A method for identifying fertile and sterile peach pollen lines based on PpMED15 gene expression
Through the detection method of PpMED15 gene expression, the problem of pollen fertility identification was solved, the accurate identification of peach pollen fertility and sterile strains was achieved, and the efficiency of peach blossom breeding was improved.
Patent Information
- Application Number
- CN202211236731.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-10-10
- Publication Date
- 2025-08-08
- Estimated Expiration
- 2042-10-10
AI Technical Summary
The existing technology has failed to clearly determine the genes that determine the fertility of peach pollen, which leads to difficulties in identifying pollen fertility and sterile lines and affects the development of the peach industry.
The PpMED15 gene was used as a marker, and the critical period of pollen development was observed through paraffin sections, and the method of detecting pollen fertile and sterile strains was used using specific primer pairs and detection kits, including extraction of RNA for reverse transcription and PCR amplification, and the expression of PpMED15 gene was used as the basis for identification.
Accurately distinguishing fertile and sterile lines has improved the accuracy and efficiency of pollen fertility identification, and solved the problem of fruitlessness caused by pollen abortion.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of plant seed breeding, and in particular to a method for identifying fertile and sterile peach pollen lines based on PpMED15 gene expression. Background Art
[0002] Pollen development is crucial for sexual reproduction in peaches. Pollen fertility is a key goal in peach variety breeding. Abnormal pollen development leads to pollen abortion, resulting in fruit failure and severely impacting the development of the peach industry. Research suggests that peach pollen sterility is determined by a single gene, designated pllen sterility (PS). Recessive homozygous Ps / PS results in pollen sterility, while dominant heterozygous Ps / PS or dominant homozygous Ps / Ps results in pollen fertility.
[0003] Currently, a single mutation in the PpABCG26 gene, resulting in a conversion of leucine (CTT) to proline (CCT), is believed to be the cause of pollen sterility. However, half of the sterile materials were either heterozygous for T / C or homozygous for T / T. This finding is inconsistent with the theory that sterile varieties are homozygous for C / C. These experimental results suggest that the PpABCG26 gene cannot be used as the sole candidate gene for distinguishing fertile from sterile lines. Further exploration of genes and molecular markers determining peach pollen fertility is needed. Summary of the Invention
[0004] The present invention provides a method for identifying fertile and sterile peach pollen lines based on PpMED15 gene expression, which is used to solve the problem that the decisive gene for peach pollen fertility has not been clearly identified in the prior art.
[0005] In a first aspect, the present invention seeks protection for the use of the PpMED15 gene in identifying fertile and sterile peach pollen lines, wherein the amino acid sequence of the PpMED15 gene is shown in SEQ ID NO.1.
[0006] Furthermore, the nucleotide sequence of the PpMED15 gene is shown in SEQ ID NO.2.
[0007] The present invention uses a fertile parent JC and a sterile parent JS as well as a group consisting of fertile and sterile plant lines generated by hybridization of the two as research materials, and analyzes the expression level of the gene in the group.
[0008] The present invention collects three key periods of flower bud development from the fertile parent JC and the sterile parent JS, and determines the key period during which the fertile parent JC and the sterile parent JS are significantly different through paraffin section observation. Secondly, flower buds from each individual plant in the hybrid population at this key period of development are collected as test materials, and the MRP II gene is used as a marker to identify fertility and infertility from the perspective of gene expression.
[0009] In a second aspect, the present invention provides a primer pair, the sequence of the primer pair is shown as SEQ ID NO.3-4; or the sequence of the primer pair is shown as SEQ ID NO.5-6.
[0010] In a third aspect, the present invention provides a detection kit comprising the above-mentioned primer pair.
[0011] The method for using the detection kit provided by the present invention is as follows: RNA is extracted from peach blossom bud tissue to be tested, the RNA is reverse transcribed to obtain cDNA, and the cDNA is amplified using a primer pair with a sequence as shown in SEQ ID NO.3-4.
[0012] If the amplification can produce an amplification product, the pollen of the peach plant to be tested is fertile. More specifically, if the amplification product is a 1900 bp band, the pollen of the peach plant to be tested is fertile.
[0013] Alternatively, in the detection kit provided by the present invention, the expression level of the PpMED15 gene in the peach plant to be tested is detected using SEQ ID NO. 5-6, with the pollen sterile line as a negative control and the pollen fertile line as a positive control.
