Genes related to Schima superba affinity and their markers and applications
By identifying and marking the affinity-related genes of the woodlori affinity, the genotype identification of the pollinating objects was carried out, and hybrid combinations with high affinity were identified, which solved the problem of yield instability caused by the affinity difference during the intra-grain hybridization pollination of the woodlori species, and achieved It improves seed yield.
Patent Information
- Application Number
- CN202211140463.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-09-20
- Publication Date
- 2025-02-11
- Estimated Expiration
- 2042-09-20
AI Technical Summary
There are differences in affinity during intra-seeding pollination of Muli, which leads to unstable fruit collection rate between asexual line pollination combinations, and the yield of hybrid seeds is large, which brings troubles to production and application.
By identifying and marking Muhe affinity-related genes, including Ssu11G00456, Ssu11G00484, Ssu13G01012 and Ssu09G00616, the genotype identification of pollinating subjects was used to identify hybrid combinations with high affinity.
Through genotype identification, hybrid combinations with easy pollination and fertilization and high seed volume can be obtained in advance, which can improve the yield of Muli seeds and solve the problem of yield instability caused by affinity differences.
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Figure CN115725618B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and in particular relates to a gene related to superba affinity, a marker and an application thereof. Background Art
[0002] Schima superba is a widely distributed tree species in subtropical areas, naturally distributed in many provinces in southern my country, with large intraspecific individual variation. More than 1,000 Schima superba superior tree resources from 37 production areas in 7 provinces have been collected and preserved. During intraspecific hybridization and pollination, Schima superba has different affinity between different superior tree asexual lines, resulting in fruit setting rates between 27.3-95% between asexual pollination combinations, and large differences in hybrid seed yields. This brings great trouble to production and application.
[0003] The study suggests that in plant hybrid incompatibility experiments, there are gametophytic (GSI) and sporophytic (SSI) incompatibility types. Generally, SSI occurs in the stigma, while GSI occurs in the style or ovary (late self-incompatibility LSI). In the GSI system, S-RNase (female determining factor) and S-locusF-box protein (SLF / SFB, male determining factor) interact with each other, thereby determining the growth of pollen tubes and sperm release.
[0004] However, there is no public report on the affinity identification of Schima superba in the prior art. Summary of the invention
[0005] In view of the problems existing in the prior art, the object of the present invention is to provide a technical solution for a gene related to Schima superba affinity and its marker and application.
[0006] The present invention is specifically implemented by the following technical solutions:
[0007] The first aspect of the present invention provides Schima superba affinity-related genes, which include Ssu11G00456, Ssu11G00484, Ssu13G01012 and Ssu09G00616. The nucleotide sequence of Ssu11G00456 is shown in SEQ ID NO.1, the nucleotide sequence of Ssu11G00484 is shown in SEQ ID NO.2, the nucleotide sequence of Ssu13G01012 is shown in SEQ ID NO.3, and the nucleotide sequence of Ssu09G00616 is shown in SEQ ID NO.4.
[0008] The second aspect of the present invention provides the affinity-related genes S-RNase of Schima superba: Ssu11G00456 (mainly expressed in the ovary, is the maternal determining factor of the GSI system, interacts with SLF / SFB at the S site, plays a role in identifying pollen and inhibiting the growth of non-affinity pollen tubes), SLF / SFB: Ssu11G00484, Ssu13G01012 and Ssu09G00616 (mainly expressed in pollen, is the paternal determining factor of the GSI system, interacts with S-RNase at the S site, affects the activity of affinity S-RNase, and promotes the growth of affinity pollen tubes). These genes are all expressed 48 hours after pollination and have a special growth regulation effect on the growth of heterologous pollen tubes ( Figure 2 ).
[0009] Among them, Ssu11G00484 and the GI factor (affinity recognition function) of Prunus are classified into one category in the systematic classification ( Figure 4 ) and has a certain expression in the whole plant ( Figure 5 ), which is linked to the Ssu11G00456 gene ( Figure 7 , 8), which has the function of locating and identifying S-RNase between genotypes.
