Method for improving genetic recombination frequency of rice

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for improving rice genetic recombination frequency. The HEI10 gene is over-expressed in rice, and a rice plant or a rice variety with improved genetic recombination frequency is obtained through screening. According to the invention, a scheme for improving the genetic recombination frequency is established in rice through a transgenosis method instead of a knockout method. Transgenic components of the gene can be screened and removed in offspring through genetic isolation, so that the genetic recombination frequency of the offspring is recovered to a normal level, and the stability of genome and maturing rate is facilitated. And the overexpression of the HEI10 basically does not influence the normal growth and the maturing rate, so that the method has practical significance.

Description

A method to increase the frequency of genetic recombination in rice Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for increasing the frequency of genetic recombination in rice. Background Technology

[0002] Genetic recombination, the core event of the third law of genetics, is the theoretical cornerstone of hybridization breeding. Since Morgan revealed the third law of genetics in the early 20th century, genetic recombination, as a major source of genetic diversity, has been widely applied in crop breeding practices. Through hybridization breeding techniques, breeders have successfully achieved gene recombination and aggregation of superior traits among different varieties or subspecies, cultivating a large number of high-yielding, high-quality, and disease-resistant crop varieties, providing an important guarantee for my country's food security. However, the inherently low recombination frequency in plants (usually only 1-2 recombination events per chromosome) severely limits the probability of generating superior gene combinations. Due to insufficient gene recombination among germplasm resources, it is difficult to break the tight linkage between superior and unfavorable genes, making the breeding of new rice varieties increasingly difficult, and the bottleneck effect of traditional breeding is becoming more and more significant, seriously hindering the further improvement of rice yield and quality.

[0003] Therefore, improving breeding efficiency, expanding genetic population diversity, and cultivating breakthrough new varieties have become major challenges for agricultural production in my country. Theoretical research shows that increasing the frequency of genetic recombination will exponentially increase the genetic diversity of the breeding population, thereby significantly improving breeding efficiency. Taking rice (2n=24) as an example, if the recombination frequency of each chromosome is increased to twice the original level, theoretically, genetic diversity can increase by approximately 4096 times (2^n=2^n). 12 Therefore, increasing the frequency of genetic recombination and genetic diversity in crops, and accelerating the breeding of superior varieties, is the key to breaking through the bottleneck of hybridization breeding and has significant economic and social benefits.

[0004] Current research on how to increase the frequency of genetic recombination mainly focuses on Arabidopsis thaliana. For example, in Arabidopsis, increasing the copy number of the cross-linking E3 ligase gene HEI10 increased its expression level, resulting in a nearly two-fold increase in recombination frequency (Ziolkowski PA, Underwood CJ, Lambing C, Martinez-Garcia M, Lawrence EJ, Ziolkowska L, Griffin C, Choi K, Franklin FC, Martienssen RA, Henderson IR. Natural variation and dosage of the HEI10 meiotic E3 ligase control Arabidopsis crossover recombination. Genes Dev. 2017 Feb 1;31(3):306-317.1); and knocking out Arabidopsis class II recombination genes recq4, fancm, and figl1 increased the recombination frequency by up to 7.8 times (Crismani W, Girard C, Froger N, Pradillo M, Santos JL, Chelysheva L, Copenhaver GP, Horlow C, Mercier R. FANCM limits). meiotic crossovers. Science.2012 Jun 22;336(6088):1588-90.; Séguéla-Arnaud M, Crismani W, Larchevêque C,Mazel J, Froger N, Choinard S, Lemhemdi A, Macaisne N, Van Leene J, GevaertK, De Jaeger G, Chelysheva L, Mercier R. Multiple limit mechanisms meioticcrossovers: TOP3α and two BLM homologs antagonize crossovers in parallel toFANCM. Proc Natl Acad Sci US A. 2015 Apr 14;112(15):4713-8.Girard C, Chelysheva L, Choinard S, Froger N, Macaisne N, Lemhemdi A, Mazel J, Crismani W, Mercier R. AAA-ATPase FIDGETIN-LIKE 1 and Helicase FANCM Antagonize Meiotic Crossovers by Distinct Mechanisms. PLoS Genet. 2015 Jul 10;11(7):e1005369.). In rice, partial mutations in the synaptonemal complex protein ZEP1 can increase the genetic recombination frequency by an average of about 1.8 times that of the wild type (Wang K, Wang C, Liu Q, Liu W, Fu Y. Increasing the Genetic Recombination Frequency by Partial Loss of Function of the Synaptonemal Complex in Rice. Mol Plant. 2015 Aug;8(8):1295-8.). In addition, the increased frequency of genetic rearrangements often leads to plant sterility, which seriously affects the seed setting rate. The ZEP1 gene was edited in hybrid rice varieties using genome editing technology. The resulting ZEP1 knockout mutant, when used as the maternal parent in hybridization, showed an approximately 1.5-fold increased frequency of genetic recombination. However, compared to the wild type, it exhibited complete pollen sterility (Liu C, Cao Y, Hua Y, Du G, Liu Q, Wei X, Sun T, Lin J, Wu M, Cheng Z, Wang K. Concurrent Disruption of Genetic Interference and Increase of Genetic Recombination Frequency in Hybrid Rice Using CRISPR / Cas9. Front Plant Sci. 2021 Oct 1;12:757152.).

