CN116064901A - Molecular marker capable of identifying allelic variation of pineapple fruit soluble acid invertase gene AcoInv-2 and application thereof - Google Patents
Molecular marker capable of identifying allelic variation of pineapple fruit soluble acid invertase gene AcoInv-2 and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of agricultural biotechnology, in particular to a molecular marker capable of identifying pineapple fruit soluble acid invertase gene AcoInv-2 allelic variation and application thereof. The molecular marker is amplified by the following primers: forward primer: 5-AGCGAGCACGCCGACATCC-3, reverse primer: 5-CGTCTATATTTGAGGACGACTAGT-3, which can identify the type of allelic variation of the soluble acid invertase gene AcoInv-2 in pineapple fruit: acoInv-2a and AcoInv-2b. Through researches, the two allelic variation types are extremely obviously related to the total sugar content of pineapple fruits, and the verification proves that the pineapple material of the AcoInv-2a type is related to high sugar content, and the pineapple material with the AcoInv-2a type and the AcoInv-2b type is related to low sugar content; the application of the molecular marker obtained by the invention in selecting pineapple varieties with high sugar content potential is helpful for accelerating the breeding selection period.
Description
Technical Field
The invention relates to the technical field of agricultural biotechnology, in particular to a molecular marker capable of identifying pineapple fruit soluble acid invertase gene AcoInv-2 allelic variation and application thereof.
Background
Pineapple is a common tropical fruit tree, and the fruit is rich in sugar and nutrient components, has unique taste and flavor, and is a fruit which is popular. Although the outside natural environment plays a certain role in regulating and controlling sugar accumulation in fruits, the sugar accumulation of pineapple fruits is mainly regulated and controlled by genetic factors. The study finds that the physical and chemical characteristics of pineapple in different harvest seasons are obviously different, and the content of soluble solids in fruits harvested in winter is higher than that in fruits harvested in other seasons. The previous research results show that the sugar content difference between different varieties of pineapple is obvious and is mainly controlled by genetic factors, the sugar content of fruits of different pineapple varieties can be 1-2 times different, and the pineapple varieties are genetically stable. Pineapple belongs to perennial plants, and hybridization is very difficult due to the problems of self-incompatibility and the like. The current breeding technology mainly comprises the steps of configuring hybridization combination and screening proper materials from offspring. However, the method has the advantages of high cost, long period, and insufficient knowledge of the genetic background of the parent, and the selection and breeding process mainly depends on manual experience, so that the selection and breeding process lacks scientific basis, and the selection of offspring materials is difficult. Along with the completion of the whole genome sequencing work of pineapple, the development of molecular markers closely related to sugar content becomes possible, and parent materials can be screened according to requirements, so that a new method and a new way for breeding new pineapple varieties with proper sugar content are provided.
Sucrose Invertase (Invertase, inv) plays a key role in sucrose metabolism in higher plants. Research shows that sucrose invertase is involved in plant growth, organ establishment, sugar transportation, phloem unloading and regulation of the composition and level of sugar in the warehouse tissue, inv can irreversibly catalyze the hydrolysis of sucrose into glucose and fructose, and is an important regulatory enzyme in sucrose decomposition. According to different classification methods, they can be classified into different families, according to their water solubility, into soluble and insoluble Inv, according to their optimum pH, and into two classes, acidic Inv and neutral/basic Inv, whose main functions are to degrade sucrose, except for the presence of sites; soluble acidic Inv, which is mainly present in vacuoles and catalyzes the hydrolysis of sucrose to hexose, plays a role in regulating accumulation of sugar in plant tissues and utilization of sucrose in vacuoles, has a structure which is very different from that of neutral invertase, but has been studied less with respect to its protein structure and properties. Inv plays a key role in sucrose accumulation in vacuoles of mature pool organs. The activity of Inv in the vacuoles has a large effect on sucrose content. The Inv gene DNA sequence or cDNA sequence has been successfully cloned from crops such as tomato, arabidopsis, carrot, potato, sugarcane, rice, sweet sorghum, and the molecular markers of tomato and sweet sorghum have been successfully developed, but no report on the cloning and molecular markers of pineapple Inv genes has been found.