[0010] The third aspect of the present invention provides a Schima superba affinity marker, which includes markers for identifying the Schima superba affinity genes Ssu11G00484 and Ssu13G01012 (SLF / SFB);
[0011] The markers for identifying the Schima superba affinity gene Ssu11G00484 include marker 8411, marker 8441, marker 8431 and marker 8421, and the markers for identifying the Schima superba affinity gene Ssu13G01012 include marker 1221 and marker 1211;
[0012] The nucleotide sequence of the upstream primer of the marker 8411 is shown as SEQ ID NO.5, and the nucleotide sequence of the downstream primer of the marker 8411 is shown as SEQ ID NO.6; the nucleotide sequence of the upstream primer of the marker 8441 is shown as SEQ ID NO.7, and the nucleotide sequence of the downstream primer of the marker 8441 is shown as SEQ ID NO.8; the nucleotide sequence of the upstream primer of the marker 8431 is shown as SEQ ID NO.9, and the nucleotide sequence of the downstream primer of the marker 8431 is shown as SEQ ID NO.10; the nucleotide sequence of the upstream primer of the marker 8421 is shown as SEQ ID NO.11, and the nucleotide sequence of the downstream primer of the marker 8421 is shown as SEQ ID NO.12; the nucleotide sequence of the upstream primer of the marker 1221 is shown as SEQ ID NO.13, and the nucleotide sequence of the downstream primer of the marker 1221 is shown as SEQ ID NO.14; the nucleotide sequence of the upstream primer of the marker 1211 is shown as SEQ ID NO.15, and the nucleotide sequence of the downstream primer of the marker 1211 is shown as SEQ ID NO.16.
[0013] The fourth aspect of the present invention provides the use of the above-mentioned Schima superba affinity marker in identifying the affinity of Schima superba.
[0014] The fifth aspect of the present invention provides a method for identifying the affinity of Schima superba using the above-mentioned Schima superba affinity marker, which specifically comprises using the marker to perform genotype identification on Schima superba.
[0015] Further, the method specifically includes:
[0016] 1) Extract plant genomic DNA;
[0017] 2) using the identified upstream and downstream primers to perform PCR amplification;
[0018] 3) Using a fully automatic nucleic acid protein analyzer (Qsep100, BIOptic. Inc. Taiwan), 5 bp resolution card clamp was used for band separation and identification;
[0019] 4) Perform genotyping based on band separation and determine parental affinity.
[0020] The beneficial effects of the present invention are:
[0021] By using the affinity gene of Schima superba to identify the genotype of the pollination object, a hybrid combination with easy pollination and fertilization and high seed yield can be obtained early, providing a method for increasing the seed yield of Schima superba. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 The growth process of pollen tube in style and ovary;
[0023] Note: A. 24h outcrossing (compatibility); B. 24h selfing (non-compatibility); C. 36h outcrossing (compatibility); D. 36h selfing (non-compatibility); E. 48h outcrossing (compatibility); F. 48h selfing (non-compatibility).
[0024] Figure 2 The expression of Ssu11G00456, Ssu11G00484, Ssu13G01012 and Ssu09G00616 in the style and ovary at 24, 48 and 72 h, respectively.
[0025] Figure 3 Phylogenetic relationship of S-RNase genes of Schima superba Ssu11G00626, Ssu11G00456 and Ssu05G01984 with species of Rosaceae, Plantaginaceae, Rutaceae, Rubiaceae and Solanaceae.
[0026] Figure 4 Systematic evolutionary relationship of SLF / SFB genes of Schima superba Ssu11G00484, Ssu13G01012, Ssu09G00616, Ssu10G01793, Ssu02G00077 and Ssu09G01602 with species of Rosaceae, Plantaginaceae and Solanaceae.
[0027] Figure 5 Expression of Ssu11G00626, Ssu11G00456, Ssu05G01984, Ssu11G00484, Ssu13G01012, Ssu09G00616, Ssu10G01793 and Ssu02G00077 in pollen, ovary, style and leaves of Schima superba.
