Application of lettuce LsGAI1 gene in lettuce growth character screening breeding

Abstract

The invention belongs to the technical field of plant genetic engineering and breeding, and particularly relates to application of a lettuce LsGAI1 gene in lettuce growth trait screening breeding. 1, and the protein coded by the LsGAI1 gene is shown as SEQ ID NO. 2. The LsGAI1 gene is knocked out from the asparagus lettuce through a CRISPR-Cas9 technology, so that internode elongation and plant height increase can be caused; and overexpression of the LsGAI1 enables internode shortening and plant height reduction. The cloning and functional verification of the LsGAI1 gene provide important gene resources and theoretical basis for plant type improvement and high-yield breeding of asparagus lettuce.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering and molecular breeding, specifically to the application of the LsGAI1 gene in lettuce growth trait screening and breeding. Background Technology

[0002] Lettuce (Lactuca sativa) is an important vegetable crop worldwide, and stem lettuce has significant economic value due to its swollen and juicy stems. Internode length is a key trait affecting the yield and marketability of stem lettuce. Currently, research on the genetic mechanisms of lettuce internode length is limited, and effective molecular breeding targets are lacking.

[0003] The DELLA protein in the gibberellin (GA) signaling pathway plays a crucial role in plant growth and development. However, the function of the DELLA protein in lettuce stem development remains unclear. This invention, through map-based cloning and functional verification, identifies for the first time the functional role of the LsGAI1 gene in regulating lettuce stem node length and plant height, providing a new gene resource for lettuce breeding. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the LsGAI1 gene in lettuce growth trait screening and breeding, wherein the growth traits include lettuce stem internode length and / or plant height, the protein encoded by the LsGAI1 gene is shown in SEQ ID NO.2, and the protein encoded by its allele lsgai1 is shown in SEQ ID NO.4.

[0005] To achieve the above objectives, the present invention adopts the following technical measures:

[0006] This invention, through map-based cloning and BSR-seq analysis, identified a QTL locus on chromosome 8 of lettuce associated with stem node length and / or plant height, and cloned its candidate gene, LsGAI1. This gene encodes a DELLA protein, and its mutant lsgai1, due to retrotransposon insertion, results in protein truncation and downregulation, thereby causing increased internode elongation and plant height. The CDS sequence of the LsGAI1 gene is shown in SEQ ID NO.1, and the encoded protein is shown in SEQ ID NO.2; the CDS sequence of its allele lsgai1 is shown in SEQ ID NO.3, and the encoded protein is shown in SEQ ID NO.4.

[0007] The scope of protection of this invention includes:

[0008] The application of the LsGAI1 gene in regulating lettuce growth traits, including lettuce stem internode length and / or plant height, wherein the protein encoded by the LsGAI1 gene is shown in SEQ ID NO.2, and the protein encoded by its allele lsgai1 is shown in SEQ ID NO.4.

[0009] The applications described above, specifically:

[0010] Application of increasing the expression level of the LsGAI1 gene in lettuce to reduce stem node length and / or plant height.

[0011] The application described above involves introducing a substance that increases the expression level of the LsGAI1 gene in lettuce into lettuce. The substance is an expression cassette containing the LsGAI1 gene, a recombinant vector, or a recombinant microorganism.

[0012] The application of knocking out or inhibiting the expression of the LsGAI1 gene in increasing the length of lettuce stem nodes and / or increasing plant height;

[0013] The application described above involves introducing a substance that reduces or eliminates the expression of the LsGAI1 gene in lettuce. This substance is an expression cassette, recombinant vector, or recombinant microorganism that reduces the expression of the LsGAI1 gene.

[0014] The above-described applications involve knockout using homologous recombination or CRISPR gene editing methods. The knockout gene translates into a protein that has no original function or cannot be translated into a protein.

[0015] The above-described applications employ CRISPR-Cas9 gene editing, with sgRNA sequences of 5'-GACGATCTACAAAGGATCCC-3' and 5'-CAGGCTTTGGCTCTTCGACC-3'.