Therefore, it is necessary to develop a molecular marker for identifying allelic variation of pineapple fruit soluble acid invertase.
Disclosure of Invention
One of the purposes of the present invention is to provide a molecular marker for identifying allelic variation of pineapple fruit soluble acid invertase gene AcoInv-2, which is selected from allelic variation of Inv gene, and is helpful for breeding workers to find excellent parent material from a plurality of pineapple varieties without carrying out identification through complicated cultivation tests and measurement.
The second object of the present invention is to provide the application of the molecular marker for identifying allelic variation of pineapple fruit soluble acid invertase gene acoInv-2 in auxiliary selection of pineapple varieties with high sugar content potential.
The aim of the invention is achieved by the following technical scheme:
providing a molecular marker capable of identifying allelic variation of pineapple fruit soluble acid invertase gene acoInv-2, wherein the molecular marker is amplified by adopting the following primers:
forward primer: 5-AGCGAGCACGCCGACATCC-3 of the total weight of the plant,
reverse primer: 5-CGTCTATATTTGAGGACGACTAGT-3.
In the above technical scheme, the molecular marker has a nucleotide sequence of 181bp as shown in Seq ID No. 1.
In the above technical scheme, the molecular marker has a nucleotide sequence of 174bp as shown in Seq ID No. 2.
The invention also provides application of the molecular marker capable of identifying the allelic variation of the pineapple fruit soluble acid invertase gene AcoInv-2 in auxiliary selection of pineapple varieties with high sugar content potential.
The specific operation method is as follows:
(1) Based on published pineapple whole genome sequence (pineapple. Angiospers. Org), a gene similar to the soluble acid invertase was found (aco017533.1), and the complete information of this sequence was downloaded and named AcoInv-2.
(2) The obtained pineapple AcoInv-2 gene sequence was subjected to genetic structural analysis using software such as DNAMAN, DNAstar. As a result, it was found that the gene was 9298bp in its entire length, acoInv-2 contained an initiation codon and a termination codon within the coding region, 8 exons and 7 introns were present, and 15 'untranslated region of 26bp and 1 3' untranslated region of 219bp, as shown in Seq ID No. 3. The AcoInv-2 sequence has high similarity to other crop Inv genes that have been reported, and it is speculated that the AcoInv-2 obtained is the full-length sequence of the soluble acid transferase gene of pineapple.
(3) The position and copy number of AcoInv-2 was determined by aligning the AcoInv-2 with the pineapple genome website (org) sequence, which showed that the AcoInv-2 gene was on chromosome 22 and only one copy of the pineapple genome.
(4) The obtained pineapple AcoInv-2 gene sequence was further analyzed bioinformatically using software such as DNAMAN, DNAstar. A pineapple AcoInv-2 gene cDNA sequence with a full length of 1914bp as shown in Seq ID No.4 was obtained, which showed high sequence similarity with rice, sugarcane, maize Inv genes. The pineapple AcoInv-2 gene cDNA sequence encodes 1 polypeptide containing 637 amino acids as shown in Seq ID No. 5; the result of the conserved domain analysis of the protein shows that the protein contains a complete glycosyl hydrolase (Glycosyl hydrolases family 32) domain which has the function of hydrolyzing sucrose.