[0028] Figure 6 Ssu11G00456 amino acid sequence and structure diagram.
[0029] Figure 7 Amino acid sequence diagram of Ssu11G00484, Ssu13G01012 and Ssu09G00616. Figure 8 Linkage relationship diagram of Ssu11G00456, Ssu11G00484, Ssu13G01012 and Ssu09G00616.
[0030] Fig. 9 Schematic diagram of the linkage position structure of Ssu11G00456 (A) and Ssu11G00484 (B).
[0031] Fig.10 Electrophoresis results of some primers in different Schima superba materials.
[0032] Fig.11 Band separation of some primers in different Schima superba materials. DETAILED DESCRIPTION
[0033] The present invention is further described below with reference to the examples.
[0034] Example 1: Affinity candidate gene screening
[0035] Previous studies have found that Schima superba is an LSI species, and self-pollination (incompatible) pollen blockage occurs in the ovary at 48h ( Figure 1 F). Through differential transcriptome identification (with reference, Schima superba genome), it was found that the S-RNase gene Ssu11G00456 and three SLF / SFB-like genes Ssu11G00484, Ssu13G01012 and Ssu09G00616 were present in the ovary. When the self-pollinated (non-compatible) pollen tube entered the ovary, the expression was significantly upregulated. The styles and ovaries after 24, 48 and 72 hours of pollination were used as materials to verify the expression of these candidate genes by qRT-PCR. The expression trend of the composite transcriptome ( Figure 2 ), and believed that these genes play an important role in the hybridization compatibility of Schima superba.
[0036] The nucleotide sequence of Ssu11G00456 is shown in SEQ ID NO.1, the nucleotide sequence of Ssu11G00484 is shown in SEQ ID NO.2, the nucleotide sequence of Ssu13G01012 is shown in SEQ ID NO.3, and the nucleotide sequence of Ssu09G00616 is shown in SEQ ID NO.4.
[0037] Example 2: Structural analysis of affinity candidate genes
[0038] (1) Ssu11G00456 is an S-RNase gene with five conserved regions C1-C5, three intronic regions, and two highly variable regions HVa and HVb (between C2 and C3) ( Figure 6 ). In the HVa region, there is a highly variable SNP site SsuSNP85 (A and T variation), when the base is A, the amino acid is non-polar alanine, and when it is T, it is polar threonine. This gene is in the same branch as Citrus and in the same sub-branch as the S-RNase gene of Prunus.
[0039] (2) Ssu11G00484, Ssu13G01012 and Ssu09G00616 are SFB genes ( Figure 7), the protein has hydrophilic polarity. Sequence identification combined with genetic map analysis revealed that there were five polymorphic SNP sites (SsuSNP3434, SsuSNP3392, SsuSNP3369, SsuSNP3362, SsuSNP3337) in the F-box domain of Ssu11G00484, which had a significant linkage relationship with the HVa region of Ssu11G00456 ( Figure 8 ,9).
[0040] Example 3: Development of affinity tags
[0041] Based on the above findings, SSR marker development was performed using gene sequences, and four SSR loci were identified for the Ssu11G00484 gene and two SSR loci for the Ssu13G01012 gene. According to the sequence and annealing temperature, 19 pairs of primers were designed for marker screening. The electrophoresis results of some primers are shown in the following figure. Fig.10 As shown. Using a fully automatic nucleic acid protein analyzer (Qsep100, BIOptic. Inc. Taiwan), 5bp resolution card clamps were further screened and 6 pairs of primers with clear bands and strong polymorphism were selected ( Fig.11 The available primers after screening are shown in Table 1.
[0042] Table 1 SSR loci information of Ssu11G00484 and Ssu13G01012
[0043]
[0044] Table 2 Affinity markers and primer sequences
[0045]
[0046] Example 4: Affinity markers for identification of Schima superba genotypes
[0047] Based on the above primers, genotype identification was performed using asexual lines of Schima superba 34, 26, 65, 30, 96, 88, 87, 5, 8, and 3.
