Detection method, liquid phase chip and kit for RppK in backcross analysis

Abstract

The present invention relates to the technical field of corn breeding, and specifically discloses a method for detecting RppK in backcross analysis, a liquid phase chip, and a kit, comprising the following steps: S1, finding the variation site of the RppK gene on R3, and screening out 23 SNP variation sites and 4 INDEL variation sites therefrom to constitute specific variation sites, as shown in Table 1 in the specification; intercepting the sequences corresponding to the 27 heterosexual variation sites respectively to obtain 24 specific site sequences, the 24 specific site sequences being shown as SEQ ID NO.1-SEQ ID NO.24 respectively; S2, comparing the 24 specific site sequences with the sequence information of the target material, and judging whether the target material contains the RppK gene according to the comparison result. The present invention can not only detect the RppK gene in corn to identify germplasm resources and screen disease-resistant germplasm; but also has the advantages of high efficiency and good predictability.

Description

Technical Field

[0001] The present invention relates to the technical field of corn breeding, and in particular to a method for detecting RppK in backcross analysis, a liquid phase chip and a kit. Background Art

[0002] Corn is a staple food crop and a major source of feed and light industrial energy. Southern corn rust is an airborne disease that can easily cause widespread outbreaks when conditions are favorable, causing devastating damage to corn production. In recent years, global warming and other factors, such as cropping systems, have led to an increasing incidence of corn rust pathogens, with increasingly severe impacts on corn production. The most cost-effective way to control the disease is to develop corn lines carrying resistance genes. Currently, 11 dominant resistance genes and 8 major resistance QTLs have been reported. The successfully cloned RppK gene confers broad-spectrum resistance to southern corn rust and can be introduced into different corn genetic backgrounds.

[0003] Currently, the development of corn varieties resistant to southern rust relies primarily on experience and phenotypic selection, resulting in long breeding cycles, low efficiency, and poor predictability. Due to the characteristics of the southern rust pathogen, resistance identification relies primarily on natural field outbreaks. However, in areas with low southern rust incidence or in years when environmental factors make it less susceptible to disease, phenotypic testing cannot be performed properly. Furthermore, each planting requires extensive genetic sequencing and analysis, significantly slowing down the selection process. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for detecting RppK in backcross analysis, which can not only detect the RppK gene in corn to identify germplasm resources and screen disease-resistant germplasm, but also has the advantages of high efficiency and good predictability.

[0005] In addition, the present invention also provides a liquid phase chip and a kit for detecting RppK.

[0006] The present invention is achieved through the following technical solutions:

[0007] The method for detecting RppK in backcross analysis comprises the following steps:

[0008] S1. Identify the mutation sites of the RppK gene on R3, and by comparing the reference genomes of various maize materials, screen 23 SNP mutation sites and 4 INDEL mutation sites from the mutation sites identified to constitute specific mutation sites, the specific mutation sites being shown in Table 1 in the Examples; intercept the sequences corresponding to the 27 heterosexual mutation sites to obtain 24 specific site sequences, the 24 specific site sequences being shown as SEQ ID NO.1 to SEQ ID NO.24, respectively;

[0009] S2. Compare the 24 specific site sequences with the sequence information of the target material, and determine whether the target material contains the RppK gene based on the comparison results.

[0010] Based on the 27 heterosexual variation sites screened out by the present invention and the design of the corresponding 24 specific site sequences, by comparing the 24 specific site sequences with the sequence information of the target material, it is possible to determine whether the target material contains the RppK gene; by determining the presence or absence of the RppK gene in the target material, it is possible to identify germplasm resources and screen disease-resistant germplasm, and also to select the prospects of improved inbred lines, quickly and efficiently improve target traits, significantly improve the breeding efficiency and predictability of excellent inbred lines resistant to southern rust, and play an important role in accelerating the breeding process of new disease-resistant varieties.

