CN115161408A - DNA methylation detection of target segments of the maize genome - Google Patents

Abstract

The application discloses DNA methylation detection of a corn genome target segment, and particularly relates to a construction method, a detection method, a kit and a system of a corn genome DNA methylation detection library containing the target segment. According to the method, the kit and the system, the DNA methylation detection library is constructed through two times of PCR amplification, and then the detection is realized through sequencing of the DNA methylation detection library and comparison of DNA methylation information. The method, the kit and the system can detect DNA methylation information of a plurality of target sections in a plurality of corn materials at one time, and can also be applied to the detection of DNA methylation of two parent alleles of hybrid materials, each target section comprises at least one or more SNP sites capable of distinguishing different corn materials or different alleles, and the method, the kit and the system not only simplify fussy experimental operation, but also greatly reduce detection cost when accurately detecting the DNA methylation level.

Description

DNA methylation detection of target segments of the maize genome

technical field

The present application relates to the technical field of maize genomic DNA methylation detection, in particular to the DNA methylation detection of target segments of the maize genome, and in particular to a construction method, detection method and reagent for a maize genomic DNA methylation detection library comprising the target segment Boxes and Systems.

Background technique

DNA methylation is a way of DNA epigenetic modification, which can affect the inheritance of phenotype without changing the DNA sequence. The methylation level of maize genomic DNA changes dynamically and is affected by the growth and development period of maize and various environmental factors, such as salt stress and drought stress. Therefore, the detection of maize DNA methylation is an important means to explore how DNA methylation affects maize growth and development regulation and responds to adversity stress.

Whole-genome sequencing is a common way to detect plant genome DNA methylation, which can detect plant genome DNA methylation on a genome-wide scale. The DNA methylation that regulates gene expression is usually located in some specific regions. For the detection of DNA methylation in specific regions, the whole genome DNA methylation sequencing method is not only expensive, even if the sequencing depth is increased for these specific regions , these specific segments are often not well covered. In addition, for the detection of DNA methylation of multiple genomes and multiple specific segments, the whole genome DNA methylation sequencing method requires multiple detections, which not only increases the detection cost, but also has a low detection efficiency.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present application is to at least provide a method, detection method, kit and system for detecting DNA methylation in the target segment of the maize genome, so as to solve one of the above technical problems to a certain extent.

In the first aspect, the examples of this application disclose a method for constructing a maize genomic DNA methylation detection library comprising a target segment, the target segment comprising at least one or more SNP sites capable of distinguishing different maize genomes, the The DNA methylation detection library is used to detect DNA mixed samples of at least two maize genomes containing the target segment, wherein the construction method includes:

converting unmethylated cytosine in DNA molecules in the DNA mixed sample into uracil;

A first amplification is performed, and the first amplification includes amplifying the DNA mixed sample in the same PCR reaction system to obtain a first amplification product, and the first amplification product comprises a DNA having the target segment. A DNA molecule and a first recognition sequence and a bridge sequence connected to the 3' end of the DNA molecule, the first recognition sequence is used to identify each DNA molecule of the target segment in the DNA mixed sample;

performing a second amplification, the second amplification comprising the step of performing PCR amplification on the first amplification product to obtain a second amplification product, the second amplification product comprising the first amplification product and the a second recognition sequence at the 3' end of the first amplification product, the second recognition sequence being used to identify the first amplification product; and

The second amplification product is used to construct the DNA methylation detection library of the maize genome comprising the target segment.

In the examples of the present application, the first amplification uses an upstream primer and a first downstream primer, and the C base in the upstream primer is designed to be a degenerate base Y; the G base in the first downstream primer is designed The base is designed as a degenerate base R, and the first downstream primer comprises the first recognition sequence and a bridge sequence connected at the 3' end of the first recognition sequence, and the bridge sequence is used to connect with the second bridge sequence. Homologous matching; the SNP site in the first amplification product is located within 150bp of the DNA molecule with the target segment and the upstream primer binding start position or the first downstream primer binding start position .

In the application examples, the reaction step of the first amplification includes:

Carry out pre-denaturation, the treatment temperature is 95 °C, and the treatment time is 5 min;

performing a first amplification cycle, the first amplification cycle comprising sequentially performing a first melting process, a first annealing process, and a first extension process;

A second amplification cycle is performed, and the second amplification cycle includes sequentially performing the second melting treatment, the second annealing treatment and the second extension treatment; the treatment time at 95°C is 30s, 60°C for 30s, and 70°C for 30s ;as well as

72℃ for 5min and 12℃ for 1s;

Wherein, the first amplification cycle includes at least 8 to 12 cycles, and the annealing temperature of the first annealing treatment in each of the first amplification cycles gradually decreases with the first amplification cycle.

