A molecular marker for identifying allelic variations in the AcoInv-2 gene, a soluble acid invertase gene in pineapple fruit, and its application.

Abstract

This invention relates to the field of agricultural biotechnology, specifically to a molecular marker and its application for identifying allelic variations of the soluble acid invertase gene AcoInv-2 in pineapple fruit. This molecular marker is amplified using the following primers: forward primer: 5-AGCGAGCACGCCGACATCC-3, reverse primer: 5-CGTCTATATTTGAGGACGACTAGT-3. These primers can identify the allelic variation types of the soluble acid invertase gene AcoInv-2 in pineapple fruit: AcoInv-2a and AcoInv-2b. Studies have shown that both allelic variation types are significantly correlated with the total sugar content of pineapple fruit. Verification has demonstrated that pineapple materials with the AcoInv-2a type are associated with high sugar content, while pineapple materials with both AcoInv-2a and AcoInv-2b types are associated with low sugar content. The application of the molecular marker obtained in this invention in selecting pineapple varieties with high sugar content potential will help 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 in the AcoInv-2 gene of soluble acid invertase 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-2 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 soluble acid invertase gene AcoInv-2 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-2 gene, a soluble acid invertase gene in pineapple fruit. This molecular marker is amplified using the following primers:

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

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

[0011] In the above technical solution, the molecular marker has a nucleotide sequence of 181 bp as shown in Seq ID NO.1.

[0012] In the above technical solution, the molecular marker has a nucleotide sequence of 174 bp as shown in Seq ID NO.2.

[0013] This invention also provides the application of the molecular markers described above, which can identify allelic variations of the soluble acid invertase gene AcoInv-2 in pineapple fruit, in assisting the selection of pineapple varieties with high sugar content potential.

[0014] The specific operating method is as follows:

[0015] (1) Based on the published pineapple genome sequence (pineapple.angiosperms.org), identify the gene similar to soluble acid invertase (Aco017533.1), download the complete information of the sequence, and name it AcoInv-2.

[0016] (2) Gene structure analysis of the obtained pineapple AcoInv-2 gene sequence was performed using software such as DNAMAN and DNAstar. The results showed that the gene is 9298 bp in length. AcoInv-2 is located within the coding region, containing a start codon and a stop codon, 8 exons and 7 introns, as well as a 26 bp 5' untranslated region and a 219 bp 3' untranslated region, as shown in Seq ID No. 3. The AcoInv-2 sequence shows high similarity to other reported Inv genes from other crops, suggesting that the obtained AcoInv-2 is the full-length sequence of the pineapple soluble acid-converting enzyme gene.

[0017] (3) By comparing the AcoInv-2 sequence with the pineapple genome website (pineapple.angiosperms.org), the location and copy number of AcoInv-2 were determined. The results showed that the AcoInv-2 gene is located on chromosome 22 of the pineapple genome and there is only one copy.

[0018] (4) Further bioinformatics analysis was performed on the obtained pineapple AcoInv-2 gene sequence using software such as DNAMAN and DNAstar. The pineapple AcoInv-2 gene cDNA sequence was obtained, with a full length of 1914 bp, as shown in Seq ID No. 4. It showed high similarity to the Inv gene sequences of rice, sugarcane, and maize. The pineapple AcoInv-2 gene cDNA sequence encodes a polypeptide containing 637 amino acids, as shown in Seq ID No. 5. Conserved domain analysis of this protein showed that it contains a complete glycosyl hydrolases family 32 domain, which has the function of hydrolyzing sucrose.

[0019] (5) This invention, by searching the AcoInv-2 gene allelic variation of different materials on the pineapple whole genome website (www.pineapple.angiosperms.org), found a 7bp insertion / deletion site at position 5192bp. Based on this information, we designed a pair of primers (AcoInv-2P) at both ends to ensure coverage of this region. Sixteen materials with significant differences in total sugar content were selected as test materials for marker screening. The DNA of young leaves of these 16 materials two weeks after emergence was amplified using these primers. The amplification results were sequenced, and partial fragments of the AcoInv-2 gene of each of the 16 materials were obtained. 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 materials were divided into two haplotypes, AcoInv-2a and AcoInv-2b. The biggest difference between these two types is that AcoInv-2b has a 7bp deletion fragment in the second intron region.