[0011] In a preferred embodiment, in step S1, the specific site sequence is intercepted as follows:

[0012] For the SNP variant site, the left and right 70 bp, a total of 141 bp, were taken as the specific site sequence;

[0013] As for the INDEL mutation sites, since the four INDELs were located close to each other, they were placed in the same sequence, resulting in a total of 313 bp of sequence.

[0014] In a preferred embodiment, in step S2, determining whether the target material contains the RppK gene comprises the following steps:

[0015] S21. Filter the comparison results and count them:

[0016] Count the number of sequencing fragments of target materials that meet the following conditions:

[0017] (A) For SNP variant sites, 100% full-length correct alignment is required;

[0018] (B) For INDEL variant sites, the sequencing fragments of the target material are required to cover the INDEL-specific site sequence and the alignment similarity is greater than 98%;

[0019] S22. Determine the presence of the RppK gene based on the SNP and INDEL counts:

[0020] Determine whether the target material contains the RppK gene based on the following conditions:

[0021] (1) For a single SNP variant site, there must be more than 5 records that meet condition (A);

[0022] (2) For all SNP variant sites, the proportion that meets condition (1) is required to be greater than or equal to 80%;

[0023] (3) For a single INDEL variant site, there must be more than 5 records meeting condition (B);

[0024] If the sequencing results of the target material do not meet the above conditions (1) and (2) at the same time, it is judged as susceptible, that is, the material does not contain the RppK gene; if the above conditions (1)-(3) are met at the same time, it is judged as resistant, that is, the material contains the RppK gene; if the above conditions (1) and (2) are met at the same time but condition (3) is not met, it is uncertain whether the material contains the RppK gene.

[0025] In a preferred embodiment, the method further comprises the following steps:

[0026] S3. Determine the homozygous RppK gene of the target material containing the RppK gene:

[0027] S31. Calculate the NADF of the donor parent;

[0028] S32. Generate a specific site set based on the receptor parent information of the target material and calculate the NADF of the progeny (i.e., the target material);

[0029] S33. If the ratio of the NADF of the offspring to the NADF of the donor parent is greater than or equal to 0.7 and less than 1.5, it is determined to be homozygous disease resistance; if the ratio of the NADF of the offspring to the NADF of the donor parent is less than 0.7, and the judgment result of step S22 is disease resistance, it is determined to be heterozygous disease resistance.

[0030] In a preferred embodiment, the specific process of step S32 is as follows:

[0031] On the basis of completing step S21, further determine whether the target material has a receptor parent; if the target material specifies a receptor parent, then count the number of SNP variation sites in the parent material that meet condition (1) in step S22, and use this part of the SNP variation sites as a specific site set to calculate the NADF of the target material; if the target material does not specify a receptor parent or has only a single receptor parent, do not count the number of SNP variation sites, and use all variation sites to calculate the NADF of the target material.

[0032] In a preferred embodiment, in step S33, if the ratio of the NADF of the offspring to the NADF of the donor parent is greater than or equal to 1.5, no determination is made and an abnormality is output.

[0033] In a preferred embodiment, the threshold value for judging the pure heterozygosity of the RppK gene is obtained by conducting experiments based on the D340, HCL645, Jing724, PH4CV, Tie9010, T12067, and Zheng58 populations, and calculating the NADF value of the progeny material and the ratio to the NADF of the donor parent of each population.

[0034] In a preferred embodiment, the calculation process of NADF is as follows:

[0035] (1) Count the number of original sequencing fragments of all 24 specific site sequences aligned;

[0036] (2) Count the number of original sequencing fragments for each of the 24 specific site sequences in the alignment;

[0037] (3) Divide the value obtained in (2) by the value obtained in (1), and then multiply by 10 to the power of 6 to normalize the data;

[0038] (4) Take the average value of each variant site.

[0039] A liquid phase chip for detecting RppK comprises a set of nucleotide probes for detecting 27 specific variation sites or 24 specific site sequences.

[0040] A kit for detecting RppK, comprising the liquid phase chip as described above.

