CN-121975862-A - Method for realizing whole genome scale mutation and germplasm creation of wheat by editing repeated sequences

Abstract

The invention discloses a method for realizing whole genome scale mutation and germplasm creation of wheat by editing repeated sequences. By screening CRISPR/Cas9 target sites with copy numbers higher than 20,000 in wheat genome, the tandem repeat sequences and the disperse repeat sequences are edited in a targeted manner, and genetable large-scale variation, chromosome structure variation and agronomic character variation of whole genome scale are induced in offspring plants. The invention can realize large-scale mutation with chromosome and subgenomic specificity by editing repeated sequences, and the created mutant library not only provides genetic variation mutant types different from the traditional physical and chemical mutagenesis methods, but also generates materials such as chromosome aneuploidy (such as monomer, tetrad and whole arm deletion) and the like, thereby providing unique genetic resources for wheat functional genomics research and germplasm improvement.

Inventors

• WANG WEI
• ZHANG YAO
• WANG XIUE
• SUN LI
• WANG HAIYAN

Assignees

• 南京农业大学

Dates

Publication Date

20260505

Application Date

20260129

Claims (8)

1. 1. A method for creating wheat germplasm by editing a repetitive sequence, comprising the steps of: (1) Screening a CRISPR/Cas9 target sequence with a copy number higher than 20,000 in a wheat genome, said target sequence targeting in tandem or dispersed repeats; (2) Constructing a virus-induced gene editing vector, wherein the vector contains a DNA sequence for encoding the sgRNA corresponding to the target sequence in the step (1); (3) Introducing the vector of step (2) into transgenic wheat expressing Cas9 protein through a viral delivery system to obtain double-strand DNA breaks in the whole genome range; (4) And screening mutants with agronomic trait variation and/or chromosome structure variation from offspring of the editing plant.
2. 2. The method according to claim 1, comprising the steps of: (1) Target screening, namely developing a computer script to scan a wheat field reference genome, screening a 23-mer sequence ending with NGG or beginning with CCN, reserving a site with the copy number of more than 20,000, and finally obtaining a plurality of full genome repeated sequence targets; (2) Constructing a virus-induced gene editing vector containing a DNA sequence encoding the sgRNA corresponding to the target sequence of step (1), (3) Delivering sgRNA to transgenic wheat Fielder-Cas9 expressing Cas9 by adopting barley streak mosaic virus BSMV, and realizing repeated sequence editing in a whole genome range in generation M 0 ; (4) Mutant screening and identification, namely selecting an M1 mutant with favorable agronomic characteristics in the field, and identifying the mutation condition of the whole gene and the chromosome structure mutation through fluorescence in situ hybridization, PCR labeling and whole genome resequencing.
3. 3. The method of claim 1 or 2, wherein the target sequence of step (1) is selected from the series-repeated sequence target REt shown in SEQ ID No.1, or the dispersed-repeated sequence targets REt5, REt and REt shown in SEQ ID nos. 2-4.
4. 4. The method of claim 3, wherein the target REt has a copy number in the genome of greater than 96,000, which targets the tandem repeat sequence set forth in SEQ ID No. 10.
5. 5. The method of claim 3, wherein target REt and target REt target the dispersed repetitive sequence shown in SEQ ID NO.11, target REt targets the dispersed repetitive sequence shown in SEQ ID NO.12, and target REt, target REt8 and target REt20 each have a copy number in the genome of greater than 100,000.
6. 6. The method of claim 2, wherein the chromosomal structural variation is selected from one or more of a short arm loss of chromosome 5B, a single chromosome elimination of chromosome 4B resulting in a single chromosome elimination of monomer-4B/a single chromosome 1A resulting in a single chromosome elimination of monomer-1A, a four-body-5A resulting from four-body formation of chromosome 5A, and a loss of weight of a long arm of chromosome 6A.
7. 7. A virus-induced gene editing vector comprising an sgRNA expression cassette corresponding to the target sequence of claim 3, said sgRNA expression cassette driven by a T7 promoter and capable of producing functional sgrnas by in vitro transcription.
8. 8. Use of the gene editing vector of claim 7 in creating wheat germplasm comprising chromosomal monomer, tetrad, whole arm deleted material and/or inducing genetic agronomic trait variation of wheat, including ear, plant height, tillering.

