tested do not contain described target DNA or the candidate is not contained described target DNA;

If detect the described row mixing sample of the genetic marker of equal value that has described target DNA has more than 2 through step (c), exist the described row mixing sample of the genetic marker of equal value of described target DNA to have more than 2 through step (c) detection simultaneously, twice the described sample to be tested of then step (d) acceptance of the bid being recorded a demerit takes out, detect respectively the genetic marker of equal value that whether has described target DNA, exist the described sample to be tested of the genetic marker of equal value of described target DNA to contain described target DNA or the candidate is contained described target DNA, all the other all described samples to be tested do not contain described target DNA or the candidate is not contained described target DNA;

If all described row mixing samples detect the genetic marker of equal value that does not have described target DNA through step (c), if and/or all described row mixing samples detect the genetic marker of equal value there is not described target DNA through step (c), then all described samples to be tested do not contain described target DNA or the candidate is not contained described target DNA;

Wherein, m and n are the integer more than 3, and N equals m * n.

One, the acquisition of sample to be tested and mixing sample

1, the structure of BC2F2 colony

Present embodiment early is female parent No. 48 take the new land of cotton (Gossypium hirsutum L.) kind, the sea island cotton chromosome segment substitution line CSSL-120(segment number of high staple length is IL024-D2-6, identify that this chromosome segment is labeled as NAU2987-320 and BNL3145-290) be male parent, hybridize and obtain F1 generation, take new land early No. 48 as recurrent parent, obtain to consist of BC2F2 colony by 147 strains.

2, the acquisition of sample to be tested and mixing sample

The genomic dna of each plant of BC2F2 colony that extraction step one obtains, therefrom select at random the genomic dna of 64 individual plants as sample to be tested, number consecutively is 1-64, it is arranged according to mode as shown in Figure 1, draw each genomic dna 5-10 μ l with the volley of rifle fire, mix the individual plant genomic dna by row respectively, 64 samples to be tested mix 8 capable mixing samples of formation, are designated as A-H; Mix the individual plant genomic dna by row, 64 samples to be tested mix 8 row mixing samples of formation, are designated as a-h.

Two, pcr amplification target DNA

Respectively to be numbered the genomic dna of 1-64 individual plant in the step 2,16 DNA mixing samples (8 capable mixing samples and 8 row mixing samples), maternal new land is No. 48 genomic dna early, the genomic dna of male parent chromosome segment substitution line CSSL-120 is masterplate, respectively with the Auele Specific Primer (referred to as the NAU2987 primer) of amplification NAU2987-320 mark, the Auele Specific Primer (referred to as the BNL3145 primer) of amplification BNL3145-290 mark, carry out pcr amplification, thereby identify whether the corresponding plant of each sample to be tested contains external source sea island cotton chromosome segment IL024-D2-6.Wherein, the Auele Specific Primer of amplification NAU2987-320 mark is that the Auele Specific Primer of two single stranded DNAs shown in sequence 1 and the sequence 2, amplification BNL3145-290 mark in the sequence table is two single stranded DNAs shown in sequence 3 and the sequence 4 in the sequence table.

The NAU2987 primer:

5 ＇-GGAAAAGGCTGCTACAAGTG-3 ＇ (sequence 1);

5 ＇-CGAAGGATGTAACCGATACC-3 ＇ (sequence 2);

The BNL3145 primer:

5 ＇-AACGAGGGAAAACGGAGAGT-3 ＇ (sequence 3);

5 ＇-CAAAACGACGCCATTTAGGT-3 ＇ (sequence 4);

PCR reaction system (20 μ l) is divided into two classes:

(1) is numbered early No. 48 genomic dna of the genomic dna of 1-64 individual plant, maternal new land, and the PCR reaction system of the DNA of male parent chromosome segment substitution line CSSL-120 is as follows: 10 * PCR buffer, 2.0 μ l, dNTPs 0.2mmol/L, Taq archaeal dna polymerase 2U, each 0.3 μ mol/L of SSR primer (NAU2987 primer or BNL3145 primer) upstream and downstream primer, masterplate DNA20ng.

The PCR reaction system of (2) 16 DNA mixing samples (8 capable mixing samples and 8 row mixing samples) is as follows: 10 * PCR buffer, 2.0 μ l, dNTPs 0.2mmol/L, Taq archaeal dna polymerase 2U, each 0.3 μ mol/L of SSR primer (NAU2987 primer or BNL3145 primer) upstream and downstream primer, masterplate DNA 60ng.

Response procedures is: 95 ℃ of denaturation 2min, a circulation; 94 ℃ of sex change 30s, 57 ℃ of annealing 45s, 72 ℃ are extended 1min, totally 35 circulations; 72 ℃ are extended 7min, a circulation; 4 ℃ of preservations are stand-by.