[0041] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0042] Based on the 27 heterosexual mutation sites screened by the present invention and the design of 24 corresponding specific site sequences, by comparing the 24 specific site sequences with the sequence information of the target material, it is not only possible to determine whether the gene RppK is introduced into the target material, but also to further refine the judgment of the pure and heterozygous nature of the gene RppK in the target material. By judging the presence or absence of the RppK gene in the target material, it is possible to identify germplasm resources and screen disease-resistant germplasm, and also to select the prospects of improved inbred lines, quickly and efficiently improve the target traits, and significantly improve the breeding efficiency and predictability of excellent inbred lines resistant to southern rust. The detection method of the present invention plays an important role in shortening the breeding cycle and improving breeding efficiency.

[0043] Compared with the existing technology KASP, which is restricted by factors such as primer selection and sample selection, this technology uses liquid phase chips for targeted sequencing, which has the characteristics of strong compatibility, high throughput and high efficiency. Experiments have shown that its accuracy is higher than KASP. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings:

[0045] Figure 1 This is a logic block diagram for determining the presence or absence of the RppK gene in Example 1 of the present invention;

[0046] Figure 2 This is a logic diagram for determining pure heterozygosity of the RppK gene in Example 2 of the present invention;

[0047] Figure 3 This is a graph showing the NADF calculation results of the PH4CV population in Example 2 of the present invention;

[0048] Figure 4 This is a graph showing the NADF calculation results for the Jing724 population in Example 2 of the present invention;

[0049] Figure 5 This is a graph showing the NADF calculation results for the Zheng58 population in Example 2 of the present invention;

[0050] Figure 6 This is a graph showing the NADF calculation results for the T12067 population in Example 2 of the present invention;

[0051] Figure 7 NADF calculation results of the Tie9010 population in Example 2 of the present invention;

[0052] Figure 8 This is a graph showing the NADF calculation results of the D340 population in Example 2 of the present invention;

[0053] Figure 9 This is a graph showing the NADF calculation results of the HCL645 population in Example 2 of the present invention;

[0054] Figure 10 This is a partial screenshot of the comparison results of multiple versions of the B73 sequence and the R1, R2, and R3 sequences of the K22 material in Example 1 of the present invention. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the examples. The exemplary embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention. The embodiments described below are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0056] In the following description, numerous specific details are set forth to provide a thorough understanding of the present invention. However, it will be apparent to one of ordinary skill in the art that these specific details are not necessarily required to practice the present invention. In other examples, well-known structures, materials, or methods are not specifically described to avoid obscuring the present invention. The materials, instruments, and reagents used in the following examples, unless otherwise specified, are commercially available. The techniques used in the examples, unless otherwise specified, are conventional techniques well known to those skilled in the art.

[0057] Example 1:

[0058] In order to determine whether the target material contains the RppK gene or whether the RppK gene is introduced into the target material, Figure 1 As shown, this embodiment provides a method for detecting RppK in backcross analysis, comprising the following steps:

[0059] S1. Perform a multiple sequence alignment of the B73V3 version of the maize reference genome sequence with the R1, R2, and R3 sequences; identify the variant sites of the RppK gene on R3. Considering that some materials may be highly similar to R3, the reference genomes of various maize materials are compared to identify the variant sites and extract the specific site sequences corresponding to the variant sites. The variant sites screened by the above method are not only located on the R3 sequence, but also have a greater number of variant sites and stronger specificity than existing site markers on the maize reference genome.

[0060] This example specifically mines variant sites in K22:

[0061] K22 is an elite corn inbred line with durable and complete resistance to southern corn rust. The RppK gene region contains three candidate genes: R1, R2, and R3, with R3 exhibiting significant resistance. The K22 sequence and the location of the R1, R2, and R3 sequences were obtained by searching the NCBI website for "MZ322317.1."

