A method for identifying functional regulatory regions during plant domestication based on ACRs

Abstract

The present application relates to the field of bioinformatics, and in particular to a method for identifying functional regulatory intervals in a plant domestication process based on ACRs; the method comprises: determining a species strain genome and a closely related species genome; obtaining ACRs of a plant to be tested; using a first analysis software and a second analysis software to match the ACRs of the plant to be tested to a closely related species genome or a species strain genome, so as to obtain conservative ACRs, missing ACRs and supplementary ACRs respectively; adding supplementary ACRs to conservative ACRs, and then counting the number of conservative ACRs matched to different genomes, so as to obtain multiple groups of regulatory interval data; judging whether the regulatory interval data is a functional regulatory interval related to plant domestication according to whether the ACRs of the plant to be tested in the regulatory interval data are successfully matched to the species strain genome and unsuccessfully matched to the closely related species genome; the method can improve the accuracy, reliability and visualization of identification.

Description

Technical Field

[0001] The present application relates to the field of bioinformatics, and in particular to a method for identifying functional regulatory intervals in a plant domestication process based on ACRs. Background Art

[0002] At present, the identification method of relevant sites of plants in the domestication process usually adopts the strategy of fine gene positioning. For example, in the prior art, a transposon (Hopscotch) was identified by comparing the genome sequence differences between corn and its ancestor plant, Teosinte zea mays. This transposon was inserted into the regulatory region of the corn domestication gene TEOSINTE BRANCHED1 (TB1), thereby completing the domestication of corn. Since TB1 is an important gene related to plant domestication, and the transposon (Hopscotch) is located 60kb upstream of the gene, it can effectively regulate the expression of TB1 during the plant domestication stage. However, the current strategy of fine gene positioning has the problems of missed detection, low resolution and low reliability. Summary of the invention

[0003] The present application provides a method for identifying functional regulatory intervals in the plant domestication process based on ACRs to solve the following technical problem: how to improve the comprehensiveness, accuracy and reliability of identifying relevant sites in the plant domestication process.

[0004] In a first aspect, the present application provides a method for identifying functional regulatory intervals in a plant domestication process based on ACRs, the method comprising:

[0005] Determine the genome of the species strain of the plant tissue sample to be tested and the genome of a closely related species related to the plant tissue sample to be tested;

[0006] Performing chromatin open sequencing on the plant tissue sample to be tested to obtain the plant ACRs of the plant tissue sample to be tested;

[0007] Using the first analysis software, the ACRs of the plant to be tested are aligned to the genome of the closely related species or the genome of the species strain, so as to obtain multiple groups of conservative ACRs and multiple groups of missing ACRs respectively;

[0008] Using a second analysis software, the ACRs of the plant to be tested are aligned to the genome of the closely related species or the genome of the species strain to obtain multiple groups of supplementary ACRs respectively;

[0009] Adding multiple groups of supplementary ACRs to multiple groups of conservative ACRs, and then counting the number of the supplemented conservative ACRs aligned to the genome of the closely related species or the genome of the species strain, to obtain multiple groups of regulatory interval data;

[0010] According to whether the ACRs of the plant to be tested in the plurality of groups of the regulatory interval data are successfully mapped to the genome of the species strain and unsuccessfully mapped to the genome of a closely related species, determining whether the regulatory interval data is a functional regulatory interval related to plant domestication of the plant tissue sample to be tested;

[0011] When the ACRs of the plant to be tested in multiple groups of the regulatory interval data are successfully mapped to the species strain genome but are not successfully mapped to the genome of a closely related species, the regulatory interval data are determined to be functional regulatory intervals related to plant domestication of the plant tissue sample to be tested.

[0012] Optionally, the method specifically includes:

[0013] Using the first analysis software, the plant ACRs to be tested are aligned to the genome of the closely related species to obtain first conserved ACRs with a first preset similarity and a first preset coverage and first missing ACRs with a second preset coverage, respectively;

[0014] Using the first analysis software, the plant ACRs to be tested are aligned to the species strain genome to obtain second conserved ACRs with a first preset similarity and a first preset coverage and second missing ACRs with a second preset coverage, respectively;

[0015] Using a second analysis software to align the plant ACRs to be tested to the genome of the closely related species, so as to obtain first supplementary ACRs having a second preset similarity and a third preset coverage, respectively;

