Molecular marker for identifying pineapple peel coloring and application

Abstract

The invention relates to the technical field of genetic engineering, in particular to a molecular marker for identifying pineapple peel coloring and application. The AcGLK gene is determined as a candidate gene for determining pineapple chlorophyll synthesis, a specific primer group is designed through molecular markers in the AcGLK gene, and the primer group can accurately distinguish genotypes of segregation population single plants under a specific genetic background, so that leaf color / peel color phenotypes are judged; the method is used in breeding practice of pineapple leaf color diversity, and the improvement process of pineapple leaf color and fruit color characters is accelerated.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, in particular to a molecular marker for identifying the color of pineapple peel and application thereof. BACKGROUND

[0002] The color of pineapple peel is an important commercial trait, and the market demand for red peel varieties has increased significantly. Studies have shown that the formation of red peel depends on the coordinated regulation of anthocyanin accumulation and chlorophyll non-synthesis in the peel. In the prior art, the identification of pineapple peel color relies on phenotypic observation. When interspecific hybridization is used for color breeding, the peel color cannot be identified until the results are obtained, which affects the efficiency of pineapple breeding. Therefore, it is urgent to use molecular marker-assisted breeding for early identification to improve the efficiency of breeding and save manpower, material resources and time. SUMMARY

[0003] In view of the above problems of the prior art, the present application provides a molecular marker for identifying the color of pineapple peel and application thereof, which realizes early identification of the color of pineapple peel and improves the efficiency of breeding.

[0004] To solve the above problems, the present application adopts the following technical scheme:

[0005] In a first aspect, the present application provides a molecular marker for identifying the color of pineapple peel, which is an AcGLK gene, and the nucleotide sequence of the coding region of the AcGLK gene is shown in SEQ ID No. 1.

[0006] In a second aspect, the present application provides a molecular marker for evaluating the chlorophyll content of pineapple, which is an AcGLK gene, and the nucleotide sequence of the coding region of the AcGLK gene is shown in SEQ ID No. 1.

[0007] Further, the AcGLK gene includes Hap1 haplotype and Hap2 haplotype; the AcGLK gene of the Hap2 haplotype has an 8515bp insertion in intron 5, and the sequence between 2858bp near exon 5 and intron 1-5 is reverse complementary, forming a hairpin structure.

[0008] Further, the protein encoded by the AcGLK gene has an amino acid sequence shown in SEQ ID No. 2.

[0009] Further, the amino acid sequence comprises a GARP-DNA binding domain and a C-terminal domain GCTbox, the GARP-DNA binding domain is the 176th-243rd amino acid of the amino acid sequence, and the C-terminal domain GCTbox is the 342nd-439th amino acid of the amino acid sequence.

[0010] In a third aspect, the present application provides a primer set for detecting the molecular marker for identifying the color of the peel of a pineapple or the molecular marker for evaluating the chlorophyll content of a pineapple, wherein the primer set comprises primers of the nucleotide sequences shown in SEQ ID No. 3-6.

[0011] In a fourth aspect, the present application provides a kit for detecting the molecular marker for identifying the color of the peel of a pineapple or the molecular marker for evaluating the chlorophyll content of a pineapple, wherein the kit comprises the primer set.

[0012] Further, the hybrid parents of the pineapple are BTH and LY.

[0013] In a fifth aspect, the present application provides the primer set or the kit for use in the preparation of a reagent for identifying or assisting in identifying the color of the peel of a pineapple or evaluating or assisting in evaluating the chlorophyll content of a pineapple or screening and breeding a pineapple.

[0014] In a sixth aspect, the present application provides the molecular marker for identifying the color of the peel of a pineapple or the molecular marker for evaluating the chlorophyll content of a pineapple or the primer set or the kit for use in screening and breeding a pineapple.

[0015] The present application has the following beneficial effects: the present application determines AcGLK as a candidate gene for determining the synthesis of chlorophyll in a pineapple and designs a specific primer set in the molecular marker in the AcGLK gene, which can accurately distinguish the genotype of a single plant in a separated population under a specific genetic background (such as BTH x LY or a hybrid offspring population with BTH as a parent) and make a judgment on the leaf color / peel color phenotype, which is used in the breeding practice of the diversity of the leaf color of a pineapple and accelerates the improvement process of the leaf color and fruit color traits of a pineapple. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Figure 4 is a comparison chart of the chlorophyll index and the anthocyanin index of green plants and light green plants according to the present application.