[0062] First, multiple versions (V3, V4, V5) of the B73 sequence (the corn reference genome with the version sequence B73V3) and the R1, R2, and R3 sequences of the K22 material were aligned using muscle software to find the variant sites on R3 of the K22 material. The screenshot of the alignment result is shown below. Figure 10As shown. Secondly, considering that some materials may be highly similar to R3, the specific sites on R3 were compared with the reference genomes of various corn materials. The comparison details are as follows: 1) 100bp on the left and right of the specific sites on R3 were cut as the comparison input sequence, and the 100bp sequences on the left and right were used to accurately locate the sites on the reference genomes of other corn varieties; 2) Specific SNPs with different bases on R3 and other reference genomes were screened out, for example, sites with a genotype of A on R3 and a genotype of G on other corn materials. Finally, a total of 27 variant sites were obtained, including 23 SNP variant sites and 4 INDEL variant sites with a close distance and within a range of 120bp. These variant sites are the key to detecting the RppK gene. The 27 variant sites are shown in Table 1; the information of the various corn materials used for the comparison is shown in Table 2.

[0063] Table 1

[0064]

[0065] Table 2

[0066]

[0067]

[0068] To ensure the accuracy of subsequent alignment with the target material sequence, the left and right 70 bp of the SNP variant site were selected, for a total of 141 bp of sequence; similarly, since the four INDELs were located close to each other, they were placed in the same sequence, resulting in a total of 313 bp of sequence. The above constitutes the FASTA file of the sequence near the variant site, which contains a total of 24 site-specific sequences (K22-specific site sequences); the 24 site-specific sequences are shown in SEQ ID NO. 1 to SEQ ID NO. 24, respectively, as shown in Table 3:

[0069] Table 3

[0070]

[0071]

[0072]

[0073] The purpose of designing the K22 specific site sequence based on the screened specific sites in this embodiment is:

[0074] According to the needs of subsequent comparison, in order to achieve a more accurate judgment of the introduction status of the RppK gene: although the liquid phase chip can be designed based on the specific sites screened out, the liquid phase chip can be used to directly test whether it contains the mutation site and then determine whether it exists, but the NADF of a single material lacks a comparison standard and cannot accurately judge pure heterozygosity. Instead, it is necessary to use the K22 specific site sequence provided in this embodiment in combination with the detection method, and use the parents in the backcross system as control samples to accurately judge the problem of pure heterozygosity.

[0075] S2. First, use BLAST software to compare the 24 specific site sequences (K22 specific site sequences) with the sequence information of the target material, and then determine whether the target material contains the RppK gene based on the comparison results. The specific determination includes the following steps:

[0076] S21. Filter the comparison results and count them:

[0077] Count the number of sequencing fragments of target materials that meet the following conditions:

[0078] (A) For SNP variant sites, 100% full-length correct alignment is required;

[0079] (B) For INDEL variant sites, the sequencing fragments of the target material are required to cover the INDEL-specific site sequence and the alignment similarity is greater than 98%;

[0080] S22. Determine the presence of the RppK gene based on the SNP and INDEL counts:

[0081] Determine whether the target material contains the RppK gene based on the following conditions:

[0082] (1) For a single SNP variant site, there must be more than 5 records that meet condition (A);

[0083] (2) For all SNP variant sites, considering the reliability of the discrimination results and combining the experimental verification data, with 23 SNP variant sites as the premise, the accuracy can be stably maintained above 99.98% when the number of SNP variant sites used for analysis reaches more than 18. Therefore, it is required that the proportion of sites that meet condition (1) is greater than or equal to 80%;

[0084] (3) For a single INDEL variant site, there must be more than 5 records meeting condition (B);

[0085] If the sequencing results of the target material do not meet the above conditions (1) and (2) at the same time, it is judged as susceptible, that is, the material does not contain the RppK gene; if the above conditions (1)-(3) are met at the same time, it is judged as resistant, that is, the material contains the RppK gene; if the above conditions (1) and (2) are met at the same time but condition (3) is not met, it is uncertain whether the material contains the RppK gene.