[0016] Using the second analysis software to align the plant ACRs to be tested to the species strain genome to obtain second supplementary ACRs having a second preset similarity and a third preset coverage;

[0017] The first supplementary ACRs are supplemented to the first conservative ACRs, and then the number of the supplemented first conservative ACRs aligned to the genome of the closely related species is counted to obtain first regulatory interval data;

[0018] The second supplementary ACRs are supplemented to the second conservative ACRs, and then the number of the supplemented second conservative ACRs aligned to the genome of the species strain is counted to obtain second regulatory interval data;

[0019] According to the types of ACRs of the plant to be tested in the first regulatory interval data and the second regulatory interval data, it is determined whether the first regulatory interval data or the second regulatory interval data is a functional regulatory interval related to plant domestication of the plant tissue sample to be tested.

[0020] Optionally, the first preset similarity is ≥ 90%, and the first preset coverage is ≥ 90%.

[0021] Optionally, the second preset coverage is ≤10%.

[0022] Optionally, the second preset similarity is ≥98%, and the third preset coverage is ≥90%.

[0023] Optionally, judging whether the first regulatory interval data or the second regulatory interval data is a functional regulatory interval related to plant domestication of the plant tissue sample to be tested according to the types of ACRs of the plant to be tested in the first regulatory interval data and the second regulatory interval data comprises the steps of:

[0024] According to whether the ACRs of the plant to be tested in the first regulatory interval data and the second regulatory interval data are successfully aligned to the genome of the species strain and unsuccessfully aligned to the genome of a closely related species, determining whether the first regulatory interval data or the second regulatory interval data is a functional regulatory interval related to plant domestication of the plant tissue sample to be tested;

[0025] If the ACRs of the plant to be tested in the first regulatory interval data and the second regulatory interval data are successfully matched to the genome of the species strain but are not successfully matched to the genome of a closely related species, then the first regulatory interval data or the second regulatory interval data is determined to be a functional regulatory interval related to plant domestication of the plant tissue sample to be tested.

[0026] Optionally, the first analysis software includes minimap2 software.

[0027] Optionally, the second analysis software includes blast software.

[0028] Optionally, the plant tissue sample to be tested is a Poaceae plant tissue sample.

[0029] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0030] The embodiment of the present application provides a method for identifying functional regulatory intervals in the plant domestication process based on ACRs, the method comprising: determining the species strain genome of the plant tissue sample to be tested and the genome of a closely related species related to the plant tissue sample to be tested; performing chromatin open sequencing on the plant tissue sample to be tested to obtain the ACRs of the plant to be tested of the plant tissue sample to be tested; using a first analysis software to align the ACRs of the plant to be tested to the genome of the closely related species or the genome of the species strain to obtain multiple groups of conserved ACRs and multiple groups of missing ACRs respectively; using a second analysis software to align the ACRs of the plant to be tested to the genome of the closely related species or the genome of the species strain to obtain multiple groups of supplementary ACRs respectively. s; adding multiple groups of the supplementary ACRs to the multiple groups of the conservative ACRs, and then counting the number of the supplemented conservative ACRs mapped to the genome of the closely related species or the genome of the species strain, so as to obtain multiple groups of regulatory interval data; judging whether the regulatory interval data is a functional regulatory interval related to plant domestication of the plant tissue sample to be tested according to whether the ACRs of the plant to be tested in the multiple groups of the regulatory interval data are successfully mapped to the genome of the species strain and unsuccessfully mapped to the genome of the closely related species; when the ACRs of the plant to be tested in the multiple groups of the regulatory interval data are successfully mapped to the genome of the species strain and unsuccessfully mapped to the genome of the closely related species, the regulatory interval data is judged to be a functional regulatory interval related to plant domestication of the plant tissue sample to be tested. Compared with the traditional method of fine gene positioning, this method determines the ACRs of the plant tissue sample to be tested through chromatin open sequencing, and then uses an analysis software to compare the genome of the closely related species of the plant tissue sample to be tested with the genome of the species strain of the plant tissue sample to be tested multiple times. It can directly and accurately screen out the conserved ACRs sequences and missing ACRs sequences on the genome of the closely related species or on the genome of the species strain. In addition, this method also uses another analysis software to verify and supplement the results of the previous analysis software to obtain a supplementary ACRs sequence. The use of the supplementary ACRs sequence can avoid the functional At the same time, the number of different ACRs sequences aligned to the genomes of closely related species or species strains is counted to obtain a variety of different regulatory intervals. Then, according to the success or failure of the alignment of these regulatory intervals in the plant tissues to be tested and closely related species, it is possible to accurately judge whether the regulatory interval is the functional regulatory interval related to the domestication of the plant to be tested during the domestication process, thereby improving the reliability and visualization of the method. In addition, the method only requires the genome sequence files and ACRs information of the plant to be tested and closely related species, so the overall process of the method is simpler and easier to implement. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0032] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0033] Figure 1 A schematic diagram of a method flow chart for identifying functional regulatory intervals in a plant domestication process based on ACRs provided in an embodiment of the present application;