[0048] Plant leaves were collected, genomic DNA was extracted, and PCR amplification was performed using the designed primers (Table 3). The amplified reaction solution was separated and identified using a fully automatic nucleic acid protein analyzer (Qsep100, BIOptic. Inc. Taiwan) with a 5 bp resolution card, and the bands were compared and classified with the corresponding genotypes.
[0049] Table 3 Amplification procedures and conditions
[0050]
[0051] Note: The amplification reagent used was 2×TaqPlusMasterMix (Novagen).
[0052] The results showed that the bands could be used to preliminarily divide the clones into five genotypes (Table 4), and were significantly correlated with affinity (Table 5). The inter-genotype hybridization, especially the combination with S1 as the female parent, had a high affinity, with an average fruit setting rate of more than 44%. The intra-genotype hybridization had a low affinity, with a fruit setting rate of less than 10%.
[0053] Table 4 Genotype classification
[0054]
[0055] Table 5 Statistical results of some cross-hybridization experiments
[0056]
Claims
1. The affinity-related gene Ssu11G00456 of Schima superba is characterized by The nucleotide sequence of the gene is shown in SEQ ID NO.
1.
2. The affinity-related gene Ssu11G00484 of Schima superba is characterized by The nucleotide sequence of the gene is shown in SEQ ID NO.
2.
3. The affinity-related gene Ssu13G01012 of Schima superba is characterized by The nucleotide sequence of the gene is shown in SEQ ID NO.
3.
4. The affinity-related gene Ssu09G00616 of Schima superba is characterized by The nucleotide sequence of the gene is shown in SEQ ID NO.
4.
5. A primer for identifying affinity markers of Schima superba, characterized in that The primers are used to identify the marker of the Schima superba affinity gene Ssu11G00484 and the marker of the Schima superba affinity gene Ssu13G01012; The nucleotide sequence of Ssu11G00484 is shown in SEQ ID NO.2, and the nucleotide sequence of Ssu13G01012 is shown in SEQ ID NO.3; The markers for identifying the Schima superba affinity gene Ssu11G00484 include marker 8411, marker 8441, marker 8431 and marker 8421, and the markers for identifying the Schima superba affinity gene Ssu13G01012 include marker 1221 and marker 1211; The nucleotide sequence of the upstream primer of identification marker 8411 is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer of identification marker 8411 is shown in SEQ ID NO.6; the nucleotide sequence of the upstream primer of identification marker 8441 is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer of identification marker 8441 is shown in SEQ ID NO.8; the nucleotide sequence of the upstream primer of identification marker 8431 is shown in SEQ ID NO.9, and the nucleotide sequence of the downstream primer of identification marker 8431 is shown in SEQ ID NO.10; the nucleotide sequence of the upstream primer of identification marker 8421 is shown in SEQ ID NO.11, and the nucleotide sequence of the downstream primer of identification marker 8421 is shown in SEQ ID NO.12; the nucleotide sequence of the upstream primer of identification marker 1221 is shown in SEQ ID NO.13, and the nucleotide sequence of the downstream primer of identification marker 1221 is shown in SEQ ID NO.14; the nucleotide sequence of the upstream primer of identification marker 1211 is shown in SEQ ID NO.15, and the nucleotide sequence of the downstream primer of identification marker 1211 is shown in SEQ ID NO. Shown in NO.
16.
6. Use of the primers as claimed in claim 5 in identifying the affinity of Schima superba.
7. The method for identifying the affinity of Schima superba using the primers described in claim 5, characterized in that The primers were used to identify the genotype of Schima superba.
8. The method according to claim 7, characterized in that Specifically include: 1) Extraction of genomic DNA from Schima superba; 2) Perform PCR amplification using upstream and downstream primers of the identification marker; 3) Use a fully automatic nucleic acid protein analyzer and 5bp resolution card clamp to separate and identify the bands; 4) Perform genotyping based on band separation and determine parental affinity.