In the embodiments of the present application, the treatment temperature of the first annealing treatment is 68-55°C, 65-53°C, 65-55°C, or 63-55°C.

In the embodiments of the present application, the second amplification uses the upstream primer and the second downstream primer, the second downstream primer includes the bridge sequence and the second recognition sequence, and the second amplification The reaction steps include:

Carry out pre-denaturation, the treatment temperature is 95 °C, and the treatment time is 5 min;

performing an amplification cycle comprising sequentially performing a melting process, an annealing process, and an extension process; and

Treat at 72°C for 5 min and at 12°C for 1 s.

In the embodiment of the present application, the second amplification product further includes a bridge sequence connected between the first recognition sequence and the second recognition sequence at the 3' end of the first amplification product, the bridge A sequence is used to link the first recognition sequence and the second recognition sequence.

In a second aspect, a method for detecting DNA methylation in a target segment of a maize genome, the target segment comprising at least one or more SNP sites capable of distinguishing different maize genomes, the method for detecting DNA methylation in a target segment using For detection of DNA mixed samples of at least two corn genomes comprising the target segment, wherein the detection method comprises:

The construction method of the first aspect, constructing the DNA methylation detection library of the maize genome comprising the target segment;

Sequencing the DNA methylation detection library to obtain the first reads library;

Carry out quality control, splitting and pairing to the first reads library to obtain the second reads library;

The second reads library is aligned, deduplicated and calculated to obtain the DNA methylation information of each maize genome in the DNA mixed sample.

In the embodiment of the present application, the first reads library includes a number of first reads corresponding to the DNA methylation detection library, and the second reads library includes second reads based on the same second recognition sequence. , the second reads are the reads from which the first recognition sequence, the second recognition sequence and the bridge sequence are removed.

In a third aspect, the embodiments of the present application disclose a kit for constructing a maize genomic DNA methylation detection library comprising a target segment, the target segment comprising at least one or more SNP sites capable of distinguishing different maize genomes , the DNA methylation detection library is used to detect DNA mixed samples of at least two maize genomes comprising the target segment, wherein the kit includes:

Maize genomic DNA processing reagent for extracting the maize genomic DNA and converting unmethylated cytosine into uracil;

an upstream primer, the C base in the upstream primer sequence is replaced with a sequence of degenerate base Y;

a first downstream primer, the G base in the downstream primer sequence is replaced with a sequence of degenerate base R, the first downstream primer comprises a first recognition sequence and a bridge sequence; and

a second downstream primer comprising the bridge sequence and the second recognition sequence sequence;

Wherein, the upstream primer and the first downstream primer are used to perform a first amplification to obtain a first amplification product, and the first amplification product includes a DNA molecule with the target segment and a DNA molecule connected to the A first recognition sequence and a bridge sequence at the 3' end of the DNA molecule;

The upstream primer and the second downstream primer can obtain a second amplification product in a second amplification, and the second amplification product includes the first amplification product and is connected to the first amplification product 3 ' end of the second recognition sequence, to construct the DNA methylation detection library;

The first identification sequence is used to identify each DNA molecule of the target segment in the mixed DNA sample, and the second identification sequence is used to identify the first amplification product.

In a fourth aspect, the embodiments of the present application disclose a system for detecting DNA methylation in maize genomes comprising a target segment, the target segment comprising at least one or more SNP sites capable of distinguishing different maize genomes, the DNA methylation The gene-based detection library is used to detect DNA mixed samples of at least two maize genomes comprising the target segment, wherein the system comprises:

The kit of the third aspect for obtaining DNA methylation information for each corn genome in the DNA mixed sample; and

A processing device, the processing device is used for running a program, and when the program is running, the detection method in the third aspect is executed.

"Sequencing the DNA methylation detection library to obtain the first reads library;

Carry out quality control, splitting and pairing to the first reads library to obtain the second reads library;

The second reads library is aligned, deduplicated and calculated to obtain the DNA methylation information of each maize genome in the DNA mixed sample".

Compared with the prior art, the present application has at least one of the following beneficial effects:

The construction method, detection method, kit and system of the maize genomic DNA methylation detection library comprising the target segment involved in the examples of the present application, for the target segment comprising the SNP site, not only can one-time detection of multiple A maize genome sample containing the target segment has a high coverage rate, does not need to perform multiple detections, improves the detection efficiency, and also improves the reliability and accuracy of DNA methylation detection.

Description of drawings

FIG. 1 is a schematic diagram of the DNA methylation detection principle of the maize genome including the target segment provided in the embodiment of the present application.

FIG. 2 is a structural diagram of primers and products in the first amplification and the second amplification in the construction of the maize genomic DNA methylation including the target segment provided in the embodiment of the present application.