[0034] Figure 2 A detailed flow chart of a method for identifying functional regulatory intervals in a plant domestication process based on ACRs provided in an embodiment of the present application;

[0035] Figure 3 A detailed flow chart of a method for identifying functional regulatory intervals in a plant domestication process based on ACRs provided in an embodiment of the present application;

[0036] Figure 4 A schematic diagram of the actual process of a method for identifying functional regulatory intervals in a plant domestication process based on ACRs provided in an embodiment of the present application;

[0037] Figure 5 The ACRs characteristic diagram of the corn tissue sample provided in the embodiment of the present application, wherein: Figure 5 a is the ranking result of transcription factors enriched in ACRs. Figure 5 b is the chromatin accessibility map of gene ra2 (Zm00001d024523) and its surrounding 100 kb region. Figure 5 c is the gene expression of genes ra1 and ra2 in various maize tissue samples. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0039] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be understood as a rigid limitation on the scope of the present application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within the range; for example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which applies regardless of the range; in addition, whenever a numerical range is indicated in this document, it is meant to include any cited numbers (fractions or integers) within the indicated range.

[0040] In this article, the terms including "including" and the like mean "including but not limited to". Relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. "At least one" means one or more, and "plurality" means two or more; "at least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of single items or plural items; for example, "at least one of a, b, or c", or "at least one of a, b, and c" can all represent: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in this application can be purchased on the market or prepared by existing methods.

[0041] It should be noted that Accessible Regions (ACRs), as potential functional elements in plant genomes, play an important regulatory role in plant gene expression. Therefore, if ACRs can be used to identify regulatory-related sites in the plant domestication process, the comprehensiveness, accuracy and reliability of the identification process can be greatly improved.

[0042] Figure 1 A method for identifying functional regulatory intervals in the plant domestication process based on ACRs provided in an embodiment of the present application is exemplarily shown;

[0043] like Figure 1 As shown, the embodiment of the present application provides a method for identifying functional regulatory intervals in the plant domestication process based on ACRs, the method comprising:

[0044] S1. Determine the genome of the species strain of the plant tissue sample to be tested and the genome of the closely related species related to the plant tissue sample to be tested;

[0045] S2. performing chromatin open sequencing on the plant tissue sample to be tested to obtain the plant ACRs of the plant tissue sample to be tested;

[0046] S3. using the first analysis software to align the ACRs of the plant to be tested to the genome of the closely related species or the genome of the species strain, so as to obtain multiple groups of conservative ACRs and multiple groups of missing ACRs respectively;

[0047] S4. using a second analysis software to align the ACRs of the plant to be tested to the genome of the closely related species or the genome of the species strain to obtain multiple sets of supplementary ACRs;

[0048] S5. adding the plurality of groups of supplementary ACRs to the plurality of groups of conservative ACRs, and then counting the number of the supplemented conservative ACRs aligned to the genome of the closely related species or the genome of the species strain, to obtain a plurality of groups of regulatory interval data;

[0049] S6. judging whether the regulatory interval data is a functional regulatory interval related to plant domestication of the plant tissue sample to be tested, according to whether the ACRs of the plant to be tested in the plurality of groups of regulatory interval data are successfully mapped to the genome of the species strain and unsuccessfully mapped to the genome of a closely related species;

[0050] When the ACRs of the plant to be tested in multiple groups of the regulatory interval data are successfully mapped to the species strain genome but are not successfully mapped to the genome of a closely related species, the regulatory interval data are determined to be functional regulatory intervals related to plant domestication of the plant tissue sample to be tested.

