阅读说明：本技术 一种适用于图位克隆的基因型快速鉴定方法 (Rapid genotype identification method suitable for map-based cloning ) 是由 关海英 汪黎明 刘铁山 何春梅 刘春晓 董瑞 刘强 王娟 于 2018-07-29 设计创作，主要内容包括：本发明属于基因遗传学技术领域,具体涉及一种适用于图位克隆的基因组DNA快速提取和基因型快速鉴定方法。本发明所述适用于图位克隆的基因型快速鉴定方法,利用作物苗为验证样本,以一般的提取DNA方法进行验证,通过设计合理的引物进行PCR扩增,可快速完成基因组DNA的快速提取和基因型的快速鉴定,大大减少了重复劳动,节约了成本和时间,加速工作进展,该方法同样可以在其他作物中予以借鉴。(The invention belongs to the technical field of genetic genetics, and particularly relates to a method for quickly extracting genome DNA and quickly identifying a genotype, which is suitable for map-based cloning. The rapid genotype identification method suitable for map-based cloning, provided by the invention, is characterized in that crop seedlings are used as verification samples, a general DNA extraction method is used for verification, PCR amplification is carried out through reasonably designed primers, rapid genome DNA extraction and rapid genotype identification can be rapidly completed, repeated labor is greatly reduced, cost and time are saved, and work progress is accelerated.)

1. A rapid genotype identification method suitable for map-based cloning is characterized by comprising the following steps:

(1) seed germination: selecting crop seeds and culturing and germinating the crop seeds by using a wet towel method;

(2) mixing and sampling: after germination, sampling above the growing point of each crop seedling;

(3) genome extraction: crushing the obtained crop seedling sample, extracting genome DNA by adopting a CTAB method, and carrying out PCR amplification;

(4) and (4) after PCR amplification, carrying out electrophoresis detection on the obtained DNA product, and extracting the DNA of the detected recombined sample by using the backup sample to divide the sample into individual strains according to the method in the step (3) to search for recombined individuals.

2. The method for rapidly identifying the genotype suitable for map-based cloning according to claim 1, wherein in the step (4), the following primers P1 and/or P2 are designed for PCR amplification:

P1-F：GTGCTCGATCGCCTCTGTAA；

P1-R: AAAGCTTGGTGGAGAGACCG, respectively; alternatively, the first and second electrodes may be,

P2-F：GCACAACCTGATTCTCCCCA；

P2-R：ATAGGACGAGCCGACCAAAC。

3. the method for rapidly identifying the genotype suitable for map-based cloning according to claim 1 or 2, wherein the culture conditions in the germination step in step (1) are light culture at 25 ℃, light culture for 16 hours and dark culture for germination at 8 hours.

4. The method for rapidly identifying genotypes suitable for map-based cloning according to any one of claims 1 to 3, wherein in the step (1), the wet towel method specifically comprises: the method comprises the steps of boiling the towel in boiling water for 15-20 minutes, cooling, taking out, spreading half of the towel at the bottom of a culture box, uniformly spreading the seeds soaked in ultrapure water on the towel, and covering the seeds with the other half of the towel.

5. The method for rapidly identifying the genotype suitable for map-based cloning according to any one of claims 1 to 4, wherein the sample is taken from a crop seedling which germinates for 7 to 10 days in the step (2).

6. The method for rapidly identifying the genotype suitable for map-based cloning according to claim 5, wherein in the step (2), the sampling is performed by selecting a sample 1cm above the growing point of the seedling.

7. The method for rapidly identifying genotypes as suitable for map-based cloning according to any one of claims 1-6, wherein in the step (3), the CTAB method for extracting genomic DNA specifically comprises: adding 500ul CTAB into the crushed sample, and placing the sample in a constant temperature water bath kettle to perform warm bath at 65 ℃ for 30 minutes; and then adding 500ul of chloroform, shaking and mixing uniformly, then centrifuging to obtain supernatant, adding the supernatant into a centrifuge tube which is added with 800ul of absolute ethyl alcohol in advance, standing, and picking out the separated white flocculent precipitate DNA to obtain the DNA.

8. The method for rapidly identifying the genotype of a map-based clone according to claim 7, wherein the chloroform in step (3) further comprises the following steps in a volume ratio of 24:1 trichloromethane and isoamyl alcohol.