(5) The invention finds that a 7bp insertion/deletion site exists at 5192bp position by searching the allelic variation condition of AcoInv-2 genes of different materials on pineapple whole genome website (www.pineapple.angiosperms.org). Based on this information we designed a pair of primers (AcoInv-2P) at both ends, guaranteeing information that can cover this area. 16 materials with larger total sugar content difference are selected as test materials for marker screening, the primers are used for amplifying DNA of tender leaves after the 16 materials emerge for two weeks, and the amplification results are sequenced to obtain AcoInv-2 gene part fragments of the 16 materials respectively. Sequence alignment was performed using the multiplex sequence alignment program in DNAMAN software, DNA polymorphism analysis and haplotype analysis were performed using the dnaSP 4.9 software, and 16 materials were divided into 2 haplotypes, acoInv-2a and AcoInv-2b, respectively. The largest difference between these two types is the presence of a 7bp deletion in the second intron region of AcoInv-2b.
(7) The present invention developed a co-dominant marker AcoInv-2X based on allelic variation sequence differences between AcoInv-2a and AcoInv-2b (forward primer: 5-AGCGAGCACGCCGACATCC-3; reverse primer: 5-CGTCTATATTTGAGGACGACTAGT-3). The pair of primers amplify 16 parts of pineapple material DNA in the embodiment, and the amplified products are subjected to denaturing polyacrylamide gel electrophoresis, so that polymorphism of the amplified products is shown.
(8) 16 parts of material were divided into 2 types according to the pineapple Inv allelic variation type. Of 16 parts of pineapple material, 12 parts of the pineapple material is of the AcoInv-2a type, and a 181bp fragment can be amplified from the material; there were 4 parts of heterozygous materials of AcoInv-2a and AcoInv-2b, and both 174bp and 181bp bands could be amplified simultaneously. The above results demonstrate that this marker can be used to identify allelic variation types of Inv genes from different pineapple materials.
Further, the invention verifies that 165 parts of pineapple material are detected with an AcoInv-2X mark:
1) 158 parts of material amplified a 181bp band (AcoInv-2 a) and 7 materials amplified 174bp and 181bp bands (AcoInv-2 a and AcoInv-2 b); 2) In AcoInv-2a, the vast majority of the total sugar content of the material is concentrated between 14 and 18%, accounting for 82% of the total; whereas in AcoInv-2a and AcoInv-2b materials, the vast majority of total sugar content is concentrated between 8-14%, accounting for 86% of the total; for 165 parts of material, the more prone to high sugar content regions, particularly at total sugar content above 14mg/g, the more significant the AcoInv-2a form is than the AcoInv-2a and AcoInv-2b forms; 3) The statistical analysis of the average value of the total sugar content of fruits of the pineapple materials of two types shows that the average value of the total sugar content of the materials of the AcoInv-2a type is 14.82, the average value of the total sugar content of the materials containing the AcoInv-2a type and the AcoInv-2b type is 11.89, and the average value of the total sugar content of the materials of the AcoInv-2a type is extremely higher than that of the materials of the AcoInv-2a type and the AcoInv-2b type. The above results indicate that the amplified fragment of the marker is very significantly correlated with the total sugar content of pineapple fruit.
Drawings
The invention will be further described with reference to the accompanying drawings, in which embodiments do not constitute any limitation of the invention, and other drawings can be obtained by one of ordinary skill in the art without inventive effort from the following drawings.
FIG. 1 is a diagram showing the structure of the pineapple AcoInv-2 gene obtained in example 1.
FIG. 2 shows the position of the pineapple AcoInv-2 gene obtained in example 2 on a chromosome.
FIG. 3 is a functional domain analysis of the encoded protein of pineapple AcoInv-2 gene obtained in example 2.
FIG. 4 shows the result of 1% agarose gel electrophoresis of pineapple DNA extract obtained in example 3.
FIG. 5 is a sequence alignment (part) of pineapple AcoInv-2a and pineapple AcoInv-2b obtained in example 3.
FIG. 6 shows the result of electrophoresis of 16 parts of pineapple material selected in example 3.
FIG. 7 shows the result of polypropylene gel electrophoresis of 165 parts of pineapple material detected by AcoInv-2X in example 4.
FIG. 8 is a statistical analysis of the allelic variation types and total sugar content of 165 parts pineapple material used in example 4.