[0016] The above-described applications involve inhibition using antisense RNA technology or interfering RNA technology.

[0017] The application of reagents for detecting the genes encoding the protein shown in SEQ ID NO.2 or SEQ ID NO.4 in the screening breeding of lettuce growth traits, wherein the breeding screening includes screening for lettuce stem node length and / or plant height.

[0018] In the above-described applications, preferably, the LsGAI1 gene is shown in SEQ ID NO.1 and the lsgai1 gene is shown in SEQ ID NO.3.

[0019] The application of a molecular marker primer combination in the screening and breeding of lettuce growth traits, wherein the primer combination is: GAI1F: 5'-CTCGCGATTTCTGTTTTTCC-3', TEF: 5'-CGTTATAACTCAGACTCAACGTC-3', GAI1R: 5'-TGGCTCAATTGGCTGATACA-3', and the growth traits include lettuce stem node length and / or plant height.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] This invention is the first to clone and functionally validate the key gene LsGAI1 related to lettuce breeding. This gene is associated with traits such as stem internode length and plant height, and its function was verified through CRISPR / Cas9 knockout and overexpression experiments. This invention also developed molecular markers for detecting LsGAI1 allelic variations, which can be used for marker-assisted selection in high-yield and disease-resistant lettuce breeding. This invention provides new gene resources for high-yield and high-quality stem lettuce breeding. Attached Figure Description

[0022] Figure 1 Location and candidate gene structure of the SSS6 site;

[0023] Where: a represents the genotypes of the F4 and F5 exchanged single plants on chromosome 8, blue represents chromosomes from the parent "Red Tip Leaf", and gray represents chromosomes from the parent "Six-Jin Stick"; b represents the gene structure of LsGAI1 and lsgai1, and the red box represents the retrotransposon.

[0024] Figure 2 A schematic diagram of phenotypic analysis of LsGAI1 knockout and LsGAI1 overexpression in CRISPR / Cas9;

[0025] a. Phenotype of LsGAI1 knockout mutant; b and c. Statistical analysis of mean internode length, stem diameter, height, and harvest weight of LsGAI1 knockout mutant and recipient.

[0026] d. Phenotype of LsGAI1 overexpression families (OX#); e. LsGAI1 expression levels in the three overexpression lines; f. Mean intersegmental length and height of LsGAI1 in LsGAI1 overexpression families and receptors were statistically analyzed using Student's t-test. * p < 0.05, * p < 0.01, *** p < 0.001.

[0027] Figure 3 This is a schematic diagram illustrating the screening of hybrid offspring using the LsGAI1-specific molecular marker.

[0028] Figure 4 Gene mutation status of the homozygous knockout line (CR#287) with deletion mutation. Detailed Implementation

[0029] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field; unless otherwise specified, the reagents or materials described are all from commercial sources.

[0030] Example 1:

[0031] Acquisition and genetic mapping of the LsGAI1 gene in lettuce

[0032] To elucidate the genetic mechanism of stem length trait in lettuce, this invention selected commercially available stem-growing lettuce variety LJB (short internodes, large stem diameter) and red-tipped leaf lettuce (HJY (long internodes, small stem diameter)) for hybridization to obtain F1 generation hybrids. After self-pollination of the F1 generation, an F2 segregating population was constructed. In the F2 population, stem length exhibited a continuous distribution, indicating that it is a quantitative trait. From this population, 15 individuals with the shortest internodes and 13 individuals with the longest internodes were selected to construct DNA pools (large stem pool and small stem pool), respectively.

[0033] Transcriptome sequencing was performed on two pooled samples using the BSR-seq (Bulked Segregant Analysis RNA-seq) method. Sequencing data were aligned to the lettuce reference genome (Lactuca sativa v8.0), and the Δ(SNP-index) value for each SNP locus was calculated. Analysis revealed a significant major-effect QTL controlling stem segment length on chromosome 8, which was named SSS6 (Succulent Swollen Stem 6).

