A molecular marker for identifying allelic variations of a soluble acid invertase gene AcoInv-1 in pineapple fruits and application thereof

Abstract

The present application relates to the field of agricultural biotechnology, and particularly relates to a molecular marker capable of identifying allelic variations of a soluble acid invertase gene AcoInv-1 in pineapple fruits and application thereof. The molecular marker is amplified by using the following primers: forward primer 5-CTGCGAAATGAACTAGTCAACTC-3 and reverse primer 5-TAGAGAACCGTTCTTGG AATGG-3. The primers can identify allelic variations of the soluble acid invertase gene AcoInv-1 in pineapple fruits, i.e. AcoInv-1a and AcoInv-1b. It is found through research that the two allelic variations are extremely significantly related to the total sugar content of the pineapple fruits. It is proved through verification that the pineapple material of the AcoInv-1a type is related to high sugar content, and the pineapple material of the AcoInv-1a and AcoInv-1b types is related to low sugar content. The application of the molecular marker obtained by the present application in selecting pineapple varieties with high sugar content potential will help to accelerate the breeding selection cycle.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to a molecular marker and its application for identifying allelic variations of the AcoInv-1 gene, a soluble acid invertase gene in pineapple fruit. Background Technology

[0002] Pineapple is a common tropical fruit tree, its fruit rich in sugar and nutrients, with a unique taste and flavor, making it a popular fruit. While the external natural environment plays a role in regulating sugar accumulation in the fruit, the accumulation of sugar in pineapple fruit is primarily regulated by genetic factors. Studies have found significant differences in the physicochemical properties of pineapples harvested in different seasons, with winter-harvested fruit having a higher soluble solids content than fruit harvested in other seasons. Previous research has shown significant differences in sugar content among different pineapple varieties, mainly controlled by genetic factors; the sugar content of different pineapple varieties can differ by 1-2 times, and this difference is genetically stable. Pineapple is a perennial plant, and due to self-incompatibility and other issues, hybridization is very difficult. Current breeding techniques mainly involve configuring hybrid combinations and selecting suitable materials from the offspring. However, this method is not only costly but also time-consuming, especially given the insufficient understanding of the genetic background of the parents. The selection process relies mainly on manual experience, lacking scientific basis, making it difficult to select suitable varieties from the offspring. With the completion of the pineapple whole genome sequencing, it will be possible to develop molecular markers closely related to sugar content. We can then screen parental materials as needed, providing new methods and approaches for breeding new pineapple varieties with suitable sugar content.

[0003] Invertase (Inv) plays a crucial role in sucrose metabolism in higher plants. Studies have shown that invertase participates in plant growth, organogenesis, sugar transport, phloem unloading, and regulates the sugar composition and levels in sink tissues. Inv irreversibly catalyzes the hydrolysis of sucrose into glucose and fructose, making it an important regulatory enzyme in sucrose decomposition. Based on different classification methods, invertases can be divided into different families. According to their water solubility, they can be divided into soluble and insoluble invertases. Based on their optimal pH, they can be divided into acidic invertases and neutral / alkaline invertases. Their main function is to degrade sucrose, but they differ in their location. Soluble acidic invertases are mainly found in vacuoles, catalyzing the hydrolysis of sucrose into hexoses, and play a role in regulating sugar accumulation in plant tissues and sucrose utilization in vacuoles. Their structure differs significantly from that of neutral invertases, but research on their protein structure and characteristics is still relatively limited. Inv plays a key role in sucrose accumulation in the vacuoles of mature sink organs. The activity of Inv in vacuoles has a significant impact on sucrose content. The Inv gene DNA or cDNA sequences have been successfully cloned from crops such as tomato, Arabidopsis, carrot, potato, sugarcane, rice, and sweet sorghum, and molecular markers for tomato and sweet sorghum have been successfully developed. However, there are no reports on the cloning and molecular markers of the pineapple Inv gene.