[0086] This example is a detection and discrimination method for the broad-spectrum southern rust resistance gene RppK in maize, based on the alignment result file of sequences near the mutation site in the superior maize inbred line K22. This example can determine whether the gene RppK has been introduced into the target material or whether the detected target material contains the broad-spectrum resistance gene RppK. Determining whether the broad-spectrum southern rust resistance gene RppK has been introduced into maize using multiple target materials can not only identify germplasm resources and screen for disease-resistant germplasm, but also select promising improved inbred lines, quickly and efficiently improving target traits, significantly improving the efficiency and predictability of breeding superior inbred lines resistant to southern rust, and playing an important role in accelerating the breeding process of new disease-resistant varieties. It also plays an important role in shortening the breeding cycle and improving breeding efficiency.

[0087] The target material in this embodiment is the progeny obtained by backcrossing. The general backcrossing process is as follows:

[0088] Using an elite inbred line containing the southern rust resistance gene RppK as the donor parent and the material to be improved in southern rust resistance as the recipient parent, the following were performed:

[0089] (1) The donor parent is hybridized with the recipient parent to obtain F1;

[0090] (2) Select F1 materials with good agronomic traits and a background recovery rate of 50% and backcross them with the recipient parent to obtain the backcross generation BC1F1;

[0091] (3) BC1F1 was tested using a liquid microarray, and heterozygous materials with good agronomic traits, a background recovery rate of 85%, and resistance to southern rust were selected and backcrossed with the recipient parent to obtain the second-generation backcross BC2F1;

[0092] (4) BC2F1 was tested using a liquid microarray, and heterozygous materials with good agronomic traits, a background recovery rate of more than 95%, and resistance to southern rust were selected and backcrossed with the recipient parent to obtain the third-generation backcross BC3F1;

[0093] (5) BC3F1 was self-pollinated for two consecutive generations, and the offspring all showed high resistance to southern rust. Single ears were harvested and harvested into ear rows. Plants were selected from the ear rows to form plant lines, and an inbred line with high resistance to southern rust was obtained.

[0094] The liquid phase chip used in this example contains a set of nucleotide probe sequences for detecting 27 specific sites.

[0095] In order to verify the accuracy of the variant sites screened in this example for detecting the maize broad-spectrum resistance gene RppK, the following experiments were performed:

[0096] X178 corn is a double-layered, heavy-eared variety with strong drought tolerance and mild resistance to southern rust. Mo17 is a classic corn inbred line known for its high and stable yields, strong tolerance to drought, and wide adaptability. Approximately 3,000 samples from the X178 population and 489 samples from the Mol17 population were selected for experimental testing. Liquid microarrays were used to capture sequences at the K22-specific locus, and the established RppK gene KASP assay was used to assist in verification. The results, as shown in Table 4, achieved concordance rates exceeding 99%.

[0097] Table 4

[0098]

[0099]

[0100] Example 2:

[0101] like Figure 2 As shown, this embodiment is based on Example 1, and further refines the judgment of the pure and impure nature of the gene RppK in the target material based on the K22 specific site sequence and judgment results obtained in Example 1.

[0102] Based on Example 1, this embodiment further includes the following steps:

[0103] S3. Determine the homozygous RppK gene of the target material containing the RppK gene:

[0104] S31. Calculate the NADF of the donor parent;

[0105] The basis for judging pure heterozygosity is the normalized abundance of DNA fragments in the sample (NADF). The NADF of a material alone, without a comparison standard, cannot measure its pure heterozygosity. Therefore, the present invention uses the parental material in the backcross system as a control sample.

[0106] The NADF calculation method for a single sample is as follows:

[0107] (1) Count the number of original sequencing fragments of all 24 specific site sequences aligned;

[0108] (2) Count the number of original sequencing fragments for each of the 24 specific site sequences in the alignment;

[0109] (3) Divide the value obtained in (2) by the value obtained in (1), and then multiply by 10 to the power of 6 to normalize the data;

[0110] (4) Take the average value of each variant site.