Figure 3 is the target segment of the four maize inbred lines involved in the examples of the application, wherein the SNP sites are marked in red.

FIG. 4 is the detection result of the methylation detection method or system provided in the mixed samples of four maize inbred line genomes provided in the embodiment of the present application respectively; CHH\CHG\CG represent the methylation C bases respectively. Three sequence environments.

Figure 5 shows the DNA methylation results obtained by using the whole-genome DNA methylation sequencing method for the two target segments provided in the embodiment of the present application; CHH\CHG\CG respectively represent three sequence environments of methylated C bases.

FIG. 6 is a gel electrophoresis image of the purified first amplification product provided in the embodiment of the present application, lane 1 is the target segment, and lane 2 is the DNA molecular weight marker.

FIG. 7 is a gel electrophoresis image of the purified second amplification product provided in the embodiment of the present application, lane 1 is the target segment, and lane 2 is the DNA molecular weight marker.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present application more clear, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application, but not to limit the present application. The reagents not described in detail and individually in this application are all conventional reagents and can be obtained from commercial sources; the methods not described in detail are all conventional experimental methods, which can be known from the prior art.

It should be noted that the terms "first", "second", etc. in the description and claims of the present invention and the above drawings are used to distinguish similar objects, and are not necessarily used to describe a specific sequence or sequence. It does not play a substantial limiting role on the subsequent technical features. It is to be understood that the data so used may be interchanged under appropriate circumstances such that the embodiments of the invention described herein can be practiced in sequences other than those illustrated or described herein. Furthermore, the terms "comprising" and "having" and any variations thereof, are intended to cover non-exclusive inclusion, for example, a process, method, system, product or device comprising a series of steps or units is not necessarily limited to those expressly listed Rather, those steps or units may include other steps or units not expressly listed or inherent to these processes, methods, products or devices.

Construction method of maize genomic DNA methylation detection library

When the specific segment of maize genomic DNA is used as the target segment for DNA methylation detection, the whole genome DNA methylation sequencing method well-known to those skilled in the art is used due to limited coverage and high cost, and its detection effect is not ideal. , even if the sequencing depth is increased, the target segment cannot be well covered. In addition, for the detection of DNA methylation in multiple genomes and multiple specific segments, the whole genome DNA methylation sequencing can only detect one genome at a time, so the price is very expensive.

To this end, the examples of the present application disclose a method for constructing a DNA methylation detection library of the maize genome comprising the target segment. The target segment contains at least one or more SNP sites capable of distinguishing different maize genomes, and the DNA methylation detection library is used to detect DNA mixed samples of at least two maize genomes comprising the target segment. The application uses SNP sites to locate the target segment, and the methylation library established in turn can be used to detect the DNA methylation information of at least one target segment in multiple maize genomes at one time, and can also be used for one-time detection. The DNA methylation information of a target segment of a maize genome greatly improves the detection efficiency of the target segment coverage.

Specifically, as shown in Figures 1-2, the method for constructing the DNA methylation detection library of the maize genome comprising the target segment disclosed in the present application includes:

S1. Convert the unmethylated cytosine in the DNA molecule in the DNA mixed sample to uracil;

S2. Perform a first amplification. The first amplification includes amplifying the mixed DNA samples in the same PCR reaction system to obtain a first amplification product. The first amplification product includes a DNA molecule with a target segment and a DNA molecule connected to the DNA. A first recognition sequence and a bridge sequence at the 3' end of the molecule, the first recognition sequence is used to identify each DNA molecule of the target segment in the DNA mixed sample;

S3. Perform a second amplification. The second amplification includes the step of performing PCR amplification on the first amplification product to obtain a second amplification product. The second amplification product includes the first amplification product and is connected to the first amplification product. a second recognition sequence at the 3' end of the product, the second recognition sequence being used to identify the first amplification product; and

S4, using the second amplification product to construct a DNA methylation detection library of the maize genome comprising the target segment.

For this reason, the above steps S1 to S4 will be described in more detail below.

S1. Acquisition of DNA Mixed Samples of Maize Genome

In the embodiment of step S1 of the present application, the DNA mixed sample is obtained by mixing the extracted corn genomic DNA samples. In more embodiments, the number of corn genomic DNA samples may be two or more.

Specifically, the "transformation" can be mutagenized using physical conditions such as chemical mutagen, ultraviolet mutagenesis, etc., as long as it is sufficient to convert the unmethylated deaminated cytosine into uracil. For the specific "transformation" "The means are not limited, for example, using bisulfite for treatment.

A specific S1 step implementation process is as follows:

(1) Extraction of maize genomic DNA

In this example, four common inbred lines of maize, B73, Mo17, W22, and SK, were selected as materials, and the third leaf at the two-leaf and one-heart stage was used as materials, and the genomic DNA was extracted by 2% CTAB method. The DNA samples of the four inbred lines were mixed together to prepare a mixed DNA sample to be tested.