[0004] Therefore, it is essential to develop molecular markers that can identify allelic variations in the soluble acid invertase gene of pineapple fruit. Summary of the Invention

[0005] One of the objectives of this invention is to address the shortcomings of existing technologies by providing a molecular marker that can identify allelic variations of the soluble acid invertase gene AcoInv-1 in pineapple fruits. This molecular marker is selected from allelic variations of the Inv gene, which helps breeders find superior parental materials from numerous pineapple varieties without having to go through complicated cultivation experiments and measurements.

[0006] The second objective of this invention is to provide the application of the molecular markers that can identify allelic variations of the AcoInv-1 gene, a soluble acid invertase gene in pineapple fruit, in assisting the selection of pineapple varieties with high sugar content potential.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A molecular marker is provided for identifying allelic variations of the AcoInv-1 gene, a soluble acid invertase gene in pineapple fruit. This molecular marker is amplified using the following primers:

[0009] Forward primer: 5-CTGCGAAATGAACTAGTCAACTC-3,

[0010] Reverse primer: 5-TAGAGAACCGTTCTTGGAATGG-3.

[0011] In the above technical solution, the molecular marker has a nucleotide sequence of 144 bp as shown in Seq ID NO.1.

[0012] In the above technical solution, the molecular marker has a nucleotide sequence of 125 bp as shown in Seq ID NO.2.

[0013] The specific preparation method is as follows:

[0014] (1) Based on the published pineapple whole genome sequence (pineapple.angiosperms.org), genes similar to soluble acid invertase (Aco016281.1, AcoInv-1) were identified. By comparing the differences in allelic variation among different varieties, segments with common nucleotide sequences upstream and downstream were selected for primer design. A total of 3 primers were designed, and the primer design ensured that each sequence contained an overlap of about 150 bp.

[0015] (2) Using the whole DNA of the pineapple variety "Barry" as a template, these three sequences were cloned. Analysis of the PCR products by 1% agarose gel electrophoresis showed a single band in the PCR products obtained from each primer pair. The obtained fragments were approximately 2300bp, 2500bp, and 1700bp, respectively, which was basically consistent with the predicted size. After recovering the three PCR products, they were cloned and sequenced, yielding three sequences with approximately 150bp of overlapping DNA. These three sequences were spliced ​​together to obtain a pineapple Inv genomic DNA fragment, named AcoInv-1.

[0016] This invention utilizes software such as DNAMAN and DNAstar to assemble three obtained sequences to obtain pineapple AcoInv-1, and its bioinformatics analysis is as follows:

[0017] The gene is 6290 bp in length, as shown in Seq ID No. 3. The location and copy number of AcoInv-1 were determined by comparing it with sequences from the pineapple genome website (pineapple.angiosperms.org). The results showed that the AcoInv-1 gene is located on chromosome 3 of the pineapple genome, and there is only one copy. Within the coding region, AcoInv-1 contains a start codon and a stop codon, six exons and five introns, as well as a 38 bp 5' untranslated region and a 58 bp 3' untranslated region. The AcoInv-1 sequence shows high similarity to other reported Inv genes from other crops, suggesting that the AcoInv-1 obtained in this invention is the full-length sequence of the soluble acid-converting enzyme gene for pineapple.

[0018] The pineapple AcoInv-1 gene cDNA sequence, with a full length of 2046 bp, is shown in Seq ID No. 4 and exhibits high similarity to the Inv gene sequences of rice, sugarcane, and maize. The pineapple AcoInv-1 gene cDNA sequence encodes a polypeptide containing 681 amino acids, as shown in Seq ID No. 5. Conserved domain analysis of this protein revealed the presence of a complete glycosyl hydrolases family 32 domain, which is capable of hydrolyzing sucrose.

[0019] This invention also provides the application of the molecular markers described above, which can identify allelic variations of the soluble acid invertase gene AcoInv-1 in pineapple fruit, in assisting the selection of pineapple varieties with high sugar content potential.