[0111] The calculation process and raw data for the NADF values ​​of the six specific loci in the two samples are shown in Table 5 to aid understanding. The final average values ​​were 10.08028723 for Sample 1 and 10.79812815 for Sample 2.

[0112] Table 5

[0113]

[0114] S32. Generate a specific site set based on the receptor parent information of the target material and calculate the NADF of the progeny (target material);

[0115] On the basis of completing step S21, further determine whether the target material has a receptor parent; if the target material specifies a receptor parent, then count the number of SNP variation sites in the parent material that meet condition (1) in step S22, and use this part of the SNP variation sites as a specific site set to calculate the NADF of the target material; if the target material does not specify a receptor parent or has only a single receptor parent, do not count the number of SNP variation sites, and use all variation sites to calculate the NADF of the target material.

[0116] S33, (1) If the ratio of the NADF of the offspring to the NADF of the donor parent is greater than or equal to 0.7 and less than 1.5, it is judged as homozygous disease resistance; (2) If the ratio of the NADF of the offspring to the NADF of the donor parent is less than 0.7, and the judgment result of step S22 is disease resistance, it is judged as heterozygous disease resistance; (3) Theoretically, the NADF value of the offspring is smaller than that of the donor parent. If the ratio of the NADF of the offspring to the NADF of the donor parent is greater than or equal to 1.5, no judgment is made and an abnormal output is output; (4) In other cases, it is judged as susceptible, that is, the material does not contain the RppK gene.

[0117] The target material of this embodiment is the progeny obtained by backcrossing.

[0118] The threshold for determining pure heterozygosity of the RppK gene was determined based on experiments conducted on populations D340, HCL645, Jing724, PH4CV, Tie9010, T12067, and Zheng58. The NADF values ​​of the progeny materials and their ratio to the NADF of the donor parent of each population were calculated. The specific process is as follows:

[0119] Materials from seven groups, including D340, HCL645, Jing724, PH4CV, Tie9010, T12067, and Zheng58, were selected for experiments using a liquid phase chip. The NADF values ​​of the materials and the NADF ratios compared to the donor parents of each group were calculated.

[0120] The liquid phase chip used in this example contains a set of nucleotide probe sequences for detecting 27 specific sites.

[0121] The actual situation shows that there are two types of situations: (1) The donor parent and the offspring materials are tested in the same batch, such as T12067, Tie9010 and HCL645 group materials. In this case, the NADF value calculated is closer to the theoretical value of the pure heterozygous ratio of 2:1, and the distribution of the pure heterozygous NADF value also has obvious differences in the numerical range; (2) The donor parent and the offspring materials are tested in different batches, such as PH4CV, Jing724, Zheng58, and D340 group materials. In this case, they will be affected by the batch effect. The distribution range of the NADF value of the heterozygous disease resistance is similar to that of the previous case, and the distribution range of the NADF value of the homozygous disease resistance is slightly lower than that of the previous case, but still in line with the theoretical value of the pure heterozygous ratio of 2:1. The results are shown in 3- Figure 9 Due to the deviation of the theoretical value, the threshold fluctuation range needs to be adjusted. After continuous trial and error, the judgment threshold is finally determined as shown above.

[0122] In order to verify the accuracy of the present invention in determining whether the RppK gene is homozygous, the following test was performed:

[0123] Finally, 25 real samples (15 homozygous positive samples, with P as the sample name prefix; 15 heterozygous positive samples, with H as the sample name prefix; 5 negative control samples, with N as the sample name prefix) were selected to verify the accuracy of the judgment logic. The experimental results showed a correct rate of 100%, as shown in Table 6.