The specific steps include: taking an appropriate amount of leaves into a 1.5mL sterile centrifuge tube, and grinding with a mortar under the freezing condition of liquid nitrogen; quickly adding 750 μL of 2% CTAB extract, quickly shaking and mixing; Gently shake several times every 10 min; take out the centrifuge tube, add 750 μL of chloroform:isoamyl alcohol (24:1), shake gently for a while; centrifuge at 8000 r/min for 10 min at room temperature, remove about 500 μL of supernatant and transfer to a new 1.5mL centrifuge tube; add 500μL of pre-cooled isopropanol (-20°C), shake and mix, and let stand for a while; centrifuge at 12,000 r/min for 2 min at room temperature, discard the supernatant; add 500 μL of 75% alcohol to wash, at room temperature Centrifuge at 12000 r/min for 1 min and discard the supernatant. Repeat once; after air-drying, add 100 μL ddH ₂ O to fully dissolve the DNA; mix the DNA of the four inbred lines with equal mass to prepare a mixed DNA sample to be tested.

(2) Bisulfite treatment.

The mixed DNA samples to be tested were treated with bisulfite using the EZ DNA Methylation-Lightning Kit (Zymo, D5031) kit, and the treated mixed DNA samples were purified. Processing steps include:

1) Add 130 μL of Zymo Lightning Conversion Reagent to 20 μL (about 1 μg) of the DNA sample to be tested. Mix by pipetting up and down for more than 10 times.

2) Transfer 75 μL of the samples in the above 1) with a total volume of 150 μL to two 200 μL PCR tubes, and follow the procedure shown in Table 1 in the PCR machine:

Table 1

temperature time 98℃ 8min 54℃ 60min 4℃ hold

3) Add 600 μL of M-Binding Buffer to the Zymo Spin IC column, transfer the two 75 μL samples after 2) to the Zymo Spin IC column containing M-Binding Buffer, and mix by inversion for more than 10 times. Centrifuge at 13,000 rpm for 30 s and discard the permeate.

4) Add 100 μL of M-Wash buffer and centrifuge at 13000rpm for 30s.

5) Add 200μL of L-DesμLfonation Buffer, and let stand at room temperature for 20min. Centrifuge at 13,000 rpm for 30 s and discard the permeate.

6) Add 200 μL of M-Wash buffer. Centrifuge at 13,000 rpm for 30 s and discard the permeate.

7) Repeat 6) once.

8) Place the Zymo Spin IC column on a new 1.5 mL centrifuge tube, add 21 μL ddH ₂ O, stand at room temperature for 2 min, and centrifuge at 13,000 rpm for 30 s to collect the flow-through fluid, that is, the purified processed maize genome sample. Mixing the corn genome samples of four common inbred lines B73, Mo17, W22 and SK can get DNA mixed samples.

S2, the first amplification

In the example of this step, the first amplification uses an upstream primer and a first downstream primer to perform PCR amplification on the target segment to obtain a first amplification product, and the first amplification product has a first recognition sequence and a bridge sequence , to identify each DNA molecule of the target segment in the DNA mixed sample. As shown in FIG. 2 , the SNP site in the first amplification product obtained by the first amplification using the upstream primer and the first downstream primer is located within 150 bp of its 3' end.

An example of a specific S2 step is as follows:

(1) Selection of the target segment

The target segment suitable for use in this application should contain at least one or more SNP loci that can distinguish between different maize genomes. The number of SNP sites in the target segment is related to the number of maize genomes that can be detected at one time and can be mixed into a DNA mixed sample that can be detected by the method provided in the embodiment of the present application. For example, if there is one SNP site in the target segment, the There are two types of polymorphisms of SNP loci, so the extracted samples of two maize genomes can be mixed to make DNA mixed samples, and so on.

In a specific embodiment, segment 1 (1:83554963-83555226) and segment 2 (7:46311503-46311767) of the maize B73 RefGen_v4 genome are used as target segments, and there are multiple segments in both segments 1 and 2 SNP loci capable of distinguishing four genome sequence information. As shown in Figure 3, the four common inbred lines of maize B73, Mo17, W22 and SK to be detected by DNA methylation all contain these two target segments, and the red dots in the figure represent SNP sites.

(2) Primer design

Primer design upstream and first downstream primers were performed on segment 1 (1:83554963-83555226) and segment 2 (7:46311503-46311767). Wherein, the C base in the upstream primer sequence is designed as a degenerate base Y; the G base in the first downstream primer sequence is designed as a degenerate base R, and the first downstream primer includes a first recognition sequence and a bridge sequence.