[0005] Currently, there are no effective methods for increasing the frequency of genetic recombination in crops that can be applied to breeding. Existing research on genetic recombination mechanisms mainly focuses on Arabidopsis thaliana, with fewer research results on crops, and the results from Arabidopsis thaliana studies may not be applicable to crops. Existing research mainly aims to increase the frequency of genetic recombination by obtaining mutants (including knockout using gene editing technology). This results in the offspring retaining the relevant mutations, leading to a consistently high frequency of genetic recombination, which can easily cause poor genetic stability and decreased seed setting rate. In the past, increasing the frequency of genetic recombination by mutating key genes often resulted in sterile plants, and the increased frequency of genetic recombination could only be detected at the cellular level, which could not be directly applied to breeding. Summary of the Invention

[0006] This invention establishes a method for driving overexpression of the HEI10 gene through a transgenic strong promoter in hybrid rice without affecting fertility and increasing the frequency of genetic recombination. This will significantly increase the genetic diversity of rice breeding populations and improve breeding efficiency. The transgenic components can be eliminated in offspring through genetic segregation and selection, thereby restoring the frequency of genetic recombination in offspring to normal levels, which is beneficial to the stability of the genome and seed setting rate.

[0007] This invention provides a method for increasing the frequency of genetic recombination in rice, which involves overexpressing the HEI10 gene in rice and screening for rice plants or varieties with increased genetic recombination frequency.

[0008] Specifically, the overexpression is performed via a constitutive promoter, preferably such as the ACTIN promoter, the Ubi promoter, or the HEI10 autopromoter.

[0009] The overexpression is achieved by gene introduction, such as Agrobacterium-mediated transformation or gene gun method; or by hybridization to achieve overexpression of the HEI10 gene in the target rice.

[0010] Specifically, the amino acid sequence encoded by the HEI10 gene is as shown in SEQ ID No: 1 or has an amino acid sequence with more than 90%, more than 95%, preferably more than 98%, more preferably more than 99% identity with it, and still maintains the same function; more specifically, its nucleotide sequence is as follows: as shown in SEQ ID No: 1 or its degenerate sequence, or has a nucleotide sequence with more than 90%, more than 95%, preferably more than 98%, more preferably more than 99% identity with it, and still maintains the same function; preferably, it is a homologous gene derived from the HEI10 gene of rice.

[0011] More preferably, rice varieties with multiple copies of the HEI10 gene introduced are screened, such as 2 copies, 3 copies, etc.

[0012] Specifically, the rice is either hybrid rice or conventional rice. Furthermore, if the method of this invention transforms hybrid rice, the offspring can directly obtain the material with increased recombination; if the method transforms conventional rice, hybrid rice can be obtained through hybridization, and the offspring can also obtain the material with increased recombination. This invention also provides the application of the HEI10 gene in increasing the frequency of genetic recombination in rice, wherein the application is achieved by overexpressing the HEI10 gene in rice.

[0013] Specifically, the amino acid sequence encoded by the HEI10 gene is as shown in SEQ ID No: 1 or has an amino acid sequence with more than 90%, more than 95%, preferably more than 98%, more preferably more than 99% identity with it, and still maintains the same function; more specifically, its nucleotide sequence is as follows: as shown in SEQ ID No: 1 or its degenerate sequence, or has a nucleotide sequence with more than 90%, more than 95%, preferably more than 98%, more preferably more than 99% identity with it, and still maintains the same function; preferably, it is a homologous gene derived from the HEI10 gene of rice.