[0051] It should be noted that the determination process of ACRs of the plant to be tested is: first, chromatin accessibility sequencing is performed on the plant tissue sample to be tested to obtain the chromatin accessibility test data (ATAC-seq data) of the plant tissue sample to be tested, and then the chromatin accessibility map of the plant tissue sample to be tested is obtained based on the ATAC-seq data.

[0052] Figure 2 A detailed flow chart of a method for identifying functional regulatory intervals in a plant domestication process based on ACRs provided in an embodiment of the present application is exemplarily shown;

[0053] Figure 4 The actual process diagram of a method for identifying functional regulatory intervals in a plant domestication process based on ACRs provided in an embodiment of the present application is exemplarily shown;

[0054] like Figure 2 and Figure 4As shown, in some optional implementations, the method specifically includes:

[0055] S301. Using the first analysis software, aligning the plant ACRs to be tested to the genome of the closely related species, to obtain first conserved ACRs with a first preset similarity and a first preset coverage and first missing ACRs with a second preset coverage, respectively;

[0056] S302. Using the first analysis software, aligning the plant ACRs to be tested to the species strain genome to obtain second conserved ACRs having a first preset similarity and a first preset coverage and second missing ACRs having a second preset coverage;

[0057] S401. Using a second analysis software, aligning the plant ACRs to be tested to the genome of the closely related species to obtain first supplementary ACRs having a second preset similarity and a third preset coverage, respectively;

[0058] S402. Using the second analysis software, aligning the plant ACRs to be tested to the species strain genome to obtain second supplementary ACRs having a second preset similarity and a third preset coverage;

[0059] S501. Supplementing the first supplementary ACRs to the first conservative ACRs, and then counting the number of the supplemented first conservative ACRs aligned to the genome of the closely related species to obtain first regulatory interval data;

[0060] S502. Supplementing the second supplementary ACRs to the second conservative ACRs, and then counting the number of the supplemented second conservative ACRs aligned to the genome of the species strain, to obtain second regulatory interval data;

[0061] S601. According to the types of the ACRs of the plant to be tested in the first regulatory interval data and the second regulatory interval data, determining whether the first regulatory interval data or the second regulatory interval data is a functional regulatory interval related to plant domestication of the plant tissue sample to be tested;

[0062] When the ACRs of the plant to be tested in multiple groups of the regulatory interval data are successfully mapped to the species strain genome but are not successfully mapped to the genome of a closely related species, the regulatory interval data are determined to be functional regulatory intervals related to plant domestication of the plant tissue sample to be tested.

[0063] In these embodiments, by using the first analysis software to analyze the genomes of closely related species and the genomes of species strains, and by using the second analysis software to analyze the genomes of closely related species and the genomes of species strains, conserved ACRs can be supplemented, thereby avoiding missing functional intervals of the tested plant tissue samples and improving the accuracy of identification.

[0064] In some optional implementations, the first preset similarity is ≥ 90%, and the first preset coverage is ≥ 90%;

[0065] In these embodiments, the first preset similarity can be ≥90%, and the first preset coverage can be ≥90%, and the first analysis software can be used to accurately screen out multiple conserved ACRs sequences from the aligned genomes of closely related species or species strains, thereby improving the accuracy and reliability of the aligned conserved ACRs sequences.

[0066] In some optional implementations, the second preset similarity is ≤10%.

[0067] In these embodiments, the second preset similarity can be ≤10%, and the first analysis software can be used to accurately screen out missing ACRs sequences from the compared genomes of closely related species or species strains, thereby improving the accuracy and reliability of the compared conserved ACRs sequences.

[0068] In some optional implementations, the second preset similarity is ≥ 98%, and the third preset coverage is ≥ 90%;

[0069] In these embodiments, the second preset similarity can be ≥98%, and the third preset coverage can be ≥90%, and the second analysis software can be used to accurately screen out supplementary ACRs sequences from the genomes of closely related species or species strains, thereby improving the accuracy and reliability of the method.