In some embodiments, the first recognition sequence is 6 random base sequences, such as CCCCCC, TTTTTT, GGGGGG, AAAAAA, AGAGGG, CCCGGG, etc., such as the underlined sequence in Table 2, the first recognition sequence is used to identify Each DNA molecule of the target segment in the DNA mixed sample.

In some embodiments, the bridge sequence acts to link the first recognition sequence and the second recognition sequence in the final second amplification product. Specifically, the length of the bridge sequence is not limited, it only needs to connect and reduce the interference to the target segment sequence. Extending the bridge sequence can increase the stability of the second PCR amplification experiment, and shortening the bridge sequence can reduce the number of primers. The synthesis cost can be, for example, a short sequence with a length of 9-18 bp (such as the bold sequence in Table 2). In some embodiments, the nucleotide sequence of the bridge sequence is shown in Table 2.

Table 2

(3) The first amplification reaction

In some embodiments, multiple target segments are set, and primers can be designed for the multiple target segments respectively, and a pair of primers can be used for each target segment to perform a first amplification reaction; After the primers of the segments are mixed, only one first amplification reaction is performed.

In a specific embodiment, the upstream primers designed for segment 1 and segment 2 and the first downstream primer are mixed and the DNA mixed sample "converted" in step S1 is added as a template at the same time, and a first amplification PCR amplification reaction to obtain the first amplification product.

In some embodiments, the reaction system of the first amplification reaction is shown in Table 3. In Table 3, the upstream primer may be a mixture of designed upstream primers for multiple target segments, and the first downstream primer may be a mixture of designed first downstream primers for multiple target segments.

Table 3 The first amplification PCR reaction system

In order to increase the specificity of amplification, some embodiments provide a first amplification reaction step comprising:

S21, carry out pre-denaturation, the treatment temperature is 95°C, and the treatment time is 5min;

S22, performing a first amplification cycle, where the first amplification cycle includes sequentially performing a first melting process, a first annealing process, and a first extension process;

S23, performing a second amplification cycle, where the second amplification cycle includes sequentially performing a second melting process, a second annealing process, and a second extension process;

S24, 72℃ for 5min and 12℃ for 1s.

In some embodiments of step S22, the first amplification cycle includes at least 8 to 12 cycles, so as to sufficiently increase the concentration of the substrate chain and improve the synthesis efficiency of the first amplification product.

In some embodiments of step S22, the condition of the first melting process is that the treatment time at 95°C is 30s.

In some embodiments of step S22, the treatment temperature in the first annealing treatment decreases successively as the cycle number of the first amplification cycle increases. In this way, not only the concentration of the substrate chain can be fully increased, the synthesis efficiency of the first amplification product can be improved, but also the probability of the target fragment not carrying the first recognition sequence in the first amplification product can be reduced in order to improve the specificity of amplification. .

In some embodiments of step S22, the treatment temperature of the first annealing treatment is 68-55°C, 65-53°C, 65-55°C, or 63-55°C. For example, if the treatment temperature of the first annealing treatment is 65-55°C, the number of times of the first amplification cycle is 11, and the treatment temperature in the first annealing treatment decreases gradually as the number of cycles of the first amplification cycle increases, For example, the treatment temperature of the first annealing treatment in the first first amplification cycle is 65°C, and the treatment temperature of the first annealing treatment in the second first amplification cycle is 64°C.

In some embodiments of step S23, the second amplification cycle includes at least 20-24 cycles to increase the concentration of the first amplification product.

In a specific example of step S23, 24 second amplification cycles are performed, and each of the second amplification cycles includes successively performing 95°C treatment for 30s, 60°C treatment for 30s, and 70°C treatment for 30s.

Table 4 A specific first amplification reaction step of S21～S24

(4) Purification of the first amplification product

In this step, well-known technical methods can be used to purify the first amplification product. For example, as shown in FIG. 6 , the first amplification product is purified by magnetic beads, purified by using Beckman magnetic beads 2.2 times the volume of the PCR product, and finally eluted with 30 μL ddH ₂ O to obtain the first amplification product.

S3, second amplification

In the example of this step, the upstream primer and the second downstream primer are used, and the first amplification product is used as a template for amplification to obtain the second amplification product. The second amplification product includes the first amplification product and a second recognition sequence connected to the 3' end of the first amplification product, and the second recognition sequence is used to identify the first amplification product.

In some embodiments, since the first downstream primer includes a bridge sequence to form the structure of the first recognition sequence, the bridge sequence and the second recognition sequence at the 3' end of the second amplification product, the second recognition sequence is used in the sequencing process The first amplification product is identified in . For example, the nucleotide sequence of the second downstream primer is shown in Table 5. In Table 5, the bold sequence is the second recognition sequence, and the rest are bridge sequences.