[0020] The specific method is as follows:

[0021] This invention selected 16 pineapple samples with significant differences in total sugar content as marker-selected test materials. DNA from the tender leaves of these 16 samples two weeks after emergence was amplified using designed primers (see Table 1). The amplification results were sequenced, and the full-length AcoInv-1 gene sequences of the 16 samples were obtained after splicing. Sequence alignment was performed using the multiple sequence alignment program in DNAMAN software, and DNA polymorphism and haplotype analysis were performed using DNA SP4.9 software. The 16 samples were classified into two haplotypes: AcoInv-1a and AcoInv-1b. The biggest difference between these two types is that AcoInv-1b contains a 19bp deletion in the second intron region.

[0022] This invention developed a co-dominant molecular marker, AcoInv-1X (forward primer: 5-CTGCGAAATGAACTAGTCAACTC-3; reverse primer: 5-TAGAGAACCGTTCTTGGAATGG-3), based on the allelic sequence differences between AcoInv-1a and AcoInv-1b. This primer pair amplified the DNA of the 16 materials mentioned above. The amplification products were subjected to denaturing polyacrylamide gel electrophoresis, and the results showed polymorphism in the amplified products. Based on the allelic variation type of the pineapple Inv gene, this invention divided the 16 materials into two types. Of the 16 pineapple materials, 12 materials were of the AcoInv-1a type, meaning a 144bp fragment could be amplified in these materials; 4 materials were heterozygous for AcoInv-1a and AcoInv-1b, simultaneously amplifying both 125bp and 144bp bands. These results demonstrate that this molecular marker can be used to identify the allelic variation type of the Inv gene in different pineapple materials.

[0023] Furthermore, this invention utilizes the AcoInv-1X label to detect 164 pineapple samples: (1) 153 samples were able to amplify a 144bp band (AcoInv-1a), and 11 samples were able to amplify two bands, 125bp and 144bp (AcoInv-1a and AcoInv-1b); (2) In AcoInv-1a, the total sugar content of most samples was concentrated between 14% and 18%, accounting for 82% of the total; while in AcoInv-1a and AcoInv-1b samples, the total sugar content of most samples was concentrated between 8% and 14%, accounting for 86% of the total; the comprehensive analysis of the total sugar content and classification of the 164 samples showed that the higher the sugar content, especially after the total sugar content exceeded 14 mg / g, the greater the proportion of AcoInv-1a was compared with AcoInv-1a and AcoInv-1b. (3) This invention statistically analyzed the average total sugar content of the fruits of two types of pineapple materials. The average total sugar content of the AcoInv-1a type material was 14.91, while the average total sugar content of the materials containing both AcoInv-1a and AcoInv-1b types was 11.29. The average total sugar content of the AcoInv-1a type material was significantly higher than that of the AcoInv-1a and AcoInv-1b types. The above results indicate that the amplified fragment of this marker is highly significantly correlated with the total sugar content of pineapple fruits. Attached Figure Description

[0024] The present invention will be further described with reference to the accompanying drawings, but the embodiments in the drawings do not constitute any limitation on the present invention. For those skilled in the art, other drawings can be obtained based on the following drawings without creative effort.

[0025] Figure 1 The results of 1% agarose gel electrophoresis of the pineapple DNA extract obtained in Example 1 are shown.

[0026] Figure 2 The gel electrophoresis results of the PCR product of the pineapple AcoInv-1 gene obtained in Example 1 are shown.

[0027] Figure 3 The structure diagram of the pineapple AcoInv-1 gene obtained in Example 2 is shown.

[0028] Figure 4 The location of the pineapple AcoInv-1 gene on the chromosome obtained in Example 2.

[0029] Figure 5 The sequence alignment (partial) of pineapple AcoInv-1a and pineapple AcoInv-1b obtained in Example 3.

[0030] Figure 6 The results of polypropylene gel electrophoresis for typing 316 pineapple materials are shown in Example 3.

[0031] Figure 7 Example 4 shows the results of polypropylene gel electrophoresis of 164 pineapple samples using AcoInv-1X.

[0032] Figure 8 Example 4 utilizes statistical analysis of allelic variation types and total sugar content of 164 pineapple samples. Detailed Implementation

[0033] The present invention will be further described in conjunction with the following embodiments and accompanying drawings.