[0070] Figure 3 A detailed flow chart of a method for identifying functional regulatory intervals in a plant domestication process based on ACRs provided in an embodiment of the present application is exemplarily shown;

[0071] like Figure 3 As shown, in some optional embodiments, judging whether the first regulatory interval data or the second regulatory interval data is a functional regulatory interval related to plant domestication of the plant tissue sample to be tested according to the type of ACRs of the plant to be tested in the first regulatory interval data and the second regulatory interval data comprises the steps of:

[0072] S611. According to whether the ACRs of the plant to be tested in the first regulatory interval data and the second regulatory interval data are successfully mapped to the genome of the species strain and unsuccessfully mapped to the genome of a closely related species, determine whether the first regulatory interval data or the second regulatory interval data is a functional regulatory interval related to plant domestication of the plant tissue sample to be tested;

[0073] If the ACRs of the plant to be tested in the first regulatory interval data and the second regulatory interval data are successfully aligned to the genome of the species strain and are not successfully aligned to the genome of a closely related species, then the first regulatory interval data or the second regulatory interval data is determined to be a functional regulatory interval related to plant domestication of the plant tissue sample to be tested;

[0074] In these embodiments, a detailed analysis of the conserved ACRs after supplementation of ACRs is performed based on the criteria of successful mapping to the genome of the species strain and unsuccessful mapping to the genome of a closely related species, and thus the functional regulatory intervals related to plant domestication of the plant tissue sample to be tested can be accurately screened.

[0075] In some optional embodiments, the first analysis software includes minimap2 software.

[0076] In some optional embodiments, the second analysis software includes blast software.

[0077] In these embodiments, minimap2 software is used in combination with blast software to align the plant ACRs to be tested to the species strain genome and the genome of closely related species, so that conservative ACRs, missing ACRs and supplementary ACRs can be accurately screened, thereby improving the accuracy and reliability of the method.

[0078] In some optional embodiments, the plant tissue sample to be tested is a grass plant tissue sample;

[0079] In these embodiments, the plant tissue sample to be tested is a Poaceae plant tissue sample. Since ACRs are commonly present in plant genomes, this can enable the method to have good universality.

[0080] It should be noted that the tissue sample of the Poaceae plant only needs to know its own plant genome and the genome of closely related species in order to accurately identify the functional regulatory intervals related to plant domestication through the method provided in the embodiments of the present application.

[0081] The present application is further described below in conjunction with specific examples. The experimental methods in the following examples that do not specify specific conditions are usually measured according to industry standards; if there are no corresponding industry standards, they are carried out according to common international standards, conventional conditions, or conditions recommended by the manufacturer.

[0082] Example 1

[0083] like Figure 1 As shown, a method for first identifying functional regulatory intervals of plant domestication process based on ACRs comprises the steps of:

[0084] S1. Determine the genome of the species strain of corn and the genome of closely related species related to corn, wherein the closely related species include two Mexican teosinte species (Zea mays ssp. Parviglumis, Zea maysssp. mexicana) of the ancestral species of corn;

[0085] S2. performing chromatin access sequencing on 15 samples of each of the 12 maize tissues to be tested, so as to obtain 80,365 maize ACRs of the maize tissues to be tested;

[0086] S301. Use minimap2 software to align the maize ACRs to be tested to the genome of closely related species, so as to obtain the first conserved ACRs with similarity ≥ 90% and coverage ≥ 90% and the first missing ACRs with coverage ≤ 10% respectively;

[0087] S302. Use minimap2 to align the maize ACRs to be tested to the genome of the species strain to obtain the second conserved ACRs with similarity ≥ 90% and coverage ≥ 90% and the second missing ACRs with coverage ≤ 10% respectively;

[0088] S401. Use blast software to align the maize ACRs to be tested to the genome of closely related species to obtain first supplementary ACRs with similarity ≥ 98% and coverage ≥ 90% respectively;

[0089] S402. Using blast software, align the maize ACRs to be tested to the genome of the species strain to obtain second supplementary ACRs with similarity ≥ 98% and coverage ≥ 90% respectively;

[0090] S501. Supplementing the first supplementary ACRs to the first conservative ACRs, and then counting the number of the supplemented first conservative ACRs aligned to the genome of the closely related species to obtain the first regulatory interval data;

[0091] S502. Supplementing the second supplementary ACRs to the second conservative ACRs, and then counting the number of the supplemented second conservative ACRs aligned to the genome of the species strain to obtain the second regulatory interval data;

[0092] S611. According to whether the ACRs of the maize to be tested in the total 1213 first regulatory interval data and the second regulatory interval data are successfully mapped to the genome of the species strain and unsuccessfully mapped to the genome of the closely related species, determine whether the first regulatory interval data or the second regulatory interval data is a functional regulatory interval related to plant domestication of the plant tissue sample to be tested;

[0093] If the ACRs of the corn to be tested in the first regulatory interval data and the second regulatory interval data are successfully mapped to the genome of the species strain but are not successfully mapped to the genome of a closely related species, the first regulatory interval data or the second regulatory interval data is determined to be a functional regulatory interval related to plant domestication of the plant tissue sample to be tested.