[0014] The present invention also provides the application of the method or HEI10 gene in rice breeding, wherein it is used to increase the recombination frequency to enable mutants for breeding screening; optionally, the transgenic component of the HEI10 gene is removed in the offspring through genetic segregation and qPCR transgenic component screening, so that the genetic recombination frequency of the offspring is restored to a normal level.

[0015] Specifically, the rice mentioned is either hybrid rice or conventional rice.

[0016] This invention establishes a scheme to increase the frequency of genetic recombination in rice through transgenic methods rather than knockout. The transgenic components can be eliminated in offspring through genetic segregation and selection, thereby restoring the frequency of genetic recombination in offspring to normal levels, which is beneficial to the stability of the genome and seed setting rate. Furthermore, overexpression of HEI10 has virtually no impact on normal growth and seed setting rate, making it of practical significance. Attached Figure Description

[0017] Figure 1. Structure of the pC1300-Actin:HEI10 vector.

[0018] Figure 2. Comparison of agronomic traits between transgenic lines and wild-type CY84.

[0019] Figure 3. Statistical analysis of seed setting rate of transgenic lines and wild-type CY84.

[0020] Figure 4. Statistics on the frequency of chromosome recombination in transgenic lines.

[0021] Figure 5. Statistical analysis of recombination frequency for different transgenic HEI10 copy numbers in F3 generation. Detailed Implementation

[0022] The present invention will be described below through specific embodiments to better understand the present invention, but this does not constitute a limitation thereof.

[0023] Example 1: Construction of HEI10 overexpression vector and acquisition of transgenic plants. HEI10 encodes E3 ubiquitin ligase, a key member of the ZMM protein family (a class of factors that ensure the correct formation of crossover) which is conserved from yeast, humans to plants. The homologous gene HEI10 (LOC_Os02g13810) in rice was identified through protein homology sequence alignment. Among them, the HEI10 amino acid sequence (SEQ ID No: 2): MKCNACWRELEGQAVSTTCGHLLCTEDAKKILSNDAACPICDQVLSKSHMRPVDTNPNDDWTNMSMAGVSPQILMKSAYRSVMFYIGQKELEMQYKMNRIVGQCRQKCELMQAKFTEKLEEVHTAYQKMAKKCQLMEQEVENLSRDKQE LQEKFAEKSRQKRKLDEMYDQLRSEYESAKRSAIQPANNYFPRAQPDLFSGVPNIMDSSDPLRQGLAGLPETPGRRDEGWAPPPRQRRSTSGPFELSAGSPAHNAAPPVDIRPRQPARPVFGTAMNNTSAALRNMIISPVKRPQLSRNRPHMFT*.

[0024] Primers were designed to amplify the promoter sequence of the housekeeping gene ACTIN (SEQ ID No: 1) and the rice HEI10 genome sequence, including the 5'UTR and 3'UTR sequences (SEQ ID No: 3). Using the Novizan ClonExpress Ultra One Step Cloning Kit V3 (catalog number C117), these two fragments were ligated into the binary vector pCAMBIA1300 (pC1300) digested with KpnI and HindIII via homologous recombination. The ligation product was then transformed into *E. coli* DH5α, and clones containing the correct insert were screened. The correctness of the vector was verified by Sanger sequencing, and the pC1300-Actin:HEI10 vector was constructed (Figure 1).

[0025] The pC1300-Actin:HEI10 vector was transformed into the indica-japonica hybrid rice variety Chunyou 84 (CY84) using the Agrobacterium tumefaciens transformation method to obtain transgenic rice plants Actin:HEI10.

[0026] Example 2, Seed setting rate and genetic recombination frequency detection Reference: Manipulation of genetic recombination by editing the transcriptional regulatory regions of a meiotic gene in hybrid rice. Plant Commun. 2023 Mar 13;4(2):100474. doi: 10.1016 / j.xplc.2022.100474. Epub 2022 Nov 10. Based on the whole genome sequence differences between the parents 16A and C84 of Chunyou 84, 130 single nucleotide polymorphism (SNP) markers distributed on 12 pairs of chromosomes were designed. Combined with multiplex PCR and second-generation high-throughput sequencing technology, they were used to analyze the genetic recombination frequency. These SNP sites are homozygous in the parents (e.g., parent 1 is A / A, parent 2 is T / T). A segregating population of parental hybrids was constructed, and whole genome SNP typing was performed on each plant in the population to obtain the genotype of each plant at each SNP site. The genetic material of offspring individuals is formed by the recombination and splicing of chromosome segments from both parents. If an individual shows a genotype transition from parental type 1 to parental type 2 at two adjacent SNP loci, then a genetic recombination event is considered to have occurred between these two loci.