Detailed Description
The invention will be further described with reference to the following examples and figures.
Example 1:
full-length acquisition of pineapple soluble acid invertase gene (aco016281.1, acoInv-2):
(1) Logging in a published pineapple whole genome website (www.pineapple.angiosperms.org) to find a Blast function module;
(2) The known sequence of the sugarcane soluble invertase gene ShinvA (AY 302083) is used as a probe, and the comparison is carried out through Blast retrieval function;
(3) The comparison result shows that 1 gene sequence (aco017533.1) with the similarity being more than 80 percent is found, and the gene is a soluble acid invertase gene according to the gene annotation information;
(4) The complete information of this gene sequence was downloaded and designated AcoInv-2.
Example 2:
the bioinformatics analysis of the pineapple soluble acid invertase gene (aco017533.1, acoInv-2) of this example is as follows:
(1) The obtained pineapple AcoInv-2 gene sequence was subjected to genetic structural analysis using software such as DNAMAN, DNAstar. As a result, it was found that the gene was 9298bp in its entire length, acoInv-2 contained an initiation codon and a termination codon within the coding region, 8 exons and 7 introns were present, and 15 'untranslated region of 26bp and 1 3' untranslated region of 219bp were present, as shown in FIG. 1 and Seq ID No. 3. The AcoInv-2 sequence has high similarity to other crop Inv genes that have been reported.
(2) The position and copy number of AcoInv-2 was determined by aligning AcoInv-2 with pineapple genomic website (pineapple. Angiospers. Org) sequences, which showed that the AcoInv-2 gene was on chromosome 22 of the pineapple genome and that there was only one copy, as shown in FIG. 2.
(3) The pineapple AcoInv-2 gene sequence obtained is further analyzed by using DNAMAN, DNAstar and other software to obtain the pineapple AcoInv-2 gene cDNA sequence, the total length of the sequence is 1914bp, and the sequence has high sequence similarity with rice, sugarcane and corn Inv genes as shown in Seq ID No. 4.
(4) Using the protein analysis function on NCBI website, it was found that the cDNA sequence of pineapple AcoInv-2 gene encoded 1 polypeptide containing 637 amino acids, as shown in Seq ID No. 5. The result of the conserved domain analysis of the protein shows that the protein contains a complete glycosylhydrolase (Glycosyl hydrolases family 32) domain which has the function of hydrolyzing sucrose, as shown in figure 3.
Example 3:
the allelic variation analysis and pineapple acoInv-2 gene molecular marker development of this example were performed as follows:
1. experimental materials
1. 165 parts of pineapple variety material are shown in the accompanying table 1.
2. Reagent and medicine:
(1)0.5M EDTA(pH 8.0)：186.1g Na 2 EDTA-2H 2 dissolving O in 800ml of water, regulating pH to 8.0 with solid NaOH, fixing volume to 1000ml, sterilizing at high temperature, and preserving at room temperature;
(2) 10 XTBE buffer: tris 216g, boric acid 110g,0.5M EDTA (pH 8.0) 74.5ml, stirring and dissolving, fixing the volume to 2000ml, and preserving at room temperature;
(3) 6% Acrylamide gum stock: UREA 420.42g,Acrylamide 60g,Bis-acrylamid 3.16g,10 XTBE 50ml, stirring to dissolve, preparing 1000ml with ultrapure water, filtering, and placing in a refrigerator at 4 ℃ or room temperature for standby;
(4) Loading Buffer:98% formamide, 10mM EDTA (pH 8.0), 0.25% Brph Blue, 0.25% XCynol;
(5) 2% repeat Silane:490ml chloroform, 10ml Repel Silane was added, and the mixture was mixed and stored at room temperature;
(6) 0.5%Binding Silane (as-prepared): mu.l of Binding Silane and 5. Mu.l of glacial acetic acid are added to 990. Mu.l of absolute ethanol;
(7) 10% ammonium persulfate (Ammonium Persulphate): 2g of ultrapure ammonium persulfate and 18ml of ultrapure water, and after dissolution, packaging 250 μl by using an EP tube, and preserving at-20 ℃;
(8) Dilution of primers: diluting the newly synthesized primer to 200 mu M by TE according to the data provided by the synthesis unit, and storing in a refrigerator at-20 ℃ for a long time; then, an appropriate amount of 200. Mu.M was used to dilute the primers to 2. Mu.M and placed in a refrigerator at 4℃for use.