Table 5 Artificial sequences

In some embodiments, if multiple first amplification reactions are performed on multiple maize genomes to obtain multiple first amplification products, then each amplification product is correspondingly designed with a second recognition sequence, and thus can be obtained Corresponding amount of the second amplification product.

A specific reaction system of the second amplification reaction is shown in Tables 6 and 7.

Some embodiments provide a step of a second amplification reaction, comprising:

S31, carry out pre-denaturation, the treatment temperature is 95°C, and the treatment time is 5min;

S32, performing an amplification cycle, wherein the amplification cycle includes sequentially performing a melting process, an annealing process, and an extension process; and

S33, treated at 72 °C for 5 min and at 12 °C for 1 s.

In the embodiment of step S32, the conditions of the melting treatment are 95°C for 30s, the annealing condition is 60°C for 30s, and the extension treatment condition is 72°C for 30s. In this way, the first downstream primer pair can be guaranteed to be the second Specific recognition of amplification products provides specificity of amplification.

Table 6 The second amplification PCR reaction system

Table 7 A specific S31～S33 second amplification PCR reaction program

(4) Purification of the second amplification product

In this step, well-known technical methods can be used to purify the second amplification product. For example, as shown in Figure 7, the second amplification product is purified by magnetic beads, using Beckman magnetic beads 2.2 times the volume of the PCR product for purification, and finally eluted with 30 μL ddH ₂ O to obtain the second amplification product .

Thus, the embodiments of the present application essentially also disclose a kit for constructing a maize genomic DNA methylation detection library comprising a target segment, the target segment comprising at least one or more SNP sites capable of distinguishing different maize genomes , the DNA methylation detection library is used to detect DNA mixed samples of at least two maize genomes comprising the target segment, wherein the kit includes:

Maize genomic DNA processing reagent for extracting the maize genomic DNA and converting unmethylated cytosine into uracil;

an upstream primer, the C base in the upstream primer sequence is replaced with a sequence of degenerate base Y;

a first downstream primer, the G base in the downstream primer sequence is replaced with a sequence of degenerate base R, the first downstream primer comprises a first recognition sequence and a bridge sequence; and

a second downstream primer comprising the bridge sequence and the second recognition sequence sequence;

Wherein, the upstream primer and the first downstream primer are used to perform a first amplification to obtain a first amplification product, and the first amplification product includes a DNA molecule with the target segment and a DNA molecule connected to the A first recognition sequence and a bridge sequence at the 3' end of the DNA molecule;

The upstream primer and the second downstream primer can obtain a second amplification product in a second amplification, and the second amplification product includes the first amplification product and is connected to the first amplification product 3 ' end of the second recognition sequence, to construct the DNA methylation detection library;

The first identification sequence is used to identify each DNA molecule of the target segment in the mixed DNA sample, and the second identification sequence is used to identify the first amplification product.

A method for detecting methylation of maize genomic DNA containing target segments

In addition, the embodiment of the present application also discloses a method for detecting the methylation of the maize genomic DNA comprising the target segment, including:

Steps S1-S3, to construct the maize genomic DNA methylation detection library comprising the target segment;

S5, sequencing the DNA methylation detection library to obtain the first reads library;

S6, performing quality control, splitting and pairing on the first reads library to obtain a second reads library;

S7. Perform alignment, deduplication and calculation on the second reads library to obtain DNA methylation information of each maize genome in the DNA mixed sample.

In the example of step S5, the second-generation high-throughput sequencing technology is used to sequence the constructed DNA methylation detection library. Wherein, the first reads library includes a corresponding number of first reads in the DNA methylation detection library, wherein the term "reads" is a short segment of sequencing fragments, which are the original sequencing generated by the second-generation high-throughput sequencer. data. The second reads library includes second reads composed based on the same second recognition sequence, and the second reads are the reads from which the first recognition sequence, the second recognition sequence and the bridge sequence are removed.

In the embodiment of step S6, it specifically includes S61-S63.

S61. Obtain the reference sequence of the target segment. For example, based on segment 1 and segment 2 described above, sequence information in the genomes of four maize inbred lines B73, Mo17, W22, SK.

S62. Perform quality control on the first reads library to remove low-quality first reads. Specifically, it can be removed based on the commonly used sequencing quality threshold Q20.

S63, splitting and pairing the first reads in the first reads library after quality control to obtain a second reads library. The term "split" is to separate the first reads with different second recognition sequences, and the term "pair" is to pool the first reads with the same second recognition sequence; for example, there are multiple second recognition sequences. sequence, the corresponding number of first reads groups can be obtained.