[0027] Thirty Actin:HEI10 transgenic lines were obtained by transforming CY84 with Agrobacterium. Three independent T0 generation lines were selected for seed setting rate statistics and plant phenotypic observation. First, the agronomic traits of the transgenic lines and wild-type CY84 were observed. It was found that during the vegetative growth stage, the transgenic lines did not show any difference from the wild type (Figure 2). Second, the seed setting rate of the transgenic lines was statistically analyzed, and the results showed no significant difference compared with wild-type CY84 (Figure 3). Third, recombination frequency was detected in the 30 transgenic lines. DNA was extracted from the harvested T1 generation seedlings (F2 population) using the CTAB method. 130 SNP molecular markers were detected in the target DNA. Multiplex PCR with 130 primer pairs was performed on each DNA sample. After library construction, next-generation sequencing (performed by Geneplus Technology Co., Ltd.) was performed. The sequencing results were analyzed by computer to split the samples and perform genotyping. The genetic recombination frequency of the T0 transgenic plants was calculated.

[0028] The three wild-type CY84 lines were statistically analyzed. The F2 population of CY84-1 consisted of 40 plants and a total of 769 recombination exchanges occurred. The average recombination per chromosome was 769 ÷ 40 (number of plants) ÷ 12 (number of chromosomes) ÷ 2 (diploid) = 0.80 times. The F2 population of CY84-2 consisted of 40 plants and a total of 797 recombination exchanges occurred. The average recombination per chromosome was 0.83 times. The F2 population of CY84-3 consisted of 49 plants and a total of 966 recombination exchanges occurred. The average recombination per chromosome was 0.82 times. The three Actin:HEI10 lines were analyzed. The F2 population of Actin:HEI10-1 consisted of 50 lines, with a total of 2010 recombination exchanges, averaging 1.68 recombinations per chromosome. The F2 population of Actin:HEI10-2 consisted of 90 lines, with a total of 3453 recombination exchanges, averaging 1.60 recombinations per chromosome. The F2 population of Actin:HEI10-3 consisted of 46 lines, with a total of 1715 recombination exchanges, averaging 1.55 recombinations per chromosome (Table 1).

[0029] The three CY84 lines had an average of 0.82 recombinations per chromosome, while the three Actin:HEI10 lines had an average of 1.61 recombinations per chromosome, representing a 1.96-fold increase, which was highly significant. Comparing the two lines with the highest recombination rates, CY84-2 had an average of 0.83 recombinations per chromosome, while Actin:HEI10-1 had an average of 1.68 recombinations per chromosome, representing a 2.02-fold increase, which was also highly significant (Figure 4).

[0030] Table 1

[0031] Example 3: Identification and Separation of Transgenic Copy Numbers. Referring to the reference (Yang Litao, Zhao Zhihui, Ding Jiayu, Zhang Chengmei, Jia Junwei, Zhang Dabing, Analysis of Exogenous Gene Copy Number in Transgenic Rice Using Real-Time Quantitative PCR, Chinese Journal of Food Hygiene, 2005, Vol. 17, No. 2), the transgenic copy number of DNA samples from T1 (F2) generation plants was identified using qPCR. BADH2 was used as the internal reference gene, and the exogenous transgenic component hygromycin gene (HYG) was the detection target. Primer sequences are shown in Table 2. The HYG / BADH2 qPCR amplification ratio determined whether the transgenic copy number consisted of two copies (homozygous (TT), one copy (heterozygous (Tt), or no copy (tt)). Among 50 T1 generation plants, 13 TT plants, 27 Tt plants, and 10 tt plants were identified. The results are shown in Table 3.

[0032] Table 2

[0033] Table 3

[0034] Example 4: Detection of Genetic Recombination Frequency in T1 Plants with and without Transgenic Components. Based on the results of Example 3, plants with homozygous (TT), heterozygous (Tt), and no transgenic component (tt) transgenic copies were selected from 50 T1 (F2) plants for genetic recombination frequency detection. Since recombination and independent assortment have already occurred in the T1 generation, not every chromosome is in a completely heterozygous state. Therefore, based on the genotyping results of 130 pairs of SNP markers, chromosomes 3, 8, and 11, whose genotypes are mostly heterozygous, were selected to detect their recombination frequency. A total of 3 tt plants, 3 Tt plants, and 3 TT plants with mostly heterozygous chromosome 3 were selected, and chromosomes 8 and 11 were identical. The selected T1 generation plants were propagated, and the seeds were germinated in a culture medium. DNA was extracted using the CTAB method. Multiplex PCR was performed on the target DNA using 52 pairs of SNP markers (numbers 3, 8, and 11). After library construction, next-generation sequencing was performed (completed by Geneplus Technology Co., Ltd.). The sequencing results were split and analyzed by computer to split the samples and perform genotyping to detect the number of chromosome crossovers.