Description

Method for realizing whole genome scale mutation and germplasm creation of wheat by editing repeated sequences Technical Field The invention belongs to the field of crop molecular genetic breeding, and in particular relates to a method for large-scale editing of wheat genome-wide repeated sequences by using a CRISPR/Cas9 gene editing technology to create new germplasm, and application of heritable agronomic character variation and chromosome structure variation generated by the method in wheat breeding. Background Gene editing (GENE EDITING), also known as genome editing (genome editing), is a technique [1] that allows targeted editing to an organism's genome-specific target with precision. CRISPR/Cas9 is currently the most common gene editing system, and its working principle is mainly that Cas9 protein has a binding domain and two cleavage domains (HNH and RuvC nuclease domains, respectively, cleaving 2 strands of DNA) [3], after specific complementary pairing of guide RNA (sgRNA) and exogenous nucleic acid [4], cas9 recognizes PAM sequence (NGG) [5] and exerts accurate cleavage activity [6,7] for the 3 nucleotide position upstream, double strand break is generated, self repair of genome is induced, and error repair occurs during repair, thereby achieving result [2] of DNA fragment deletion, insertion, substitution or chromosomal rearrangement, etc., and finally finishing gene editing [8]. Wheat breeding programs require continuous input of genetic diversity to improve yield potential and stress resistance. Traditional mutagenesis methods such as EMS or irradiation treatment can produce extensive variation in genome but the mutation type and location are unpredictable. While CRISPR/Cas9 technology can achieve precise gene editing, most research has focused on a few target sites, the potential of which in whole genome-scale mutagenesis is not fully explored. The repeat sequence (REPETITIVE ELEMENTS, RES) accounts for about 90% of the wheat genome, including tandem arrangement of satellite DNA and dispersed transposons, and plays an important role in gene expression regulation and maintenance of genomic structure. However, repeated sequence editing may cause massive double-stranded DNA breaks (DSBs) that trigger lethal effects, limiting its use. The prior art lacks a method to edit repeated sequences safely and efficiently in crops to create usable variations for breeding. The virus-induced gene editing technology (reference) realizes gene editing in a mode independent of genetic transformation, can avoid plant regeneration difficulty caused by a large number of DSBs, and has the opportunity to realize the creation of genome-wide mutants of wheat through RE editing. The Long terminal repeat (Long TERMINAL REPEATS, LTRS) is a structural feature of a class of important retrotransposons (LTR-Retrotransposons) in the genome of eukaryotic organisms. They are located at both ends of the transposon and repeat in the same direction, and the genes encoding proteins required for reverse transcription and transposition are enclosed in the middle. In large genomic plants such as wheat, LTR retrotransposons are one of the main driving forces for genome expansion and evolution, with very large copy numbers, occupying a large proportion of the genome capacity. These sequences are not only key factors for structural variations (e.g., amplifications, rearrangements) in the genome, but their promoter activity also regulates the expression of neighboring genes, affecting agronomic traits [9]. Tandem repeat sequences are more a unique class of DNA sequences that are made up of multiple repeats of the same core unit. Such sequences are an important component of eukaryotic genomes, and in common wheat the total amount of all types of repeated sequences is even more than 80% of their genome. Tandem repeat sequences (e.g., specific oligonucleotide probe sequences) are chromosome location specific and can be directly observed on the chromosome by Fluorescence In Situ Hybridization (FISH) techniques, thus becoming an effective cytological marker [10,11] for identifying the chromosome, tracking its structure and rearrangement (e.g., translocation, inversion). The two types of repeated sequences contain 23bp sequences with CCN beginning and NGG ending, and can be used as targets for gene editing, so that Cas9 can target a large number of chromosome positions by using one target to perform interrupt repair, thereby realizing creation of chromosome fragment insertion/deletion mutant, and providing a basis for creating mutation on the whole genome scale by utilizing VIGE technology and screening wheat germplasm with improved agronomic characteristics. Disclosure of Invention The invention aims to provide a method for creating new germplasm by editing wheat whole genome repetitive sequences, overcomes the defects of poor targeting of traditional mutagenesis and limited CRISPR/Cas9 editing sites, realizes large-scale mutation with subgenom