[0020] (7) Based on the allelic sequence differences between AcoInv-2a and AcoInv-2b, this invention developed a codominant marker, AcoInv-2X (forward primer: 5-AGCGAGCACGCCGACATCC-3; reverse primer: 5-CGTCTATATTTGAGGACGACTAGT-3). This primer pair amplified the DNA of 16 pineapple materials in this example. The amplification products were subjected to denaturing polyacrylamide gel electrophoresis, and the results showed polymorphism in the amplification products.

[0021] (8) Based on the allelic variation type of the pineapple Inv gene, the 16 materials were divided into two types. Of the 16 pineapple materials, 12 were of the AcoInv-2a type, meaning a 181bp fragment could be amplified in these materials; 4 materials were heterozygous for both AcoInv-2a and AcoInv-2b, simultaneously amplifying both 174bp and 181bp bands. These results demonstrate that this marker can be used to identify the allelic variation type of the Inv gene in different pineapple materials.

[0022] Furthermore, this invention was validated by testing 165 pineapple samples using AcoInv-2X labeling:

[0023] 1) 158 materials amplified a 181bp band (AcoInv-2a), and 7 materials amplified both 174bp and 181bp bands (AcoInv-2a and AcoInv-2b); 2) In AcoInv-2a, 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-2a and AcoInv-2b materials, the total sugar content of the vast majority was concentrated between 8% and 14%, accounting for 86% of the total; the comprehensive analysis of the total sugar content and classification of 165 materials showed a tendency towards higher sugar content regions, especially... When the total sugar content exceeded 14 mg / g, the proportion of AcoInv-2a was significantly higher than that of AcoInv-2a and AcoInv-2b. 3) Statistical analysis of the average total sugar content of the two types of pineapple materials showed that the average total sugar content of AcoInv-2a type materials was 14.82, while the average total sugar content of materials containing both AcoInv-2a and AcoInv-2b types was 11.89. The average total sugar content of AcoInv-2a type materials was significantly higher than that of AcoInv-2a and AcoInv-2b types. These 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 structure diagram of the pineapple AcoInv-2 gene obtained in Example 1 is shown.

[0026] Figure 2 The location of the pineapple AcoInv-2 gene on the chromosome obtained in Example 2.

[0027] Figure 3 Analysis of the functional domains of the protein encoded by the pineapple AcoInv-2 gene obtained in Example 2.

[0028] Figure 4 The results of 1% agarose gel electrophoresis of the pineapple DNA extract obtained in Example 3 are shown.

[0029] Figure 5 The sequence alignment (partial) of pineapple AcoInv-2a and pineapple AcoInv-2b obtained in Example 3.

[0030] Figure 6 The results of polypropylene gel electrophoresis of the 16 pineapple materials selected in Example 3 are shown.

[0031] Figure 7 Example 4 shows the results of polypropylene gel electrophoresis of 165 pineapple samples using AcoInv-2X.

[0032] Figure 8 Example 4 utilizes statistical analysis of allelic variation types and total sugar content of 165 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] Obtaining the full length of the pineapple soluble acid invertase gene (Aco016281.1, AcoInv-2):

[0036] (1) Log in to the published pineapple genome website (www.pineapple.angiosperms.org) and find the Blast function module;

[0037] (2) Using the known sugarcane soluble invertase gene ShinvA (AY302083) sequence as a probe, the sequence was compared using the Blast search function;

[0038] (3) The comparison results revealed a gene sequence with a similarity greater than 80% (Aco017533.1). According to its gene annotation information, this gene is a soluble acid invertase gene.

[0039] (4) Download the complete information of this gene sequence and name it AcoInv-2.

[0040] Example 2:

[0041] The bioinformatics analysis of the pineapple soluble acid invertase gene (Aco017533.1, AcoInv-2) in this embodiment is as follows:

[0042] (1) The obtained pineapple AcoInv-2 gene sequence was analyzed using software such as DNAMAN and DNAstar. The results showed that the gene is 9298 bp in length. AcoInv-2 is located within the coding region, containing a start codon and a stop codon, 8 exons and 7 introns, as well as a 26 bp 5' untranslated region and a 219 bp 3' untranslated region. Figure 1 As shown in SeqID No. 3, the AcoInv-2 sequence has a high similarity to other reported crop Inv genes.