[0034] Example 1:

[0035] The cloning of the full-length DNA of the pineapple soluble acid invertase gene (Aco016281.1, AcoInv-1) in this embodiment includes the following steps:

[0036] (1) DNA extraction:

[0037] The DNA extraction method and reagents were based on those used by Sambrook et al. (Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual, Edition. Cold Spring Habour Laboratory Press, New York, 1989). The DNA extraction results are as follows: Figure 1 As shown, a single, distinct band is visible, indicating that the DNA quality is reliable and can be used for subsequent experiments.

[0038] (2) Primer design:

[0039] Based on the published pineapple genome sequence (pineapple.angiosperms.org), genes similar to soluble acid invertase (Aco016281.1, AcoInv-1) were identified. By comparing the differences in allelic variation among different varieties, segments with common upstream and downstream nucleotide sequences were selected for primer design. A total of 3 primers were designed, and the primer design ensured that each sequence contained an overlap of about 150 bp, as shown in Table 1.

[0040] Table 1. Primers used for cloning and allelic typing of the AcoInv-1 gene in pineapple

[0041]

[0042] (3) Using the whole DNA of the pineapple variety "Barry" as a template, these three sequences were cloned:

[0043] There are no special requirements for the reagents used in PCR; ordinary, commonly used reagents are sufficient.

[0044] PCR program: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 61℃ annealing for 30 s, 72℃ extension for 90 s; 30 cycles in total, 72℃ extension for 10 min, store at 4℃.

[0045] Gel electrophoresis: 5 μl of gel was deposited on a 1% agarose gel for electrophoresis and analyzed using a gel imaging system from Bio-Rad.

[0046] PCR product recovery: The PCR product recovery was performed in accordance with the instructions of the Tiangen Biotech DNA Recovery Kit.

[0047] Validation of ligation, transformation and cloning of the recovered products: Refer to the pGEM-T EasyVector System instruction manual from TransGen Biotech Ltd.

[0048] Sequencing: Positive clones were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Each PCR product was sequenced 2-4 times to ensure the correct nucleotide sequence was obtained.

[0049] Taq DNA polymerase was purchased from TaKaRa's EX Taq, the cloning kit was purchased from Promega's pGEM-TEasy Vector System, agarose was purchased from Genetech, and DNA recovery kit, dNTPs, and DNA markers were purchased from Tiangen Biotech.

[0050] (4) Using the genomic DNA of the pineapple variety "Barry" as a template, PCR amplification was performed. Analysis by 1% agarose gel electrophoresis showed that each primer pair produced a single band in its PCR product, with fragments approximately 2300bp, 2500bp, and 1700bp, respectively, consistent with the predicted sizes. Figure 2 As shown. After recovering the three PCR products, they were cloned and sequenced, yielding three DNA sequences with approximately 150 bp of overlap. These three sequences were then spliced ​​together to obtain a pineapple Inv genomic DNA fragment, named AcoInv-1.

[0051] Example 2:

[0052] The bioinformatics analysis of the pineapple soluble acid invertase gene (Aco016281.1, AcoInv-1) in this embodiment is as follows:

[0053] The pineapple AcoInv-1 gene was obtained by assembling three sequences using software such as DNAMAN and DNAstar. The full-length gene is 6290 bp, as shown in Seq ID No. 3. The location and copy number of AcoInv-1 were determined by comparing it with sequences from the pineapple genome website (pineapple.angiosperms.org). The results showed that the AcoInv-1 gene is located on chromosome 3 of the pineapple genome and has only one copy. Figure 3 As shown; AcoInv-1 contains a start codon and a stop codon within its coding region, has 6 exons and 5 introns, as well as a 38bp 5' untranslated region and a 58bp 3' untranslated region, as shown. Figure 4 As shown, the AcoInv-1 sequence has a high similarity to other reported Inv genes in crops, suggesting that the obtained AcoInv-1 is the full-length sequence of the soluble acid-converting enzyme gene of pineapple.