[0034] To locate the SSS6 locus, we selected a heterozygous plant (A3) with the SSS6 locus from the F2 segregating population of the "Liujinbang" and "Hongjianye" hybrids. This plant was self-crossed twice, maintaining SSS6 heterozygosity in each generation, resulting in an F4 family with segregation at the SSS6 locus. Within the F4 family, we found a single exchanged plant. Resequencing results showed that this plant was heterozygous between 58.08 and 62.68 on chromosome 8. F5 plants from this family exhibited segregation in internode length and plant height. Therefore, we preliminarily located the SSS6 locus at 4.6 Mbs. Simultaneously, a series of markers (61.03, 61.25, 61.51) were developed to scan F5 generation plants within the initially located heterozygous region. One plant was found to have exchanged between 61.03 and 61.25. F6 generation plants planted with this exchanged individual showed segregation in internode length and plant height. Therefore, the candidate gene was ultimately located within the 61,032,329-62,684,537 bp region on chromosome 8, with a physical distance of approximately 1.65 Mb. Gene annotation of this region yielded 28 predicted genes. Sequence alignment and functional prediction, along with DNA resequencing analysis, revealed the insertion of a retrotransposon in the coding region (+1,515 bp) of the Lsat_1_v5_gn_8_44501 gene of the parental HJY. This resulted in a truncated transcript, a loss of 194 C-terminal amino acids in the encoded protein, and the introduction of 71 aberrant amino acids derived from the transposon. Furthermore, its mRNA expression level was significantly downregulated. This mutation resulted in lettuce exhibiting a phenotype of longer internodes and increased plant height. Therefore, we named this gene LsGAI1, and the mutant allele in HTS lsgai1.

[0035] The CDS sequence of the LsGAI1 gene is shown in SEQ ID NO.1, and its encoded amino acid sequence is shown in SEQ ID NO.2. An example of the CDS sequence of the transposon insertion mutant allele lsgai1 is shown in SEQ ID NO.3, and its encoded amino acid sequence is shown in SEQ ID NO.4.

[0036] The amplification primers used to identify the two alleles were GAI1F: 5'-CTCGCGATTTCTGTTTTTCC-3', TEF: 5'-CGTTATAACTCAGACTCAACGTC-3', and GAI1R: 5'-TGGCTCAATTGGCTGATACA-3'. An amplified fragment of 744 bp (shown in SEQ ID NO. 6) indicates the presence of the LsGAI1 allele; an amplified fragment of 480 bp (shown in SEQ ID NO. 7) indicates the presence of the lsgai1 allele.

[0037] Example 2:

[0038] Application of LsGAI1 in regulating lettuce stem node length and plant height:

[0039] (1) Using CRISPR-Cas9 gene editing technology, the LsGAI1 gene was knocked out in the LsGAI1 homozygous haplotype (Liujinbang genotype background, short internodes) lettuce background in F6 generation plants. sgRNAs targeting the LsGAI1 gene exons were designed: 5'-GACGATCTACAAAGGATCCC-3' and 5'-CAGGCTTTGGCTCTTCGACC-3', and constructed into the PL2B vector (GRF–GIF chimeric proteins enhance in vitro regeneration and Agrobacterium-mediated transformation efficiencies of lettuce (Lactucaspp.)). Wild-type lettuce cotyledon explants (i.e., Liujinbang genotype background, short internodes) were transformed using Agrobacterium-mediated transformation (strain EHA105). After resistance selection and regeneration culture, T0 generation transgenic plants were obtained. By detecting the target sequence amplified by PCR using primers (5'-GGGAACGATGATGGGTTGTCTCAGC-3 and 5'-GCCCGGTCTAGCCAACAACT-3') and sequencing, a homozygous knockout strain (CR#287) with a deletion mutation at the target site was identified (containing the polynucleotide shown in SEQ ID NO.5). This mutant strain had a one-base insertion at the target site, as shown in the image. Figure 4 As shown.