[0094] Related experiments and effect data:

[0095] TB1 is an important domestication-related gene. There is an insertion site of the functional transposon Hopscotch 60kb upstream of it. This functional transposon can be used as a newly acquired regulatory element (enhancer) during the domestication process to regulate the expression of TB1. In addition, an ACRs was found in the enhancer region 60kb upstream of TB1 that is open in most tissues. This ACRs is highly conserved in 45 maize inbred lines (alignments ≥ 90%), but can only be aligned to less than 37% in the two teosinte genomes. This shows that the upstream functional transposon inserted into TB1 is the latest acquired during the maize domestication process, and this functional transposon can be fixed in almost all inbred lines.

[0096] Next, in order to identify similar newly acquired ACRs that were fixed in all maize inbred lines during maize domestication, the screening process was further stringent, and a total of 1,213 ACRs that were highly conserved in 43 or more maize inbred lines and did not exist in the two teosinte (alignments ≤ 10%) were screened out. This result was consistent with the expected assumption. In addition, these domestication-related ACRs were mainly enriched in TFBS related to inflorescence, such as RAMOSA and TCP (such as Figure 5 As shown in a, Figure 5 Different transcription factor families in a are represented by dots of different colors, and the enrichment P value of each transcription factor is estimated by AME software). It is worth noting that the ACRs related to maize domestication and the most enriched ACRs is RAMOSA1, which can control the branching structure of maize inflorescence and play an important role in inflorescence development.

[0097] Figure 5 The ACRs characteristic graph of the corn tissue sample provided in the embodiment of the present application is exemplarily shown;

[0098] The prior art discloses that the gene ra1 encoding RAMOSA1 is a domestication gene, and that the expression of ra1 is also regulated by the upstream gene ra2. The present application also identified domestication-related ACRs upstream of both ra2 and rel2. Figure 5 As shown in Figure 2, (1) the domestication-associated ACR 70 kb upstream of rel2 is a constitutive ACR that is open in all maize tissues; (2) the domestication-associated ACR 1 kb upstream of ra2 is only open in tassel and ear (e.g. Figure 5 c). According to Figure 5 The results suggest that the ACRs upstream of ra2 may serve as a potential cis-regulatory element selected by domestication, which not only regulates the specific high expression of ra2 in maize flower organs (such as Figure 5 As shown in b, a total of 11 RNA-seq samples were used to estimate the expression level. Figure 5 The red region in b indicates the domestication-related chromatin open region), which can also promote the significantly higher expression of ra2 in maize ear relative to teosinte. In general, the domestication-related ACRs upstream of ra2 ultimately regulate the expression of the domestication gene ra1 by regulating the expression of ra2.

[0099] The present application provides a method for identifying functional regulatory intervals in the plant domestication process based on ACRs, which combines high-throughput sequencing technology and bioinformatics analysis to accurately identify potential regulatory intervals in plant tissue samples to be tested, and further verify their regulatory effects on domestication gene expression. This method also helps to further study the molecular mechanism of corn domestication and provides important theoretical guidance and practical support for optimizing plant breeding technology and agricultural production.

[0100] In addition, the embodiment of the present application provides a method for identifying functional regulatory intervals in the plant domestication process based on ACRs. Based on the ubiquitous characteristics of ACRs in plant genomes, this method can also be applied to other important food crops, and provides a powerful tool for the identification of new regulatory intervals during the domestication of these crops, which helps to promote the improvement of food crop yield and quality.

[0101] The above is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. It will be apparent to those skilled in the art that various modifications to these embodiments are possible, and the general principles defined in the present application can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown in the present application, but will conform to the widest scope consistent with the principles and novel features applied for by the present application.