[0043] (2) By comparing the AcoInv-2 sequence with the sequence from the pineapple genome website (pineapple.angiosperms.org), the location and copy number of AcoInv-2 were determined. The results showed that the AcoInv-2 gene is located on chromosome 22 of the pineapple genome, and there is only one copy. Figure 2 As shown.

[0044] (3) The obtained pineapple AcoInv-2 gene sequence was further analyzed using software such as DNAMAN and DNAstar, and the pineapple AcoInv-2 gene cDNA sequence was obtained. The full length of the sequence is 1914bp, as shown in Seq ID No.4, and it has a high similarity to the Inv gene sequences of rice, sugarcane and maize.

[0045] (4) Using the protein analysis function on the NCBI website, the cDNA sequence of the pineapple AcoInv-2 gene was found to encode a polypeptide containing 637 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, as shown in... Figure 3 As shown.

[0046] Example 3:

[0047] The allelic variation analysis and the development of molecular markers for the pineapple AcoInv-2 gene in this embodiment are performed as follows:

[0048] I. Experimental Materials

[0049] 1. The 165 pineapple varieties are shown in Appendix 1.

[0050] 2. Reagents and medicines:

[0051] (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;

[0052] (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;

[0053] (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.

[0054] (4) Loading Buffer: 98% Formamide, 10mM EDTA (pH 8.0), 0.25% BrphBlue, 0.25% X Cynol;

[0055] (5) 2% Repel Silane: Add 10ml Repel Silane to 490ml chloroform, mix and store at room temperature;

[0056] (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;

[0057] (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℃.

[0058] (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.

[0059] 3. DNA extraction from pineapple samples:

[0060] The DNA extraction methods and reagents were based on those of Sambrook et al. (Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual, Edition. Cold Spring Habour Laboratory Press, New York, 1989).

[0061] 4. PCR procedure:

[0062] Pre-denaturation at 94℃ for 3 min, denaturation at 94℃ for 30 s, annealing at 61℃ for 30 s, extension at 72℃ for 90 s. A total of 30 cycles were performed, with a final extension at 72℃ for 10 min, followed by storage at 4℃. Gel electrophoresis: 5 μl was transferred to a 1% agarose gel for electrophoresis and analyzed using a Bio-Rad gel imaging system.

[0063] 5. PCR product recovery:

[0064] PCR product recovery was performed according to the instructions for the Tiangen Biotech DNA Recovery Kit. Ligation, transformation, and cloning verification of the recovered products were performed according to the instructions for the pGEM-T Easy Vector System from TransGen Biotech Ltd. Taq DNA polymerase was TaKaRa's EX Taq, the cloning kit was Promega's pGEM-T Easy Vector System, agarose was purchased from Genentech, and the DNA recovery kit, dNTPs, and DNA marker were purchased from Tiangen Biotech Ltd.

[0065] 6. Sequencing:

[0066] 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.

[0067] 7. 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] 8. 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 sample DNA and electrophoresis for 1 hour.

[0078] 9. 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] Appendix Table 1: Total sugar content and detection type of 165 pineapple samples used in this invention

[0089]

[0090]

[0091]

[0092]

[0093]

[0094] Note: A represents type AcoInv-2a; H represents type that contains both AcoInv-2a and AcoInv-2b.

[0095] II. Experimental Methods:

[0096] 1. Determine the total sugar content:

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] Calculate: C = AN / W

[0102] C—Total sugar content of the sample (mg / g)

[0103] W — Sample weight (g)

[0104] A—Sugar content (mg) obtained from the standard curve.

[0105] N—The ratio of sample extract to sample reaction solution

[0106] 2. Development of molecular markers:

[0107] DNA was extracted from pineapple material. The DNA extraction results are as follows: Figure 4 As shown, a clear single band is visible, indicating that the DNA quality is reliable and can be used for subsequent experiments. Sixteen materials with significant differences in total sugar content as measured in step 1 were selected as test materials for label screening. DNA from the tender leaves of these 16 materials two weeks after emergence was amplified using the primers (AcoInv-2P) in Table 1. The amplification results were sequenced to obtain partial fragment sequences of the AcoInv-2 gene for each of the 16 materials. DNA extraction, PCR procedures, sequencing, and sequence alignment were performed using the same methods as above.