[0014] According to the understanding of those skilled in the art, the present invention seeks to protect the use of the above primer pair or the above detection kit in improving the accuracy of detecting fertile and sterile lines of peach pollen.
[0015] In a fourth aspect, the present invention claims protection for a method for identifying the fertility and sterility of peach pollen, wherein RNA is extracted from the flower bud tissue of the peach plant to be tested and reverse transcribed to obtain cDNA; amplification is performed using the primer pair shown in SEQ ID NO.5-6 and the cDNA as a template; when the amplified product of the flower bud of the peach plant to be tested is higher than the expression level of the internal reference gene RPL13, the peach plant to be tested is a pollen fertile plant.
[0016] In the method provided by the present invention, the expression level of the PpMED15 gene in the sterile peach pollen plant is used as a negative control, and the expression level of the PpMED15 gene in the fertile peach pollen plant is used as a positive control.
[0017] The beneficial effects of the present invention are:
[0018] The present invention first discovered that the PpMED15 gene, whose amino acid sequence is shown in SEQ ID NO. 1, can be used to identify fertile and sterile peach pollen lines.
[0019] The experimental results of the present invention demonstrate that PpMED15 gene expression can accurately distinguish fertile and sterile lines in the fertile parent JC and the sterile parent JS, as well as in hybrid populations of the two. This gene expression profile can be used as an effective method for identifying fertile and sterile lines in peach pollen. Specifically, using the primer pairs provided by the present invention, fertile and sterile pollen can be accurately and efficiently identified through simple PCR amplification. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0021] Figure 1 This is a phenotypic comparison between fertile lines and infertile plants in the present invention.
[0022] Figure 2 These are paraffin section observation images of fertile strains and infertile plants in the present invention.
[0023] Figure 3 This is the electrophoresis diagram of the PpMED15 gene of the fertile plant line and the infertile plant in the present invention.
[0024] Figure 4 It is the relative expression level of the PpMED15 gene in the fertile lines and the infertile plants in the present invention. DETAILED DESCRIPTION
[0025] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the present invention are clearly and completely described. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.
[0026] Example 1 Comparison of fertile lines and infertile plants
[0027] (1) Phenotypic comparison
[0028] Figure 1Among them, the pollen-fertile parent 'Jiucui' (JC) and the pollen-sterile parent 'Jiushuo' (JS) are both self-pollinated offspring of 'Dajiubao'. 'Jiucui' and 'Jiushuo' differ in anther morphology, size and color. JC's anthers are mature, full, and purple-red in color, while JS's anthers are short, small, and light yellow in color. When JC anthers are fully anthesised, the filaments are elongated, the pollen grains are abundant, and the anthers open to release pollen. In contrast, JS's filaments are short, the anthers turn white, the anthers do not open, and no pollen is produced (see the results). Figure 1 ).
[0029] (2) Paraffin section observation
[0030] In order to determine the cause of pollen abortion, paraffin sections of the anthers of pollen fertile JC and pollen sterile JS were observed (see the results). Figure 2 ), and found the critical period during which the fertile parent JC and the sterile parent JS were significantly different.
[0031] Figure 2 During mid-flower bud development, the anthers of the fertile parent, JC, formed free haploid microspores, the tapetum cells solidified and degenerated, and pollen walls formed on the microspore surfaces. In the sterile cultivar, JS, the tapetum cells did not degenerate, but the microspores showed deformation and degeneration, uneven cytoplasm, abnormal vacuolization, and no pollen walls. These results suggest that the primary cause of pollen sterility is the failure of the tapetum to degenerate, resulting in the inability to form pollen walls on the microspore surfaces and the degeneration of the microspores.
[0032] Example 2 Acquisition of PpMED15 gene
[0033] 1. The present invention uses the EASYspin plant RNA rapid extraction kit of Bomade Bio to extract RNA from peach flower buds at the single-core pollen stage. The specific steps are as follows:
[0034] 1) Peach blossom bud tissue was ground into a fine powder in liquid nitrogen. 500 μL of lysis buffer RLT (to which β-mercaptoethanol had been added) and 50 μL of PLANTaid were added. The mixture was vortexed for 20 seconds and incubated in a 56°C water bath for 2 minutes.
[0035] 2) The lysate was centrifuged at 13,000 rpm for 10 min. 400 μL of the supernatant was added to 200 μL of anhydrous ethanol and immediately mixed by pipetting.
[0036] 3) The mixture was added to an adsorption column RA placed in a collection tube, centrifuged at 12,000 rpm for 60 seconds, and the waste liquid was discarded.