3. Extraction of pineapple sample DNA:
methods and reagents for DNA extraction are described in Sambrook et al (Sambrook J, fritsch E.F., maniatis T.molecular cloning: a laboratory manual, edition. Cold Spring Habour Laboratory Press, new York, 1989).
4. PCR procedure:
pre-denatured at 94℃for 3min, denatured at 94℃for 30s, renatured at 61℃for 30s, and extended at 72℃for 90s. For 30 cycles, the mixture was extended at 72℃for 10min and stored at 4 ℃. Gel electrophoresis: mu.l of the gel was subjected to electrophoresis on a 1% agarose gel, and analyzed by a gel imaging system of Bio-Rad Co.
5. And (3) PCR product recovery:
PCR product recovery was referred to the day root biochemical DNA recovery kit instructions. Ligation, transformation and cloning validation of recovered product: reference is made to pGEM-T Easy Vector System specification of full gold biotechnology Co. The Taq DNA polymerase adopts EXTaq from TaKaRa, the cloning kit adopts pGEM-T Easy Vector System from Promega, agarose is purchased from Gene tech, and the DNA recovery kit, dNTP and DNA Marker are purchased from Tiangen biochemical technology.
6. Sequencing:
positive clones were sent to Shanghai Biotechnology Co.Ltd for sequencing. Each PCR product sequencing was repeated 2-4 times to ensure that the correct nucleotide sequence was obtained.
7. Preparation before electrophoresis detection:
(1) Cleaning of the glass plate: repeatedly scrubbing the glass plate with a detergent, and drying with alcohol. One of the glass plates was coated with 2% repeat Silane and then scrubbed and dried. The other glass plate was coated with 0.5%Binding Silane. In the operation process, the two glass plates are prevented from being polluted by each other, and the glass plates are assembled and glued after being thoroughly dried;
(2) Assembling a vertical electrophoresis plate and detecting by a level meter;
(3) Preparing polyacrylamide modified glue: 6% of PA glue, 10% of ammonium persulfate and TEMED;
(4) And (3) glue filling: after shaking up the glue, the glue is gently poured in along the edge of the glue pouring opening, and is gently beaten at the same time, so that air bubbles are prevented. When the glue flows to the bottom, the comb is inserted into the glue pouring port lightly, and the flat port faces downwards. Allowing them to polymerize sufficiently; if the glue is allowed to stand overnight, wet filter paper or preservative film should be paved at the two ends of the glue to prevent the glue from drying;
(5) Denaturation of amplified products: adding 5-8ml Loading Buffer,95 ℃ denaturation for 5-10 minutes into the amplified product, and immediately placing into an ice-water mixture for later use.
8. Electrophoresis of amplified products:
preparation of 1×tbe: 200ml of 1X 10TBE was added to 1800ml of deionized water and mixed well. Wherein 800ml is added into the positive electrode tank, 1200ml is preheated to 60 ℃, and added into the negative electrode tank;
adding a sample comb to remove bubbles;
pre-electrophoresis: 100W constant power electrophoresis for 30 minutes. After the pre-electrophoresis is finished, urea and bubbles deposited on the rubber surface are removed, and a sample comb is inserted;