[0040] Phenotypic identification was performed on the T1 generation homozygous knockout lines. Compared with the recipient material, the homozygous knockout mutants showed a significant increase in stem segment length (approximately 62.4%) and overall plant height (approximately 67.5%). Figure 2 a, b, and c. These phenotypes are highly consistent with those of the HJY parents carrying the natural lsgai1 allele, confirming that the loss of LsGAI1 gene function is the direct cause of these phenotypic changes.

[0041] (2) Use overexpression technology to verify the function of LsGAI1 gene.

[0042] The complete CDS sequence of LsGAI1 (shown in SEQ ID NO.1) was cloned downstream of the promoter of the plant overexpression vector pRI101 to construct the recombinant plasmid pRI101:LsGAI1. F6 generation materials carrying the natural lsgai1 mutant allele (HYJ background, long internodes) were transformed using Agrobacterium-mediated transformation. T0 generation transgenic plants were obtained, and lines with significantly upregulated LsGAI1 gene expression were confirmed as positive overexpression lines by qRT-PCR.

[0043] The LsGAI1 quantitative PCR primers are GAI1qPCRF: 5'-TGCCCATTTTACGGCGAATCAAGCTA-3', GAI1qPCRR: 5'-CCTGCATCAGAGCCGGCCAC-3'.

[0044] The quantitative primers for the internal control were ubiquitin F: 5'-GAAGAAGACCTACACCAAGCCAAAG-3', ubiquitin R: 5'-ACTCAGCATTAGGGCACTCTTTCC-3'.

[0045] Three families with hyperexpression of LsGAI1 (GAI1-OE#70, GAI1-OE#115, and GAI1-OE#131) were identified, with LsGAI1 expression levels 42.3, 32.2, and 14.3 times that of the receptor, respectively. Figure 2 (d and e).

[0046] Phenotypic identification of the T1 generation overexpression line (OX#) showed that, compared with the recipient material (HJY background), the LsGAI1 overexpression line had significantly shorter internode lengths (approximately 35%) and reduced plant height (approximately 30%). Figure 2 (f). This result further confirms from the perspective of functional gain that the LsGAI1 gene negatively regulates internode elongation and plant height.

[0047] Example 3:

[0048] Application of dominant molecular markers developed based on LsGAI1 and its allele lsgai1 in the selection and breeding of lettuce growth traits:

[0049] HJY, carrying the naturally occurring loss-of-function lsgai1 allele, was used as the donor parent and crossed with Liujinbang, a stem lettuce cultivar with excellent overall traits but shorter internodes. In the hybrid offspring, dominant molecular markers based on transposon insertion sequences in the lsgai1 allele were used. The marker primers were: GAI1F: 5'-CTACAAGATCGGCAACGT-3'; TE-F: 5'-CGTTATAACTCAGACTCAACGTC-3'; GAI1R: 5'-TGGCTCAATTGGCTGATACA-3'. The GAI1-F / GA1-R combination amplified a band of 744 bp; the TE-F / GAI1-R combination amplified a band of 480 bp.

[0050] Using the aforementioned dominant molecular markers for marker-assisted selection (MAS), individuals carrying the lsgai1 allele were screened. The amplified band of the LsGAI1 gene from the *L. 6* parent was 744 bp (shown in SEQ ID NO. 6), while the amplified band of the lsgai1 gene from the HJY parent was 480 bp (shown in SEQ ID NO. 7).

[0051] Twelve individual plants were tested. Among them, the genes of plants 3, 5, 8, and 11 were amplified to 480 bp, and the phenotype showed long internodes; the genes of plants 1, 6, 7, and 12 were amplified to 744 bp, and the phenotype showed short internodes; the genes of plants 2, 4, 9, and 10 could be amplified to 480 bp and 744 bp bands, and the phenotype showed short internodes.