[0124] Table 6

[0125]

[0126]

[0127]

[0128] KASP technology is limited by factors such as primer selection and sample selection. This example utilizes liquid-phase microarrays for targeted sequencing, which offers strong compatibility, high throughput, and high efficiency. Experiments have shown that this method offers higher accuracy than KASP. The experiment involved 22 BC1F1 cells from two populations, DH351 and PH4CV. Excluding three samples with undetectable genotypes, the KASP test showed one false positive, resulting in an accuracy rate of approximately 94.74%. The results are shown in Table 7.

[0129] Table 7

[0130]

[0131] The same sample was tested using the judgment method of this embodiment, and the detection rate and accuracy rate were both 100%, as shown in Table 8:

[0132] Table 8

[0133]

[0134] Example 3:

[0135] A liquid phase chip for detecting RppK comprises a set of nucleotide probes for detecting 27 specific variation sites or 24 specific site sequences.

[0136] After screening the variant sites, probe sequences capable of detecting the variant sites are designed based on the principle of base complementarity. The design principles are as follows:

[0137] (1) Probe length: 110 bp; (2) GC content ratio: 30%-70%; (3) Maximum number of similar fragments ≤ 5; (4) Coverage multiplier: 2X; (5) Maximum distance between the probe and the designed region: 10; (6) Maximum length of the SSR covered by the probe: 120.

[0138] The maximum number of similar fragments refers to the upper limit of the number of specific similar fragments of the probe on the reference genome.

[0139] The detection process is as follows:

[0140] a. DNA sample extraction and detection

[0141] DNA samples were extracted using a high-throughput DNA extraction kit. The extracted DNA samples were then subjected to two tests: (1) 1% agarose gel electrophoresis to analyze DNA purity and integrity; and (2) Qubit assay to accurately quantify DNA concentration.

[0142] b. GenoBaits Experimental Procedure

[0143] DNA that has passed quantitative quality control is randomly physically fragmented using an ultrasonic disruptor, with the fragment peak size controlled at 200-300bp. The fragmented DNA undergoes end-repair and ligation with A tails. The A-tailed DNA fragments are ligated to sequencing adapters using ligase. The library is then purified and fragments are selected using carboxyl-modified magnetic beads, retaining ligation products with inserts between 200-300bp. The ligation products are then amplified using barcoded sequencing primers and a high-fidelity PCR reaction system. Different barcodes are used to distinguish different samples. After purification using carboxyl magnetic beads, the amplified products are ready for probe hybridization experiments.

[0144] Take 500ng of the constructed sequencing library, freeze-dry it, add the probe and hybridization reagent, denature it, and incubate it at 65°C for 2 hours to complete the hybridization reaction. After the hybridization product is washed with washing solution, another round of PCR is performed to complete the construction of the hybridization capture library.

[0145] c. Library construction and sequencing

[0146] For samples that pass the aforementioned DNA quality inspection, targeted sequencing libraries are constructed using the corresponding products, ultimately completing library preparation for all samples in the project. After library construction is complete, preliminary quantification is performed using Qubit 2.0, followed by accurate quantification of the effective concentration using qPCR to ensure library quality. Once the library passes the inspection, sequencing begins.

[0147] d. Information analysis process

[0148] Information analysis includes: data quality control (removal of connectors and low-quality data), comparison with the reference genome, variation detection and annotation, and other analyses.

[0149] Example 4: A kit for detecting RppK, comprising the liquid phase chip as described in Example 3.