In some embodiments of step S63, as shown in Table 5, a total of 17 second recognition sequences are designed, thereby generating 17 first read groups, and each specific first read group is aggregated to form a group fq file, and remove the first recognition sequence, bridge sequence and second recognition sequence of each first read, thereby obtaining a second reads library; at the same time, output the data of the first recognition sequence corresponding to each first read.

In a specific example of step S7, aligning the second reads library includes performing the composition of the second reads with the reference sequence respectively, except that the C site allows the mismatch of C and T to compare that the remaining sites are free of mismatches. The reference sequence is the maize genome sequence comprising the target segment. For example, take the sequences of 1:83554963-83555226 and 7:46311503-46311767 in B73, Mo17, W22, SK four maize inbred lines as reference sequences, arrange them into fa format, and use Bsmap alignment software to compare the split The reads are strictly aligned with the sorted reference sequence, and no mismatch is allowed (-v 0), and the aligned bam file is obtained, and the samtools software is used to build an index; note: mismatched alignment is not allowed here. It is necessary. For the new fq format file processed by the second recognition sequence, it contains reads from a mixture of four genome samples. Based on the SNPs between different genomes, the reads can be assigned to the corresponding reads during the alignment process. Genome.

In a specific embodiment of step S7, the deduplication of the second reads library includes removing duplicate reads caused by PCR amplification from the aligned reads. For example, based on the first identification sequence, duplicate data caused by PCR amplification in the preliminary DNA methylation alignment information is removed to obtain deduplicated DNA methylation alignment information.

In a specific embodiment of step S7, the calculation of the second reads library includes comparing the deduplicated reads on the pair and counting the number of C bases and T bases paired at each C site in the target segment of each sample, The DNA methylation level of each C site was calculated: the number of C bases per C site/(the number of C bases per C site + the number of T bases per C site).

A specific result obtained by the detection method in steps S1 to 7 is shown in FIG. 4 . In addition, the known maize inbred lines B73 and Mo17 leaf tissue whole-genome DNA methylation sequencing data were obtained from the NCBI public database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?id=20000000 acc=GSE128859), the DNA methylation levels of target segments 1 and 2 obtained by the whole genome DNA methylation sequencing method (conventional method) were calculated, as shown in Figure 5. Comparing Figures 4 and 5, it can be seen that the DNA methylation levels of B73 and Mo17 target segments 1 and 2 determined by the method of the present application are consistent with the methylation levels of the target segments detected by conventional methods.

From the above, the embodiments of the present application also substantially disclose a system for DNA methylation detection of maize genomes comprising a target segment, the target segment comprising at least one or more SNP sites capable of distinguishing different maize genomes, DNA methylation The detection library is used to detect DNA mixed samples of at least two maize genomes comprising the target segment, wherein the system comprises:

The kits provided in the preceding embodiments are used to obtain DNA methylation information of each maize genome in a mixed DNA sample; and

A processing device, the processing device is used to run a program, and when the program runs, executes "sequencing the DNA methylation detection library to obtain the first reads library" in the detection method provided in the above embodiment;

Carry out quality control, splitting and pairing to the first reads library to obtain the second reads library;

The second reads library is aligned, deduplicated and calculated to obtain the DNA methylation information of each maize genome in the DNA mixed sample".

The above are only the preferred specific embodiments of the present application, but the protection scope of the present application is not limited to this. Substitutions should be covered within the protection scope of this application.

sequence listing

<110> Huazhong Agricultural University

<120> DNA methylation detection of target segments of the maize genome

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Claims (10)