6. Mu.l of denatured amplified sample DNA was added thereto, and the mixture was electrophoresed for 1 hour.
9. Silver staining and color development of amplified products:
decoloring and fixing: after electrophoresis, the two glass plates are carefully separated, and the adhesive is tightly adhered to the glass plates coated with Binding Silane; placing in 1L fixing solution (895 ml distilled water +100ml absolute ethanol +5ml glacial acetic acid), and shaking on a shaker until the indicator is colorless for about 10min;
flushing: rinsing the rubber plate with deionized water for 3-5 minutes;
dyeing: 1 liter of staining solution (containing 1.5g of AgNO3) was added, and the mixture was gently shaken for staining for 10 minutes;
flushing: rinsing the glue plate with deionized water for no more than 10 seconds;
developing: the plate was placed in 1 liter of cold developer (1000 ml of ionized water +15g naoh +3.5ml formaldehyde) and gently shaken until the streaks clearly appeared;
stop display/fixation: transferring the rubber plate to 10% fixing solution for stopping display/fixation for 3-5 min;
flushing: rinsing the rubber plate with deionized water for 3-5 minutes;
and (3) drying: naturally drying the rubber plate at room temperature;
recording and analyzing experimental results: the glue plate can be permanently stored or photographed.
Table 1 the total sugar content and detection type of 165 parts pineapple material used in the present invention
Note that: a represents AcoInv-2a type; h represents a type containing both AcoInv-2a and AcoInv-2b.
2. The experimental method comprises the following steps:
1. determining the total sugar content:
soluble sugar extraction: accurately weighing 10.0 g of pineapple fruit sample, placing into a centrifuge tube, adding 8ml of 80% ethanol, leaching in a water bath at 80 ℃ for 30 minutes, cooling, centrifuging at 4000 rpm for 5 minutes, collecting supernatant, adding 8ml of 80% ethanol into residues, leaching again, repeating twice, combining the three extracted supernatants into a 100ml volumetric flask, and fixing the volume to 100 ml.
Determination of total sugar: taking 1 ml of the extract, adding 5ml of anthrone reagent into a test tube, shaking uniformly, and colorizing at 620nm wavelength after cooling.
Anthrone reagent: 1 g of anthrone is dissolved in 72% of H 2 SO 4 And (3) in the solution, the volume is fixed to 1000ml, and the solution is stored in a brown bottle refrigerator for 2 to 3 weeks.
Drawing a standard curve: taking 6 dry test tubes, sequentially adding glucose standard solution (100 mug/ml) 0, 0.2, 0.4, 0.6, 0.8, 1.0ml and distilled water 1.0, 0.8, 0.6, 0.4, 0.2 and 0ml, respectively adding anthrone reagent 5ml, heating in a boiling water bath for 10 minutes, cooling, comparing colors at 620nm wavelength, recording OD value, and drawing a standard curve.
And (3) calculating: c=an/W
C-Total sugar content of sample (mg/g)
W-weight of sample (g)
A-sugar amount (mg) found by standard curve
N-sample extracting solution is a multiple of sample reaction solution
2. Development of molecular markers:
the pineapple material DNA is extracted, the DNA extraction result is shown in figure 4, and a single obvious band can be seen, which shows that the DNA has reliable quality and can be used for the subsequent experiments. The 16 materials with larger difference of total sugar content measured in the step 1 are selected as test materials for marker screening, the DNA of tender leaves after two weeks of emergence of the 16 materials is amplified by using the primer (AcoInv-2P) in the table 1, and the amplified results are sequenced to obtain the partial fragment sequences of the AcoInv-2 genes of the 16 materials respectively. DNA extraction, PCR procedure, sequencing, sequence comparison and equivalent method.
TABLE 1 allelic variation analysis and marker development primers for pineapple AcoInv-2 gene
Sequence alignment was performed using the multiplex sequence alignment program in DNAMAN software, DNA polymorphism analysis and haplotype analysis were performed using the dnaSP 4.9 software, and 16 materials were divided into 2 haplotypes, acoInv-2a and AcoInv-2b, respectively. The largest difference between these two types is the presence of a 7bp deletion in the second intron region of AcoInv-2b, as shown in FIG. 5.