[0052] Example 4:

[0053] Application of dominant molecular markers developed based on LsGAI1 and its allele lsgai1 in lettuce growth trait screening breeding (universality verification):

[0054] Using 77 domestically collected lettuce quality lines as experimental materials (all collected by the applicant), a stem internode length trait survey was conducted in Wuhan, Hubei Province in 2023 (the internode length of the 8th node of the main stem above ground was uniformly measured as the phenotypic value of the internode length trait). The results showed that the longest internode length was 4.2 cm and the shortest internode length was 0.4 cm. Among them, 15 materials with internode lengths greater than 3.36 cm were defined as long internode materials. Primer analysis results showed that among the above 77 lettuce varieties, 20 materials had the genotype A (base A insertion) and 57 materials had the genotype W (wild) (Table 1).

[0055] The developed molecular marker was used to genotype 77 lettuce materials, and association analysis was performed with the field internode length phenotype. Chi-square test results showed a highly significant association between the molecular marker genotype (A / W) and the internode phenotype (long / short) (χ² = 35.63, df = 1, p < 0.001), indicating co-segregation of the marker with the target trait. Further analysis showed that the marker had extremely high predictive accuracy for the short internode phenotype, with a negative predictive value of 96.5%; its predictive accuracy for the long internode phenotype was 65.0%. Overall, the genotyping accuracy of this marker was 88.3%. These results confirm that the marker is a functional marker closely linked to the short internode trait and can be used for efficient screening of short internode plants in marker-assisted breeding of lettuce.

[0056] Table 1. Internode phenotypes of the two genotypes at the GAI1 marker SSS6 locus in 77 lettuce varieties.

[0057]

[0058] This marker is applicable to screening lettuce based on the length of its internodes. It can be used to select lettuce with longer internodes during the seedling stage for later improvement of superior quality, and has significant value for promotion and application.

Claims

1. Lettuce LsGAI1 The application of genes in regulating lettuce growth traits, including lettuce stem internode length and / or plant height. LsGAI1 The protein encoded by the gene is shown in SEQ ID NO.2, and its alleles are... lsgai1 The encoded protein is shown in SEQ ID NO.

4.

2. The application according to claim 1, characterized in that: Improve lettuce LsGAI1 The expression level of genes is used to reduce the length of lettuce stem segments and / or decrease plant height.

3. The application according to claim 2, characterized in that: The process described is to improve the lettuce LsGAI1 The substance that increases gene expression is introduced into lettuce; the substance is a substance containing... LsGAI1 Gene expression cassettes, recombinant vectors, or recombinant microorganisms.

4. The application according to claim 1, characterized in that: Knockout or inhibition of lettuce LsGAI1 Gene expression increases lettuce stem internode length and / or plant height.

5. The application according to claim 4, characterized in that: The process described involves reducing the lettuce's... LsGAI1 The gene is expressed or inhibited by a substance introduced into lettuce, wherein the substance is a substance that reduces gene expression. LsGAI1 Gene expression cassettes, recombinant vectors, or recombinant microorganisms.

6. The application according to claim 5, characterized in that: The knockout method used is homologous recombination or CRISPR gene editing. The protein translated from the knocked-out gene has no original function or cannot be translated into a protein.

7. The application according to claim 6, characterized in that: The knockout was performed using the CRISPR-Cas9 gene editing method, and the sgRNA sequences were: 5'-GACGATCTACAAAGGATCCC-3' and 5'-CAGGCTTTGGCTCTTCGACC-3'.

8. The application of a reagent for detecting the gene encoding the protein shown in SEQ ID NO.2 or SEQ ID NO.4 in the screening breeding of lettuce growth traits, wherein the breeding screening includes screening for lettuce stem node length and / or plant height.

9. The application according to claim 1 or 8, characterized in that, The gene encoding the protein shown in SEQ ID NO.2 is shown in SEQ ID NO.1, and the gene encoding the protein shown in SEQ ID NO.4 is shown in SEQ ID NO.

3.

10. The application of a molecular marker primer combination in the screening and breeding of lettuce growth traits, wherein the primer combination is: GAI1F: 5'-CTCGCGATTTCTGTTTTTCC-3', TEF: 5'-CGTTATAACTCAGACTCAACGTC-3', GAI1R: 5'-TGGCTCAATTGGCTGATACA-3', and the growth traits include lettuce stem node length and / or plant height.

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