[0150] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for detecting RppK in backcross analysis, characterized in that: The following steps are involved: S1. Identify the variant sites of the RppK gene on R3, and by comparing the reference genomes of various maize materials, screen 23 SNP variant sites and 4 INDEL variant sites from the found variant sites to constitute specific variant sites, the specific variant sites are shown in Table 1; intercept the sequences corresponding to the 27 specific variant sites respectively, and obtain 24 specific site sequences, the 24 specific site sequences are shown in SEQ ID NO.1 to SEQ ID NO.24 respectively; S2. Compare the 24 specific site sequences with the sequence information of the target material, and determine whether the target material contains the RppK gene based on the comparison results; In step S1, the method of intercepting the specific site sequence is as follows: For the SNP variant site, the left and right 70 bp, a total of 141 bp, were taken as the specific site sequence; For INDEL variant sites, the four INDEL variant sites were placed in the same sequence, obtaining a total of 313 bp of sequence; In step S2, it is determined whether the target material contains the RppK gene The following steps are involved: S21. Filter the comparison results and count them: Count the number of sequencing fragments of target materials that meet the following conditions: (A) For SNP variant sites, 100% full-length correct alignment is required; (B) For INDEL variant sites, the sequencing fragments of the target material are required to cover the INDEL-specific site sequence and the alignment similarity is greater than 98%; S22. Determine the presence of the RppK gene based on the SNP and INDEL counts: Determine whether the target material contains the RppK gene based on the following conditions: (1) For a single SNP variant site, there must be more than 5 records that meet condition (A); (2) For all SNP variant sites, the proportion that meets condition (1) is required to be greater than or equal to 80%; (3) For a single INDEL variant site, there must be more than 5 records meeting condition (B); If the sequencing results of the target material do not meet the above conditions (1) and (2) at the same time, it is judged as susceptible, that is, the material does not contain the RppK gene; if the above conditions (1)-(3) are met at the same time, it is judged as resistant, that is, the material contains the RppK gene; if the above conditions (1) and (2) are met at the same time but condition (3) is not met, it is uncertain whether the material contains the RppK gene; Table 1 2. The method for detecting RppK in backcross analysis according to claim 1, wherein The following steps are also included: S3. Determine the homozygous RppK gene of the target material containing the RppK gene: S31, calculating the standardized sample DNA abundance value NADF of the donor parent; S32. Generate a specific site set based on the recipient parent information of the target material, and calculate the standardized sample DNA abundance value NADF of the progeny; S33. If the ratio of the standardized DNA abundance value NADF of the progeny sample to the standardized DNA abundance value NADF of the donor parent is greater than or equal to 0.7 and less than 1.5, the progeny is determined to be homozygous for disease resistance; if the ratio of the standardized DNA abundance value NADF of the progeny sample to the standardized DNA abundance value NADF of the donor parent is less than 0.7, and the result of step S22 is disease resistance, the progeny is determined to be heterozygous for disease resistance; The calculation process of the standardized sample DNA abundance value NADF is as follows: (1) Count the number of original sequencing fragments of all 24 specific site sequences aligned; (2) Count the number of original sequencing fragments for each of the 24 specific site sequences in the alignment; (3) Divide the value obtained in (2) by the value obtained in (1), and then multiply by 10 to the power of 6 to normalize the data; (4) Take the average value of each variant site.

3. The method for detecting RppK in backcross analysis according to claim 2, wherein The specific process of step S32 is as follows: On the basis of completing step S21, further determine whether the target material has a receptor parent; if the target material specifies a receptor parent, then count the number of SNP variation sites in the parent material that meet condition (1) in step S22, and use this part of the SNP variation sites as the specific site set to calculate the standardized sample DNA abundance value NADF of the target material; if the target material does not specify a receptor parent or has only a single receptor parent, do not count the number of SNP variation sites, and use all variation sites to calculate the standardized sample DNA abundance value NADF of the target material.

4. The method for detecting RppK in backcross analysis according to claim 2, wherein In step S33, if the ratio of the standardized sample DNA abundance value NADF of the offspring to the standardized sample DNA abundance value NADF of the donor parent is greater than or equal to 1.5, no determination is made and an abnormality is output.

5. The method for detecting RppK in backcross analysis according to claim 2, wherein The threshold value for judging pure heterozygosity of the RppK gene was obtained by conducting experiments on the D340, HCL645, Jing724, PH4CV, Tie9010, T12067 and Zheng58 populations, and calculating the standardized sample DNA abundance value NADF of the progeny material and its ratio to the standardized sample DNA abundance value NADF of the donor parent of each population.

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