Electrophoresis and silver dye detection: pcr amplification product adopts 6% sex change polyacrylamine gel electrophoresis, constant voltage 180V electrophoresis 45～60min.At the fixing 10min of stationary liquid (ethanol of 10% volume content, the Glacial acetic acid of 0.5% volume content); Utilize the quick rinsing of distilled water 2 times; Then 0.2%(0.2g/100ml) AgNO ₃Solution-dyed 12min; The quick rinsing of distilled water 2 times; Developing solution (1.5% (1.5g/100ml) NaOH, the formaldehyde of 0.5% volume content) develops; In stationary liquid (ethanol of 10% volume content, the Glacial acetic acid of 0.5% volume content) photographic fixing, then use distilled water rinsing 1-2 time.Take a picture with digital camera, and the statistics banding pattern.

Three, results and analysis

1, individual plant DNA sample amplification

SSR primer NAU2987 sees Fig. 2 (swimming lane 1-64 in the DNA sample amplification situation of individual plant 1-64, the new land of cotton variety early No. 48, chromosome segment substitution line CSSL-120, and P1 and P2), primer BNL3145 sees Fig. 3 (swimming lane 1-64, and P1 and P2) in individual plant 1-64, new land early No. 48, substitution line CSSL-120DNA sample amplification situation.As shown in the figure, primer NAU2987 and BNL3145 all sample 9,30 and chromosome segment substitution line CSSL-120 in the special band of amplification sea island cotton chromosome segment IL024-D2-6 (the special band size of the sea island cotton chromosome segment IL024-D2-6 of primer NAU2987 amplification is about 320bp, the special band size of the sea island cotton chromosome segment IL024-D2-6 of primer BNL3145 amplification is about 290bp), and the new land of all the other 62 individual plants and cotton variety early in No. 48 the special band without sea island cotton chromosome segment IL024-D2-6 occur.

2, hybrid dna sample amplification

SSR primer NAU2987 and the BNL3145 mixing sample A-H that is expert at, row mixing sample a-h, totally 16 DNA sample amplification situations are seen respectively Fig. 2 (swimming lane A-H and a-h) and Fig. 3 (swimming lane A-H and a-h).Row mixing sample B, D are found in amplification, and above-mentioned two pairs of primers all increase among row mixing sample a, the f, and (the special band size of the sea island cotton chromosome segment IL024-D2-6 of primer NAU2987 amplification is about 320bp for the special band of sea island cotton chromosome segment IL024-D2-6, the special band size of the sea island cotton chromosome segment IL024-D2-6 of primer BNL3145 amplification is about 290bp), and the special band without sea island cotton chromosome segment IL024-D2-6 occurs in all the other 14 mixing samples.

3, analyze

Will be in the gene DNA Pareto diagram of as shown in Figure 1 64 individual plants, can amplify the special row with the DNA sample of sea island cotton chromosome segment IL024-D2-6, row are drawn respectively straight line, and (described sample to be tested all carries out mark before namely pcr amplification being obtained mixing corresponding to all described row mixing samples of positive findings, described sample to be tested all carries out mark before simultaneously pcr amplification being obtained mixing corresponding to all described row mixing samples of positive findings), article 4, intersect to obtain 4 intersection points (be that the described row mixing sample that pcr amplification obtains positive findings has 2 to straight line, the described row mixing sample that the while pcr amplification obtains positive findings also has 2), numbering is respectively 9,14,25 and 30(be that mark is crossed twice described sample to be tested) (as shown in Figure 4).The individual plant genomic dna sample that is numbered 9,14,25 and 30 is carried out one by one PCR with reference to (1) in the above-mentioned steps two described PCR reaction system, DNA sample 9,30 can amplify the special band of sea island cotton chromosome segment IL024-D2-6 among the analysis discovery sample 1-64, and DNA sample 14,25 fails to amplify respective strap.Above result shows, is numbered in 64 individual plants of 1-64, be numbered 9 and 30 two individual plants and carry exogenous chromosome fragment IL024-D2-6, and remaining 62 individual plant does not carry exogenous chromosome fragment IL024-D2-6.

Four, pricing

64 individual plant DNA are mixed into 16 samples among the present invention, and direct-detection goes out to contain the plant of foreign gene.Take the colony that formed by 1000 individual plants as example, be 60 yuan of calculating according to the testing cost of each DNA sample, if single sample directly carries out the evaluation of exogenous chromosome fragment or gene, then need 1000 individual plant DNA samples * 60 yuan/DNA sample=6.0 ten thousand yuan.And adopting the method for arranged hybrid dna sample of the present invention, the testing cost of 1000 individual plants only is: ten thousand yuan of 1000 DNA sample * 60 yuan/DNA sample * 16/64=1.5.Therefore 6.0 ten thousand yuan-1.5 ten thousand yuan=4.5 ten thousand yuan can be saved, the experiment process can be accelerated simultaneously.