Claims

1. A method for identifying functional regulatory intervals in plant domestication based on ACRs, characterized in that: The method comprises: Determine the genome of the species strain of the plant tissue sample to be tested and the genome of a closely related species related to the plant tissue sample to be tested; Performing chromatin open sequencing on the plant tissue sample to be tested to obtain the plant ACRs of the plant tissue sample to be tested; Using the first analysis software, the ACRs of the plant to be tested are aligned to the genome of the closely related species or the genome of the species strain, so as to obtain multiple groups of conservative ACRs and multiple groups of missing ACRs respectively; Using the first analysis software, the plant ACRs to be tested are aligned to the genome of the closely related species to obtain first conserved ACRs with a first preset similarity and a first preset coverage and first missing ACRs with a second preset coverage, respectively; Using the first analysis software, the plant ACRs to be tested are aligned to the species strain genome to obtain second conserved ACRs with a first preset similarity and a first preset coverage and second missing ACRs with a second preset coverage, respectively; Using a second analysis software, the ACRs of the plant to be tested are aligned to the genome of the closely related species or the genome of the species strain to obtain multiple groups of supplementary ACRs respectively; Using the second analysis software, the plant ACRs to be tested are aligned to the genome of the closely related species to obtain first supplementary ACRs having a second preset similarity and a third preset coverage, respectively; Using the second analysis software to align the plant ACRs to be tested to the species strain genome to obtain second supplementary ACRs having a second preset similarity and a third preset coverage; Adding multiple groups of supplementary ACRs to multiple groups of conservative ACRs, and then counting the number of the supplemented conservative ACRs aligned to the genome of the closely related species or the genome of the species strain, to obtain multiple groups of regulatory interval data; According to whether the ACRs of the plant to be tested in the plurality of groups of the regulatory interval data are successfully mapped to the genome of the species strain and unsuccessfully mapped to the genome of a closely related species, determining whether the regulatory interval data is a functional regulatory interval related to plant domestication of the plant tissue sample to be tested; When the ACRs of the plant to be tested in multiple groups of the regulatory interval data are successfully mapped to the species strain genome but are not successfully mapped to the genome of a closely related species, the regulatory interval data are determined to be functional regulatory intervals related to plant domestication of the plant tissue sample to be tested.

2. The method according to claim 1, characterized in that The method specifically comprises: The first supplementary ACRs are supplemented to the first conservative ACRs, and then the number of the supplemented first conservative ACRs aligned to the genome of the closely related species is counted to obtain first regulatory interval data; The second supplementary ACRs are supplemented to the second conservative ACRs, and then the number of the supplemented second conservative ACRs aligned to the genome of the species strain is counted to obtain second regulatory interval data; According to the types of ACRs of the plant to be tested in the first regulatory interval data and the second regulatory interval data, it is determined whether the first regulatory interval data or the second regulatory interval data is a functional regulatory interval related to plant domestication of the plant tissue sample to be tested.

3. The method according to claim 2, characterized in that The first preset similarity is ≥ 90%, and the first preset coverage is ≥ 90%.

4. The method according to claim 2, characterized in that: The second preset coverage is ≤10%.

5. The method according to claim 2, characterized in that: The second preset similarity is ≥98%, and the third preset coverage is ≥90%.

6. The method according to claim 2, characterized in that The method of judging whether the first regulatory interval data or the second regulatory interval data is a functional regulatory interval related to plant domestication of the plant tissue sample to be tested according to the types of the ACRs of the plant to be tested in the first regulatory interval data and the second regulatory interval data comprises the steps of: According to whether the ACRs of the plant to be tested in the first regulatory interval data and the second regulatory interval data are successfully aligned to the genome of the species strain and unsuccessfully aligned to the genome of a closely related species, determining whether the first regulatory interval data or the second regulatory interval data is a functional regulatory interval related to plant domestication of the plant tissue sample to be tested; If the ACRs of the plant to be tested in the first regulatory interval data and the second regulatory interval data are successfully matched to the genome of the species strain but are not successfully matched to the genome of a closely related species, then the first regulatory interval data or the second regulatory interval data is determined to be a functional regulatory interval related to plant domestication of the plant tissue sample to be tested.

7. The method according to claim 1, characterized in that The first analysis software includes minimap2 software.

8. The method according to claim 1, characterized in that The second analysis software includes blast software.

9. The method according to claim 1, characterized in that: The plant tissue sample to be tested is a Gramineae plant tissue sample.

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