[0054] The full-length cDNA sequence of the pineapple AcoInv-1 gene is 2046 bp, as shown in Seq ID No. 4, and shows high similarity to the Inv gene sequences of rice, sugarcane, and maize. The pineapple AcoInv-1 gene cDNA sequence encodes a polypeptide containing 681 amino acids, as shown in Seq ID No. 5. Conserved domain analysis of this protein revealed that it contains a complete glycosyl hydrolases family 32 domain, which has the function of hydrolyzing sucrose.

[0055] Example 3:

[0056] The allelic variation analysis and the development of molecular markers for the pineapple AcoInv-1 gene in this embodiment are specifically performed as follows:

[0057] I. Experimental Materials

[0058] 1. Reagents and medicines:

[0059] (1) 0.5M EDTA (pH 8.0): Dissolve 186.1g Na2EDTA-2H2O in 800ml of water, adjust the pH to 8.0 with solid NaOH, bring the volume to 1000ml, sterilize at high temperature, and store at room temperature;

[0060] (2) 10×TBE buffer: Tris 216g, Boric acid 110g, 0.5M EDTA (pH 8.0) 74.5ml, stir to dissolve, bring to a final volume of 2000ml, store at room temperature;

[0061] (3) 6% Acrylamide gel stock solution: UREA 420.42g, Acrylamide 60g, Bis-acrylamide 3.16g, 10×TBE 50ml, stir to dissolve, then prepare 1000ml with ultrapure water, filter and store at 4℃ or room temperature for later use.

[0062] (4) Loading Buffer: 98% Formamide, 10mM EDTA (pH 8.0), 0.25% BrphBlue, 0.25% X Cynol;

[0063] (5) 2% Repel Silane: Add 10ml Repel Silane to 490ml chloroform, mix and store at room temperature;

[0064] (6) Preparation of 0.5% Binding Silane (prepare fresh for use): Add 5 μl of Binding Silane and 5 μl of glacial acetic acid to 990 μl of anhydrous ethanol;

[0065] (7) 10% Ammonium Persulphate: 2g of ultrapure ammonium persulphate and 18ml of ultrapure water are dissolved and dispensed into 250μl vials using EP tubes and stored at -20℃.

[0066] (8) Primer dilution: The newly synthesized primers were diluted to 200 μM with TE according to the data provided by the synthesis unit and stored in a -20℃ refrigerator for long-term storage; then an appropriate amount of 200 μM was taken and diluted to 2 μM and stored in a 4℃ refrigerator for later use.

[0067] 2. Preparations before electrophoresis testing:

[0068] (1) Cleaning the glass plates: Use hot water and dish soap to repeatedly wipe the glass plates clean, then wipe them dry with alcohol. Apply 2% Repel Silane to one glass plate, then wipe and dry it. Apply 0.5% Binding Silane to the other glass plate. During the operation, prevent the two glass plates from contaminating each other. After they are completely dry, proceed with the glass plate assembly and glue application.

[0069] (2) Assembly of vertical electrophoresis plates and level testing;

[0070] (3) Preparation of polyacrylamide modified adhesive: 6% PA adhesive, 10% ammonium persulfate, TEMED;

[0071] (4) Glue pouring: After shaking the glue well, gently pour it into the glue container along the edge of the nozzle while tapping it lightly to prevent air bubbles. Once the glue has flowed to the bottom, gently insert a comb into the nozzle with the flat end facing down. Allow it to fully polymerize; if leaving the glue overnight, place damp filter paper or plastic wrap on both ends to prevent it from drying out.

[0072] (5) Denaturation of amplification products: Add 5-8 ml of Loading Buffer to the amplification products, denature at 95°C for 5-10 minutes, and then immediately place in an ice-water mixture for later use.

[0073] 3. Electrophoresis of amplification products:

[0074] Preparation of 1×TBE: Add 200ml of 1×10TBE to 1800ml of deionized water and mix well. Add 800ml to the positive electrode tank and preheat 1200ml to 60℃ and add it to the negative electrode tank.