9. The method for rapid genotype identification suitable for map-based cloning according to any one of claims 1 to 8, wherein the crop is a maize crop.

10. The method of claim 9, wherein the corn plant seed is embryo shrunken kernel in a segregating population.

Technical Field

The invention belongs to the technical field of genetic genetics, and particularly relates to a method for quickly extracting genome DNA and quickly identifying a genotype, which is suitable for map-based cloning.

Background

The acquisition of the draft maize genome at month 2 in 2008 makes maize the second successfully sequenced crop following rice. In 2009, american scientists completed genome sequencing work on maize inbred line B73, with coverage reaching more than 95% (Schnable et al 2009). It is predicted that the maize genome size is about 2500MB, the number of genes is about 5-6 million, the sequencing team has stored maize genome draft information into the public database of internet gene sequencing maizeGDB, and the data is also constantly being updated.

The large-scale sequencing of the corn genome is beneficial to developing enough molecular markers by using genome sequence information, and meanwhile, the development of the corn genome information and a large number of molecular markers provides great convenience for map-based cloning of corn genes, thereby greatly accelerating the process of gene positioning. At present, the map-based cloning work of maize genes is very vigorous, and a plurality of laboratories at home and abroad clone maize important genes by using a map-based cloning method.

Map-based cloning, also known as positional cloning, was first proposed in 1986 by Alan coulson of Cambridge university, and the isolation of genes by this method was performed based on the location of the target gene on the chromosome, without prior knowledge of the DNA sequence of the gene and information about its expression product, and with only one genetic segregation population established based on the presence or absence of the target gene, such as F, DH, BC, RI, etc. The principle of using the method to separate the genes is that the functional genes have relatively stable gene loci in a genome, and on the basis of accurately positioning the target genes by using a molecular marker technology, a DNA library is screened by using molecular markers which are closely linked with the target genes, so that a physical map of a target gene region is constructed, and then the physical map is used for gradually approaching the target genes through chromosome walking or through a chromosome landing method, so that the target genes are finally cloned, and the functions of the target genes can be researched through a genetic transformation experiment.

Map-based cloning requires construction of mapping populations, which are mainly F2, BC1, DH (double hash), RIL (recombinant expressed lines), NIL (Near-isogenic lines) and other types, and the population size generally increases as well as the population size increases. At present, the number of the populations for cloning target genes by a map-based cloning method in corn is generally 3000-5000. Some target genes are located close to the centromere region, the recombination rate is low, and a large mapping population (10000 or more) is required to search for recombinant individuals. The extraction of the genome DNA and the identification of the genotype are repetitive hard labor, so that the method for quickly extracting the genome DNA and identifying the genotype is labor-saving and time-saving, can accelerate the experiment progress, and has very important significance.

Disclosure of Invention

Therefore, the technical problem to be solved by the present invention is to provide a method for rapid extraction of genomic DNA and rapid identification of genotype suitable for map-based cloning, so as to solve the problem that the methods for extraction of genomic DNA and identification of genotype are repetitive and time-consuming in the prior art.

In order to solve the above technical problems, the rapid genotype identification method suitable for map-based cloning, provided by the invention, comprises the following steps:

(1) seed germination: selecting crop seeds and culturing and germinating the crop seeds by using a wet towel method;

(2) mixing and sampling: after germination, sampling above the growing point of each crop seedling;

(3) genome extraction: crushing the obtained crop seedling sample, extracting genome DNA by adopting a CTAB method, and carrying out PCR amplification;

(4) and (4) after PCR amplification, carrying out electrophoresis detection on the obtained DNA product, and extracting the DNA of the detected recombined sample by using the backup sample to divide the sample into individual strains according to the method in the step (3) to search for recombined individuals.

In the step (4), the following primers P1 and/or P2 are designed for PCR amplification:

P1-F：GTGCTCGATCGCCTCTGTAA；

P1-R: AAAGCTTGGTGGAGAGACCG, respectively; alternatively, the first and second electrodes may be,

P2-F：GCACAACCTGATTCTCCCCA；

P2-R：ATAGGACGAGCCGACCAAAC。

in the step (1), the culture conditions in the germination step are illumination culture at 25 ℃, illumination is kept for 16 hours, and culture germination is carried out in 8 hours of darkness.