Claims (2)

1. one kind is detected the method that whether contains target DNA in the sample to be tested, comprises the steps:

(a) N sample to be tested arranged according to matrix A as the formula (1);

$A=\left(\begin{array}{cccc}{a}_{11}& a_{12}& \&CenterDot;\&CenterDot;\&CenterDot;& a_{1n}\\ a_{21}& a_{22}& \&CenterDot;\&CenterDot;\&CenterDot;& a_{2n}\\ \&CenterDot;\&CenterDot;\&CenterDot;& \&CenterDot;\&CenterDot;\&CenterDot;& \&CenterDot;\&CenterDot;\&CenterDot;& \&CenterDot;\&CenterDot;\&CenterDot;\\ a_{m1}& a_{m2}& \&CenterDot;\&CenterDot;\&CenterDot;& a_{\mathrm{mm}}\end{array}\right)$

Formula (1)

(b) mix all described samples to be tested that are arranged in the every delegation of described matrix A, obtain altogether m capable mixing sample; Mix all described samples to be tested that are arranged in described each row of matrix A, obtain altogether n row mixing sample;

(c) whether there is respectively the genetic marker of equal value of described target DNA in m capable mixing sample of detecting step (b) gained and n the row mixing sample;

(d) will before detecting mixing corresponding to all described row mixing samples of the genetic marker of equal value have described target DNA, step (c) all carry out mark by described sample to be tested; To before detecting mixing corresponding to all described row mixing samples of the genetic marker of equal value have described target DNA, step (c) all carry out mark by described sample to be tested;

(e) according to the mark result of step (d), determine as follows whether a described N sample to be tested contains target DNA:

There is and only has 1 if detect the described row mixing sample of the genetic marker of equal value that has described target DNA through step (c), and/or there is and only has 1 through the described row mixing sample that step (c) detects the genetic marker of equal value there is described target DNA, then step (d) acceptance of the bid is recorded a demerit, and twice described sample to be tested contains described target DNA or the candidate is contained described target DNA, and all the other all described samples to be tested do not contain described target DNA or the candidate is not contained described target DNA;

If detect the described row mixing sample of the genetic marker of equal value that has described target DNA has more than 2 through step (c), exist the described row mixing sample of the genetic marker of equal value of described target DNA to have more than 2 through step (c) detection simultaneously, twice the described sample to be tested of then step (d) acceptance of the bid being recorded a demerit takes out, detect respectively the genetic marker of equal value that whether has described target DNA, exist the described sample to be tested of the genetic marker of equal value of described target DNA to contain described target DNA or the candidate is contained described target DNA, all the other all described samples to be tested do not contain described target DNA or the candidate is not contained described target DNA;

If all described row mixing samples detect the genetic marker of equal value that does not have described target DNA through step (c), if and/or all described row mixing samples detect the genetic marker of equal value there is not described target DNA through step (c), then all described samples to be tested do not contain described target DNA or the candidate is not contained described target DNA;

Wherein, m and n are the integer more than 3, and N equals m * n.

2. one kind is detected the method that whether contains target DNA in the sample to be tested, comprises the steps:

(a) N sample to be tested arranged according to matrix A as the formula (1);

$A=\left(\begin{array}{cccc}{a}_{11}& a_{12}& \&CenterDot;\&CenterDot;\&CenterDot;& a_{1n}\\ a_{21}& a_{22}& \&CenterDot;\&CenterDot;\&CenterDot;& a_{2n}\\ \&CenterDot;\&CenterDot;\&CenterDot;& \&CenterDot;\&CenterDot;\&CenterDot;& \&CenterDot;\&CenterDot;\&CenterDot;& \&CenterDot;\&CenterDot;\&CenterDot;\\ a_{m1}& a_{m2}& \&CenterDot;\&CenterDot;\&CenterDot;& a_{\mathrm{mm}}\end{array}\right)$

Formula (1)

(b) mix all described samples to be tested that are arranged in the every delegation of described matrix A, obtain altogether m capable mixing sample; Mix all described samples to be tested that are arranged in described each row of matrix A, obtain altogether n row mixing sample;

(c) whether there is described target DNA in m capable mixing sample of detecting step (b) gained and n row mixing sample respectively;

(d) will before detecting mixing corresponding to all the described row mixing samples have described target DNA, step (c) all carry out mark by described sample to be tested; To before detecting mixing corresponding to all the described row mixing samples have described target DNA, step (c) all carry out mark by described sample to be tested;

(e) according to the mark result of step (d), determine as follows whether a described N sample to be tested contains target DNA:

If exist the described row mixing sample of described target DNA to have and only have 1 through step (c) detection, and/or detect through step (c) and to exist the described row mixing sample of described target DNA to have and only have 1, then step (d) acceptance of the bid is recorded a demerit, and twice described sample to be tested contains described target DNA or the candidate is contained described target DNA, and all the other all described samples to be tested do not contain described target DNA or the candidate is not contained described target DNA;

If exist the described row mixing sample of described target DNA to have more than 2 through step (c) detection, detecting through step (c) simultaneously exists the described row mixing sample of described target DNA to have more than 2, twice the described sample to be tested of then step (d) acceptance of the bid being recorded a demerit takes out, detect respectively and whether have described target DNA, exist the described sample to be tested of described target DNA to contain described target DNA or the candidate is contained described target DNA, all the other all described samples to be tested do not contain described target DNA or the candidate is not contained described target DNA;

If detecting through step (c), all described row mixing samples all do not have described target DNA, if and/or all described row mixing samples detect through step (c) and all do not have described target DNA, then all described samples to be tested do not contain described target DNA or the candidate is not contained described target DNA;

Wherein, m and n are the integer more than 3, and N equals m * n.

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