[0037] 4) Add 350 μL of deproteinization buffer (RWI), incubate at room temperature for 30 seconds, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid. Return the adsorption column (RA) to the collection tube.
[0038] 5) Add 80 μL of DNasel working solution (mix 10 μL of DNasel stock solution with 70 μL of RDD solution) to the center of the adsorption column RA and incubate at 37°C for 15 min.
[0039] 6) Add 350 μL of deproteinization solution RW1, let it stand at room temperature for 30 seconds, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.
[0040] 7) Add 500 μL of Rinse Buffer RW (prepared with anhydrous ethanol), centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, add 500 μL of Rinse Buffer RW, and repeat.
[0041] 8) Place the adsorption column RA back into the empty collection tube, centrifuge at 12,000 rpm for 2 minutes, and place the adsorption column at room temperature for 3 minutes.
[0042] 9) Remove the RA adsorption column and place it in an RNase-free centrifuge tube. Add 30 μL of RNase-free water (heated in a 70°C water bath) to the center of the adsorption membrane. Incubate at room temperature for 1 minute. Centrifuge at 12,000 rpm for 1 minute. Add the eluate back to the adsorption column and repeat the process.
[0043] 2. RNA purity test and electrophoresis test:
[0044] Purity test: 1 μL RNA sample was taken with 1 μL RNase-free water as a control, and the OD value was measured on a micro-spectrophotometer (BioDrop, Cambridge, UK). An A260 / A280 ratio greater than 1.8 indicated that the prepared RNA was relatively pure and free of protein contamination.
[0045] RNA reverse transcription:
[0046] 1) Add template RNA and primer mixture to a PCR tube, totaling 12 μL. The reaction system is shown in Table 1:
[0047] Table 1 RNA reverse transcription reaction system
[0048]
[0049]
[0050] After incubation at 65°C for 5 min, the mixture was cooled on ice.
[0051] 2) Add the reagents listed in Table 2 to the PCR tube in the specified order:
[0052] Table 2 Reagents added to PCR tubes
[0053]
[0054] The above 20 μL solution was reacted in the following procedure: 42° C. for 60 min; 70° C. for 5 min; and stored at −20° C. for later use.
[0055] The PpMED15 gene was cloned using the following primers:
[0056] Forward:5'-GTCCGTATTGTTCACTTCAAGTCCC-3'(SEQ ID NO.3);
[0057] Reverse: 5'-TTGGACTAACTTTTTCCGGTCTGAA-3' (SEQ ID NO. 4).
[0058] The amino acid sequence shown in Sequence 1 of the sequence listing was named PpMED15. The gene encoding the PpMED15 protein was named PpMED15 gene.
[0059] Example 3 Analysis of PpMED15 gene expression pattern
[0060] This example provides analysis of the PpMED15 gene expression pattern, and the steps are as follows:
[0061] 1) F1 generation and phenotypic analysis of pollen fertile and pollen sterile hybrid populations
[0062] The pollen-fertile variety JC was crossed with the pollen-sterile variety JS to generate an F1 population of 20 lines. Phenotypic observation revealed that lines with 1-12 individual plants producing pollen normally were considered pollen-fertile, while lines with 13-20 individual plants not producing pollen normally were considered pollen-sterile.
[0063] 2) PCR amplification of the cDNA fragments of the PpMED15 gene parent and progeny using the following primers
[0064] Forward: 5′-GTCCGTATTGTTCACTTCAAGTCC-3′ (SEQ ID NO. 3);
[0065] Reverse: 5′-TTGGACTAACTTTTTCCGGTCTGAA-3′ (SEQ ID NO. 4). The reaction system is shown in Table 3.
[0066] Table 3 Reaction system
[0067]
[0068] Reaction conditions: refer to Bio-Rad (Bio-Rad Laboratories, Hercules, CA, USA), see Table 4.
[0069] Table 4 Reaction conditions
[0070]
[0071] Electrophoresis pattern analysis:
[0072] The amplified product was detected by 1.0% agarose gel electrophoresis. The electrophoresis bands on the gel were observed on a gel imager (Bio-Rad Company) and the size of the electrophoresis bands was determined by comparing with the DNA marker. The size of the electrophoresis band of the PpMED15 gene should be 1900 bp. Figure 3 .