[0075] Add the sample comb and remove air bubbles;

[0076] Pre-electrophoresis: Electrophoresis at a constant power of 100W for 30 minutes. After pre-electrophoresis, remove any urea and air bubbles deposited on the gel surface and insert the sample comb;

[0077] Add 6 μl of denatured amplified DNA sample and electrophoresis for 1 hour;

[0078] 4. Silver staining of amplification products:

[0079] Decolorization and fixation: After electrophoresis, carefully separate the two glass plates. The gel will adhere tightly to the glass plate coated with BindingSilane. Place it in 1L of fixative (895ml distilled water + 100ml anhydrous ethanol + 5ml glacial acetic acid) and shake on a shaker until the indicator is colorless, about 10 minutes.

[0080] Rinse: Rinse the rubber sheet with deionized water for 3-5 minutes;

[0081] Staining: Add 1 liter of staining solution (containing 1.5g AgNO3), gently shake and stain for 10 minutes;

[0082] Rinse: Rinse the rubber sheet with deionized water for no more than 10 seconds;

[0083] Developing: Place the film plate in 1 liter of cold developing solution (1000ml deionized water + 15g NaOH + 3.5ml formaldehyde) and gently shake until the banding appears clearly;

[0084] Stop development / fix: Transfer the film to 10% fixative and stop development / fix for 3-5 minutes;

[0085] Rinse: Rinse the rubber sheet with deionized water for 3-5 minutes;

[0086] Drying: Allow the glued sheets to air dry at room temperature;

[0087] Experimental results recording and analysis: The adhesive sheet can be permanently preserved or photographed.

[0088] 5. 164 pineapple materials are listed in Appendix 1.

[0089] Appendix Table 1: Total sugar content and detection type of 164 pineapple samples used in this invention

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096] Note: A represents type AcoInv-1a; H represents a type that contains both AcoInv-1a and AcoInv-1b.

[0097] II. Experimental Methods:

[0098] 1. Determine the total sugar content:

[0099] Soluble sugar extraction: Accurately weigh 10.0 g of pineapple fruit sample, place it in a centrifuge tube, add 8 mL of 80% ethanol, and extract in an 80°C water bath for 30 minutes. After cooling, centrifuge at 4000 rpm for 5 minutes, collect the supernatant, add another 8 mL of 80% ethanol to the residue, and extract again. Repeat twice. Combine the supernatants from the three extractions in a 100 mL volumetric flask and bring the volume up to 100 mL.

[0100] Determination of total sugar: Take 1 ml of the extract into a test tube, add 5 ml of anthrone reagent, shake well, and measure the color at a wavelength of 620 nm after cooling.

[0101] Anthrone reagent: Dissolve 1 gram of anthrone in 72% H2SO4 solution, bring the volume up to 1000 ml, and store in a brown bottle in the refrigerator for 2-3 weeks.

[0102] Standard curve preparation: Take 6 clean test tubes and add 0, 0.2, 0.4, 0.6, 0.8, and 1.0 ml of glucose standard solution (100 μg / ml) and 1.0, 0.8, 0.6, 0.4, 0.2, and 0 ml of distilled water, respectively. Then add 5 ml of anthrone reagent to each tube. Heat in a boiling water bath for 10 minutes. After cooling, measure the color at a wavelength of 620 nm and record the OD value to plot the standard curve.

[0103] Calculate: C = AN / W

[0104] C—Total sugar content of the sample (mg / g)

[0105] W — Sample weight (g)

[0106] A—Sugar content (mg) obtained from the standard curve.

[0107] N—The ratio of sample extract to sample reaction solution

[0108] 2. Development of molecular markers:

[0109] Sixteen materials with significant differences in total sugar content as measured in step 1 were selected as test materials for marker screening. The DNA of the tender leaves of these 16 materials two weeks after emergence was amplified using the primers in Table 1 of Example 1. The amplification results were sequenced and spliced ​​to obtain the full-length AcoInv-1 gene sequences of the 16 materials.