1. the construction method of the DNA methylation detection library of the maize genome comprising the target segment, the target segment comprises at least one or more SNP sites capable of distinguishing different maize genomes, and the DNA methylation detection library uses For detection of DNA mixed samples of at least two maize genomes comprising the target segment, wherein the construction method comprises: converting unmethylated cytosine in DNA molecules in the DNA mixed sample into uracil; A first amplification is performed, and the first amplification includes amplifying the DNA mixed sample in the same PCR reaction system to obtain a first amplification product, and the first amplification product comprises a DNA having the target segment. A DNA molecule and a first recognition sequence and a bridge sequence connected to the 3' end of the DNA molecule, the first recognition sequence is used to identify each DNA molecule of the target segment in the DNA mixed sample; performing a second amplification, the second amplification comprising the step of performing PCR amplification on the first amplification product to obtain a second amplification product, the second amplification product comprising the first amplification product and the a second recognition sequence at the 3' end of the first amplification product, the second recognition sequence being used to identify the first amplification product; and The second amplification product is used to construct the DNA methylation detection library of the maize genome comprising the target segment. 2. construction method according to claim 1, wherein, described first amplification uses upstream primer and first downstream primer, and the C base in described upstream primer is designed to be degenerate base Y; The G base in the first downstream primer is designed to be a degenerate base R, and the first downstream primer comprises the first recognition sequence and the bridge sequence; The SNP site in the first amplification product is located within 150 bp of the starting position of the DNA molecule of the target segment and the upstream primer binding or with the first downstream primer binding start position, so as to ensure the SNP position. Spots can be detected in subsequent sequencing. 3. construction method according to claim 2, wherein, the reaction step of described first amplification comprises: Carry out pre-denaturation, the treatment temperature is 95 °C, and the treatment time is 5 min; performing a first amplification cycle, the first amplification cycle comprising sequentially performing a first melting process, a first annealing process, and a first extension process; performing a second amplification cycle comprising sequentially performing a second melting process, a second annealing process, and a second extension process; and 72℃ for 5min and 12℃ for 1s; Wherein, the first amplification cycle includes at least 8 to 12 cycles, and the treatment temperature of the first annealing treatment decreases successively as the cycle number of the first amplification cycle increases. The construction method according to claim 3, wherein the treatment temperature of the first annealing treatment is 68-55°C, 65-53°C, 65-55°C or 63-55°C. 5. The construction method according to claim 1, wherein the second amplification uses the upstream primer and the second downstream primer, the second downstream primer comprises the second recognition sequence, the second The amplification reaction steps include: Carry out pre-denaturation, the treatment temperature is 95 °C, and the treatment time is 5 min; performing an amplification cycle comprising sequentially performing a melting process, an annealing process, and an extension process; and Treat at 72°C for 5 min and at 12°C for 1 s. 6. The construction method according to claim 5, wherein the second amplification product further comprises between the first recognition sequence and the second recognition sequence connected at the 3' end of the first amplification product The bridge sequence is used to connect the first recognition sequence and the second recognition sequence. 7. A method for detecting DNA methylation in a target segment of a maize genome, the target segment comprising at least one or more SNP sites capable of distinguishing different maize genomes, and the method for detecting DNA methylation in a target segment is used to detect A DNA mixed sample of at least two corn genomes comprising the target segment, wherein the detection method comprises: The construction method according to any one of claims 1 to 6, wherein a DNA methylation detection library of the maize genome comprising the target segment is constructed; Sequencing the DNA methylation detection library to obtain the first reads library; Carry out quality control, splitting and pairing to the first reads library to obtain the second reads library; The second reads library is aligned, deduplicated and calculated to obtain the DNA methylation information of each maize genome in the DNA mixed sample. 8. The detection method according to claim 7, wherein the first reads library comprises the first reads corresponding to the number of the DNA methylation detection library, and the second reads library comprises the second reads based on the same The second reads composed of the recognition sequence, the second reads are the reads from which the first recognition sequence, the second recognition sequence and the bridge sequence are removed. 9. A kit for constructing a DNA methylation detection library of a maize genome comprising a target segment comprising at least one or more SNP sites capable of distinguishing different maize genomes, the DNA methylation The detection library is used to detect DNA mixed samples of at least two maize genomes comprising the target segment, wherein the kit includes: Maize genomic DNA processing reagent for extracting the maize genomic DNA and converting unmethylated cytosine into uracil; an upstream primer, the C base in the upstream primer sequence is replaced with a sequence of degenerate base Y; a first downstream primer, the G base in the sequence of the first downstream primer is replaced with a sequence of degenerate base R, the first downstream primer comprises a first recognition sequence and a bridge sequence; and a second downstream primer comprising the bridge sequence and the second recognition sequence sequence; Wherein, the upstream primer and the first downstream primer are used to perform a first amplification to obtain a first amplification product, and the first amplification product includes a DNA molecule with the target segment and a DNA molecule connected to the A first recognition sequence and a bridge sequence at the 3' end of the DNA molecule; The upstream primer and the second downstream primer can obtain a second amplification product in a second amplification, and the second amplification product includes the first amplification product and is connected to the first amplification product 3 ' end of the second recognition sequence, to construct the DNA methylation detection library; The first identification sequence is used to identify each DNA molecule of the target segment in the mixed DNA sample, and the second identification sequence is used to identify the first amplification product. 10. A system for detection of maize genome DNA methylation comprising a target segment comprising at least one or more SNP sites capable of distinguishing between different maize genomes, the DNA methylation detection library being used for detection of comprising A mixed sample of DNA from at least two maize genomes of the target segment, wherein the system comprises: The kit of claim 9, for obtaining DNA methylation information of each corn genome in the DNA mixed sample; and A processing device, the processing device is used for running a program, and when the program is running, the detection method of claim 7 or 8 is executed. "Sequencing the DNA methylation detection library to obtain the first reads library; Carry out quality control, splitting and pairing to the first reads library to obtain the second reads library; The second reads library is aligned, deduplicated and calculated to obtain the DNA methylation information of each maize genome in the DNA mixed sample".

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