[0073] Figure 3 In the figure, the marker is 5000, JC is a pollen fertile variety, and JS is a pollen sterile variety. 1-20 are the hybrid offspring of JC and JS, of which 1-12 is a pollen fertile line and 13-20 is a pollen sterile line. Figure 3 It can be seen that the pollen fertile sample has an obvious band below 2000bp, while the pollen sterile sample has no obvious band.
[0074] 3) The expression level of the PpMED15 gene was analyzed using the following primers. The base sequence is shown below:
[0075] PpMED15
[0076] Forward:5'-CCAGACGGTACTCATGTTAAT-3'(SEQ ID NO.5)
[0077] Reverse:5′-CACCGCTTTAATCAACCGTTC-3'(SEQ ID NO.6)
[0078] Internal reference RPL13
[0079] Forward:5′-GCAGCGACTGAAGACATACAAG-3′(SEQ ID NO.7)
[0080] Reverse:5'-GGTGGCATTAGCAAGTTCCTC-3'(SEQ ID NO.8)
[0081] 1) First, dilute the cDNA concentration to 500 mg / mL. See Table 5 for the reaction system.
[0082] Table 5 Reaction system
[0083]
[0084] 2) Reaction conditions were based on the Bio-Rad CFX Connect Real-Time System (Bio-Rad Laboratories, Hercules, CA, USA) and were as follows: 95°C for 30 min;
[0085] 95°C for 5 seconds, 60°C for 30 seconds (collecting fluorescence signals); 40 cycles.
[0086] Draw a melting curve: from 65℃ to 95℃, increase by 0.5℃ every 5 seconds.
[0087] Figure 4 The horizontal axis represents different samples, and the vertical axis represents the relative expression of the PpMED15 gene. The expression of the PpMED15 gene in JS is normalized to 1, and the expression of other samples is based on this. Figure 4 The results show that the expression levels of the PpMED15 gene in the pollen-sterile variety JS and its individual pollen-sterile plants 13-20 were around 1, while the expression levels in the pollen-fertile variety JC and its fertile offspring were greater than 5, a difference of 5 times. These results indicate that the expression level of the PpMED15 gene in the pollen-fertile variety and its offspring was significantly higher than that in the pollen-sterile variety and its offspring.
[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.
Claims
1. PpMED15 The application of the gene in identifying fertile and sterile peach pollen lines is characterized in that: described PpMED15 The amino acid sequence of the gene is shown in SEQ ID NO.
1.
2. The use according to claim 1, characterized in that described PpMED15 The nucleotide sequence of the gene is shown in SEQ ID NO.
2.
3. The use of primer pairs in identifying fertile and sterile peach pollen lines, characterized in that: The sequence of the primer pair is shown as SEQ ID NO.3-4; or the sequence of the primer pair is shown as SEQ ID NO.5-6.
4. Use of a detection kit for identifying fertile and sterile peach pollen lines, characterized in that: The detection kit contains a primer pair having a sequence shown in SEQ ID NO. 3-4; or a primer pair having a sequence shown in SEQ ID NO. 5-6.
5. The use according to claim 4, characterized in that RNA is extracted from flower bud tissue of the peach plant to be tested, and after reverse transcription of the RNA to obtain cDNA, the cDNA is amplified using a primer pair with a sequence as shown in SEQ ID NO. 3-4.
6. The use according to claim 5, wherein if the amplification can produce an amplification product, the pollen of the peach plant to be tested is fertile.
7. The use according to claim 4, characterized in that The primer pair shown in SEQ ID NO.5-6 was used to detect the PpMED15 The expression level of the gene, in the kit, the pollen sterile line is used as a negative control, and the pollen fertile line is used as a positive control.
8. Use of a primer pair having the sequences shown in SEQ ID NOs. 3-4 or 5-6, or a detection kit according to any one of claims 4-7, in improving the accuracy of detecting fertile and sterile peach pollen lines.
9. A method for identifying fertility and sterility of peach pollen, characterized in that: RNA is extracted from flower bud tissue of the peach blossom plant to be tested and reverse transcribed to obtain cDNA; amplification is performed using the primer pair shown in SEQ ID NO. 5-6 and the cDNA as a template. When the amplified product of the flower bud of the peach blossom plant to be tested is higher than the expression level of the internal reference gene RPL13, the peach blossom plant to be tested is a pollen-fertile plant.
10. The method according to claim 9, characterized in that: Peach pollen sterile plants PpMED15 The gene expression level was used as negative control, and the PpMED15 Gene expression levels served as positive controls.