[0110] DNA extraction, PCR procedures, sequencing, and sequence alignment were performed using the same methods as in Example 1. Multiple sequence alignment was performed using the DNAMAN software, and DNA polymorphism and haplotype analysis were conducted using DNA SP4.9 software. The 16 materials were classified into two haplotypes: AcoInv-1a and AcoInv-1b. The biggest difference between these two types is that AcoInv-1b contains a 19bp deletion fragment in the second intron region. Figure 5 As shown.

[0111] Based on the allelic sequence differences between AcoInv-1a and AcoInv-1b, a co-dominant marker, AcoInv-1X, was developed. Figure 5 .

[0112] The primers designed based on this marker are: forward primer: 5-CTGCGAAATGAACTAGTCAACTC-3; reverse primer: 5-TAGAGAACCGTTCTTGGAATGG-3. These primers amplified the DNA from 16 pineapple samples in this example. The amplification products were subjected to denaturing polyacrylamide gel electrophoresis, and the results showed polymorphism in the amplification products.

[0113] 3. Data Analysis:

[0114] Based on the allelic variation type of the pineapple Inv gene in step 2, the 16 materials were divided into two types. Of the 16 pineapple materials, 12 were of the AcoInv-1a type, meaning a 144bp fragment could be amplified in these materials; the other 4 materials were heterozygous for both AcoInv-1a and AcoInv-1b, capable of simultaneously amplifying both 125bp and 144bp bands. Figure 6 As shown above, the results demonstrate that this molecular marker can be used to identify the allelic variation types of the Inv gene in different pineapple materials.

[0115] Example 4:

[0116] This embodiment uses 164 samples of pineapple material for label verification, including the following steps:

[0117] 1) Detection of 164 pineapple samples using AcoInv-1X labeling showed that 153 samples amplified a 144bp band (AcoInv-1a), and 11 samples amplified both 125bp and 144bp bands (AcoInv-1a and AcoInv-1b). Figure 7 As shown.

[0118] 2) In AcoInv-1a, the total sugar content of the vast majority of materials was concentrated between 14% and 18%, accounting for 82% of the total; while in AcoInv-1a and AcoInv-1b materials, the total sugar content of the vast majority was concentrated between 8% and 14%, accounting for 86% of the total. The comprehensive analysis results of the total sugar content and classification of the 164 materials are shown below. Figure 8 As the sugar content increases, especially when the total sugar content exceeds 14%, the proportion of AcoInv-1a is significantly higher than that of AcoInv-1a and AcoInv-1b.

[0119] 3) Statistical analysis of the average total sugar content also showed that the average total sugar content of materials of type AcoInv-1a was 14.91, while the average total sugar content of materials containing both types AcoInv-1a and AcoInv-1b was 11.29. The average total sugar content of materials of type AcoInv-1a was significantly higher than that of materials containing both types AcoInv-1a and AcoInv-1b (see Table 2). These results indicate that the amplified fragment of this marker is highly significantly correlated with the total sugar content of pineapple fruit.

[0120] Table 2. Types and number of allelic variations and total sugar content of 164 pineapple samples

[0121]

[0122] Note: a and b indicate highly significant differences (P<0.01).

[0123] The above experimental results show that the molecular markers obtained in this invention can be used to select pineapple varieties with high sugar content potential by identifying allelic variations in soluble acid invertase genes.

[0124] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. The application of a primer pair for detecting AcoInv-1a and AcoInv-1b molecular markers in the assisted selection of pineapple varieties with high total sugar content, characterized in that: The nucleotide sequence of the AcoInv-1a molecular marker is shown in SEQ ID NO. 1; The nucleotide sequence of the AcoInv-1b molecular marker is shown in SEQ ID NO.2; Pineapple varieties that only detected the AcoInv-1a molecular marker had higher total sugar content than pineapple varieties that detected both the AcoInv-1a and AcoInv-1b molecular markers.

2. The application as described in claim 1, characterized in that: The forward primer of the primer pair is: 5-CTGCGAAATGAACTAGTCAACTC-3. The reverse primer of the primer pair is: 5-TAGAGAACCGTTCTTGGAATGG-3.

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