[0108] Table 1. Primers used for allelic variation analysis and marker development in the pineapple AcoInv-2 gene

[0109]

[0110] 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 materials were classified into two haplotypes: AcoInv-2a and AcoInv-2b. The biggest difference between these two types is that AcoInv-2b contains a 7bp deletion fragment in the second intron region. Figure 5 As shown.

[0111] A co-dominant marker, AcoInv-2X, was developed based on the allelic sequence differences between AcoInv-2a and AcoInv-2b. Figure 5 As shown.

[0112] Primers were designed based on this marker: forward primer: 5-AGCGAGCACGCCGACATCC-3; reverse primer: 5-CGTCTATATTTGAGGACGACTAGT-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, 14 were of the AcoInv-2a type, meaning a 181bp fragment could be amplified in these materials; the other two materials were heterozygous for AcoInv-2a and AcoInv-2b, capable of simultaneously amplifying both 174bp and 181bp bands. Figure 6 As shown above, the results demonstrate that this 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 165 samples of pineapple material for label verification, including the following steps:

[0117] 1) Detection of 165 pineapple samples using AcoInv-2X labeling showed that 158 ​​samples amplified a 181bp band (AcoInv-2a), and 7 samples amplified two bands (AcoInv-2a and AcoInv-2b) at 174bp and 181bp respectively. Figure 7 As shown.

[0118] 2) In AcoInv-2a, the total sugar content of the vast majority of materials was concentrated between 14% and 18%, accounting for more than 80% of the total; while in AcoInv-2a and AcoInv-2b materials, the total sugar content of the vast majority was concentrated between 12% and 14%, accounting for 84% of the total. The comprehensive analysis results of the total sugar content and classification of the 165 materials are shown below. Figure 8 As the sugar content increases, especially when the total sugar content exceeds 14%, the proportion of AcoInv-2a is significantly higher than that of AcoInv-2a and AcoInv-2b.

[0119] 3) Statistical analysis of the average total sugar content also showed that the average total sugar content of materials of type AcoInv-2a was 14.82, while the average total sugar content of materials containing types AcoInv-2a and AcoInv-2b was 11.89. The average total sugar content of materials of type AcoInv-2a was significantly higher than that of materials containing types AcoInv-2a and AcoInv-2b (see Table 2). The above 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 165 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. A molecular marker for the identification of a gene for soluble acid invertase in the fruit of a pineapple AcoInv-2 an allelic variant, characterized in that: The molecular marker is amplified by using the following primers: the allelic variation is AcoInv-2a and AcoInv-2b, wherein AcoInv-2b has a 7bp deletion in the second intron segment; Forward primer: 5-AGCGAGCACGCCGACATCC-3, Reverse primer: 5-CGTCTATATTTGAGGACGACTAGT-3; The molecular marker is a 181bp nucleotide sequence as shown in Seq ID NO.1, corresponding to AcoInv-2a allelic variation; The molecular marker is a 174bp nucleotide sequence as shown in Seq ID NO.2, corresponding to AcoInv-2b allelic variation; When the amplification product is a 181bp nucleotide sequence as shown in Seq ID NO.1, corresponding to AcoInv-2a allelic variation, it corresponds to a high-sugar-content pineapple variety; When the amplification product is a 181bp nucleotide sequence as shown in Seq ID NO.1 and a 174bp nucleotide sequence as shown in Seq ID NO.2, corresponding to AcoInv-2b allelic variation, it corresponds to a low-sugar-content pineapple variety.

2. Use of a molecular marker capable of discriminating allelic variations of the soluble acid invertase gene AcoInv-2 in the assisted selection of pineapple varieties with high potential for high sugar content, according to claim 1, characterized in that: wherein, When the amplification product is a 181bp nucleotide sequence as shown in Seq ID NO.1, corresponding to AcoInv-2a allelic variation, it corresponds to a high-sugar-content pineapple variety; When the amplification product is a 181bp nucleotide sequence as shown in Seq ID NO.1 and a 174bp nucleotide sequence as shown in Seq ID NO.2, corresponding to AcoInv-2b allelic variation, it corresponds to a low-sugar-content pineapple variety.

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