In the step (1), the wet towel method specifically includes: the method comprises the steps of boiling the towel in boiling water for 15-20 minutes, cooling, taking out, spreading half of the towel at the bottom of a culture box, uniformly spreading the seeds soaked in ultrapure water on the towel, and covering the seeds with the other half of the towel.

In the step (2), the sample is taken from a crop seedling which germinates for 7-10 days.

In the step (2), the sampling is to select a sample at a position 1cm above the growing point of the crop seedling.

In the step (3), the step of extracting genomic DNA by the CTAB method specifically comprises: adding 500ul CTAB into the crushed sample, and placing the sample in a constant temperature water bath kettle to perform warm bath at 65 ℃ for 30 minutes; and then adding 500ul of chloroform, shaking and mixing uniformly, then centrifuging to obtain supernatant, adding the supernatant into a centrifuge tube which is added with 800ul of absolute ethyl alcohol in advance, standing, and picking out the separated white flocculent precipitate DNA to obtain the DNA.

In the step (3), the chloroform further contains a solvent with a volume ratio of 24:1 trichloromethane and isoamyl alcohol.

The step (3) further comprises the steps of adding 75% ethanol into the picked flocculent precipitate DNA for washing, centrifuging at 7500rpm for 5 minutes, then pouring out the supernatant, taking the precipitate, drying in the air, and adding 200ul ddH ₂And O, mixing uniformly, and performing PCR amplification for later use.

The crop is a corn crop.

The corn crop seeds are embryo shrinkage seeds in the separated group.

The rapid genotype identification method suitable for map-based cloning, provided by the invention, is characterized in that crop seedlings are used as verification samples, a general DNA extraction method is used for verification, PCR amplification is carried out through reasonably designed primers, rapid genome DNA extraction and rapid genotype identification can be rapidly completed, repeated labor is greatly reduced, cost and time are saved, and work progress is accelerated.

The method adopts a wet towel method to culture the seeds, and adopts a plastic box germination method, so that time and labor are saved compared with the method of directly planting the seeds in soil, and the method saves time and labor, and generally can plant two plastic boxes at a time, thereby saving trouble and labor.

Drawings

In order that the present disclosure may be more readily and clearly understood, the following detailed description of the present disclosure is provided in connection with specific embodiments thereof and the accompanying drawings, in which,

FIG. 1 is an electrophoretogram of P1 primer test samples in example 1, wherein A in FIG. 1 is an electrophoretogram of 10 mixed samples, 1-10 correspond to 10 mixed samples, 11 is a mutant, 12 is F1 strain, and 13 is B73 strain; in FIG. 1, B is an electrophoretogram of 10 individual plants corresponding to mixed sample 9, 1-10 correspond to 10 mixed samples, 11 is a mutant, 12 is F1 strain, and 13 is B73 strain;

FIG. 2 is an electrophoretogram of P2 primer test samples in example 2, wherein A in FIG. 2 is an electrophoretogram of 10 mixed samples, 1-10 correspond to 10 mixed samples, 11 is a mutant, 12 is F1 strain, and 13 is B73 strain; in FIG. 2, B is an electrophoretogram of 10 individuals corresponding to the mixed sample 2, 1-10 correspond to 10 mixed samples, 11 is a mutant, 12 is F1 strain, and 13 is B73 strain;

FIG. 3 is an electrophoretogram of P1 and P2 primer detection samples in example 3, wherein, in FIG. 3, A is an electrophoretogram of 10 mixed samples for P1 primer detection, 1-10 correspond to 10 mixed samples, 11 is a mutant, 12 is F1 strain, and 13 is B73 strain; in FIG. 3, B is an electrophoretogram of 10 individuals corresponding to the P1 primer detection mixed sample 5, 1-10 correspond to 10 mixed samples, 11 is a mutant, 12 is F1 strain, and 13 is B73 strain; FIG. 3C is an electrophoretogram of P2 primer for detecting 10 mixed samples, where 1-10 correspond to 10 mixed samples, 11 is a mutant, 12 is F1 strain, and 13 is B73 strain; in FIG. 3, D is an electrophoretogram of 10 individuals corresponding to P2 primer detection mixed sample 5, 1-10 correspond to 10 mixed samples, 11 is a mutant, 12 is F1 strain, and 13 is B73 strain.

Detailed Description