A co-dominant marker AcoInv-2X was developed based on allelic variation sequence differences between AcoInv-2a and AcoInv-2b, as shown in FIG. 5.
Primers were designed based on this label, forward primer: 5-AGCGAGCACGCCGACATCC-3; reverse primer: 5-CGTCTATATTTGAGGACGACTAGT-3. The pair of primers amplify 16 parts of pineapple material DNA in the embodiment, and the amplified products are subjected to denaturing polyacrylamide gel electrophoresis, so that polymorphism of the amplified products is shown.
3. Data analysis:
through the experiment of step 2, 16 parts of material were divided into 2 types according to the allelic variation type of pineapple Inv gene. Of 16 parts of pineapple materials, 14 parts of materials are of the AcoInv-2a type, and a 181bp fragment can be amplified from the materials; there were 2 parts of heterozygous materials of AcoInv-2a and AcoInv-2b, and both 174bp and 181bp bands could be amplified simultaneously, as shown in FIG. 6. The above results demonstrate that this marker can be used to identify allelic variation types of Inv genes from different pineapple materials.
Example 4:
the verification of the mark using 165 parts pineapple material in this example includes the following steps:
1) 165 parts of pineapple material were detected with the AcoInv-2X label, 158 parts of material amplified a 181bp band (AcoInv-2 a) and 7 materials amplified 174bp and 181bp bands (AcoInv-2 a and AcoInv-2 b), as shown in FIG. 7.
2) In AcoInv-2a, the total sugar content of most materials is concentrated between 14 and 18 percent, and accounts for more than 80 percent of the total weight; whereas in AcoInv-2a and AcoInv-2b materials, the vast majority of total sugar content is concentrated between 12-14%, accounting for 84% of the total; as a result of comprehensive analysis of total sugar content and classification of 165 parts of material, see FIG. 8, the more tends to be in the region of high sugar content, particularly after the total sugar content is higher than 14, acoInv-2a type is significantly more than AcoInv-2a and AcoInv-2b type.
3) Statistical analysis of the total sugar content mean also showed that the average total sugar content of AcoInv-2a type material was 14.82, while the average total sugar content of AcoInv-2a and AcoInv-2b type containing materials was 11.89, the average total sugar content of AcoInv-2a type material was significantly higher than AcoInv-2a and AcoInv-2b type materials, see table 2. The above results indicate that the amplified fragment of the marker is very significantly correlated with the total sugar content of pineapple fruit.
TABLE 2 allelic variation type, number and total sugar content of 165 parts pineapple material
Note that: a and b represent very significant differences (P < 0.01)
The experimental result shows that the molecular marker obtained by the invention can select pineapple varieties with high sugar content potential by identifying allelic variation of the soluble acid invertase gene.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.
Claims (4)
1. A molecular marker for identifying allelic variation of pineapple fruit soluble acid invertase gene AcoInv-2, characterized in that: the molecular marker is amplified by adopting the following primers:
forward primer: 5-AGCGAGCACGCCGACATCC-3 of the total weight of the plant,
reverse primer: 5-CGTCTATATTTGAGGACGACTAGT-3.
2. A molecular marker for identifying allelic variation of pineapple fruit soluble acid invertase gene AcoInv-2 as claimed in claim 1, wherein: the molecular marker has a nucleotide sequence of 181bp as shown in Seq ID No. 1.
3. A molecular marker for identifying allelic variation of pineapple fruit soluble acid invertase gene AcoInv-2 as claimed in claim 1, wherein: the molecular marker has a nucleotide sequence of 174bp as shown in Seq ID No. 2.
4. Use of a molecular marker for identifying allelic variation of pineapple fruit soluble acid invertase gene AcoInv-2 as claimed in any of claims 1 to 3 in assisting selection of pineapple varieties with high sugar content potential.
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