[0035] The test results showed that on chromosome 3, the tt plants with the transgenic component removed had an average recombination of 1.01 times, which was not significantly higher than the wild type's 1.14 times. The recombination frequency returned to the level of the wild type. The homozygous TT plants with two transgenic copies had an average recombination of 2.49 times, and the heterozygous Tt plants with one transgenic copy had an average recombination of 2.06 times.

[0036] On chromosome 8, the average recombination frequency of tt plants without transgenic components was 0.91 times, which was not significantly higher than the wild type's 0.88 times, and the recombination frequency returned to the wild type level. Homozygous TT plants with two transgenic copies had an average recombination frequency of 1.79 times, and heterozygous Tt plants with one transgenic copy had an average recombination frequency of 1.45 times.

[0037] On chromosome 11, tt plants with the transgene removed averaged 0.70 recombinations, which was not significantly higher than the wild-type 0.74 recombinations, and the recombination frequency returned to the wild-type level. Homozygous TT plants with two transgene copies averaged 1.3 recombinations, and heterozygous Tt plants with one transgene copy averaged 1.08 recombinations. The recombination frequency of all tt plants with the transgene removed returned to the wild-type level. The recombination frequency of Tt and TT transgene plants containing the transgene was significantly higher than that of the wild-type, and TT plants had a higher recombination frequency than Tt plants. This indicates that the more HEI10 copies and the higher the expression level, the higher the genetic recombination frequency (Figure 5).

Claims

1. A method for increasing the frequency of genetic recombination in rice, characterized in that, Overexpression of the HEI10 gene in rice was used to screen for rice plants or varieties with increased genetic recombination frequency.

2. The method as described in claim 1, characterized in that, The overexpression is achieved through constitutive promoters such as the ACTIN promoter, the Ubi promoter, and the HEI10 autopromoter.

3. The method as described in claim 1, characterized in that, The overexpression is achieved by gene introduction, such as Agrobacterium-mediated transformation or gene gun method; or by hybridization to achieve overexpression of the HEI10 gene in the target rice.

4. The method as described in claim 1, characterized in that, The amino acid sequence encoded by the HEI10 gene is as shown in SEQ ID No: 1 or has an amino acid sequence with more than 90%, more than 95%, preferably more than 98%, more preferably more than 99% identity with it, and still maintains the same function; more specifically, its nucleotide sequence is as follows: as shown in SEQ ID No: 1 or its degenerate sequence, or has a nucleotide sequence with more than 90%, more than 95%, preferably more than 98%, more preferably more than 99% identity with it, and still maintains the same function; preferably, it is a homologous gene derived from the HEI10 gene of rice.

5. The method as described in claim 1, characterized in that, Rice varieties with multiple copies of the HEI10 gene were screened.

6. The method according to any one of claims 1 to 4, characterized in that, The rice mentioned is either hybrid rice or conventional rice.

7. Application of the HEI10 gene in increasing the frequency of genetic recombination in rice, wherein the application is achieved by overexpressing the HEI10 gene in rice.

8. The application as described in claim 7, characterized in that, The amino acid sequence encoded by the HEI10 gene is as shown in SEQ ID No: 1 or has an amino acid sequence with more than 90%, more than 95%, preferably more than 98%, more preferably more than 99% identity with it, and still maintains the same function; more specifically, its nucleotide sequence is as follows: as shown in SEQ ID No: 1 or its degenerate sequence, or has a nucleotide sequence with more than 90%, more than 95%, preferably more than 98%, more preferably more than 99% identity with it, and still maintains the same function; preferably, it is a homologous gene derived from the HEI10 gene of rice.

9. The method of any one of claims 1 to 6 or the application of the HEI10 gene in rice breeding, wherein the method is used to increase the recombination frequency to enable mutants for breeding screening; optionally, the transgenic component of the HEI10 gene is removed in the offspring through genetic segregation and qPCR transgenic component screening, so that the genetic recombination frequency of the offspring returns to a normal level.

10. The application as described in claim 9, characterized in that, The rice mentioned is either hybrid rice or conventional rice.

Images