[0017] Figure 2 Figure 5 is a diagram of the positioning of the GL gene of a green leaf color of a pineapple by QTL technology according to the present application.

[0018] Figure 3 Figure 6 is a diagram of haplotype analysis of the AcGLK gene of a green leaf trait germplasm according to the present application.

[0019] Figure 4 Figure 7 is a hairpin structure in the 8515 bp insertion of Hap2 haplotype according to the present application.

[0020] Figure 5 Figure 8 is an electrophoresis detection chart of 10 F1 single plants according to the present application. DETAILED DESCRIPTION

[0021] The application will be further described in detail below with reference to specific examples.

[0022] It should be noted that the examples are only used to illustrate the present application, but not to limit the present application, and simple improvements of the present application under the concept of the present application are within the scope of the present application.

[0023] Example 1

[0024] 1. Materials

[0025] The hybrid parents are fresh pineapple variety 'Bingtanghong' (A. comosus cv 'Bingtanghong', hereinafter referred to as 'BTH') leaf surface (including bracts and calyx) with several light green and green interlaced stripes, the total width of the light green stripes accounts for about 80% of the leaf width; the fruit skin is red during the development period and gradually fades to pale yellow during the ripening process; 'Ziye' is a cultivated variety of ornamental species A. lucidus (A. lucidus cv 'Ziye'; hereinafter referred to as 'ZY'), with purple red and uniform leaf color; the fruit is red during the growth and development period, and gradually fades to pale yellow during the ripening process. The tender leaves of green leaf pineapple variety 'LZZ' were used as materials, and total RNA was extracted using the CTAB method, and cDNA was synthesized by reverse transcription. The F1 hybrid population of BTH x LY, and two materials 'HZZ' (light green leaves) and 'LZZ' (green leaves) separated by tissue culture of BTH.

[0026] 2. Phenotype analysis of hybrid offspring

[0027] Through visual observation and pigment spectrum index, it was found that the chlorophyll index (ChlIdx) and anthocyanin index (AriIdx) of the leaves and fruits of green plants (F178, E051, F410, F179) were significantly higher than those of light green plants (E005, E003, F455, E350), while the fruits of light green plants could present red color, indicating that high concentration of chlorophyll content could mask the color of anthocyanin, and when the chlorophyll content was low, the fruits would present pink phenotype due to the accumulation of anthocyanin, as shown in Figure 1 .

[0028] 3. Extraction method of pineapple leaf genomic DNA

[0029] Take 0.05 g of pineapple leaf base tissue, put it into a 2 mL centrifuge tube and add 2 steel balls, pre-cool in liquid nitrogen and put it into a sample grinder at 60 Hz for 5 min; add 1 mL of 2xCTAB, 65°C water bath for 40 min, turn it up and down several times; 12000g centrifuge for 8 min, take 600 μL of supernatant into a new 2 mL centrifuge tube, add 1 mL of chloroform-isoamyl alcohol mixture (24:1) and mix well; precipitate at -20°C overnight; 12000g centrifuge for 8 min, discard the supernatant and dry on a water absorption paper, add 700 μL of 70% alcohol and rinse, then 12000g centrifuge for 5 min, discard the supernatant and rinse again with 700 μL of 70% alcohol; stand at room temperature until the centrifuge tube is completely dry; add 100 μL of ddH2O, stand at 37°C for 30 min and then at 65°C for 5 min; use a spectrophotometer to detect the DNA concentration, use agarose gel electrophoresis to detect the DNA quality, and store at -20°C for standby.

[0030] 4. QTL mapping of GL gene of pineapple green leaf

[0031] 100 F1 plants in the BTHxLY hybrid population were resequenced, the chlorophyll index was determined by a chlorophyll fluorometer, and QTL technology was used to map the GL gene of pineapple green leaf color. In the initial mapping interval, 11 genes in the chlorophyll synthesis, degradation, and chloroplast structure pathways were extracted. Further transcriptome data of 'HZZ' and 'LZZ' leaves showed that only one of the above 11 genes had an expression trend consistent with the leaf color phenotype, as shown in Table 1. Figure 2 Therefore, Aco00309.1 (AcGLK) was determined as the key candidate gene for the pineapple green leaf color trait.

[0032] 5. Coding region and amino acid structural characteristics of AcGLK gene

[0033] Through analysis of the AcGLK gene, the results showed that the AcGLK gene consists of 6 exons and 5 introns. The CDS is 1371 bp in length (SEQ ID No. 1), encoding 456 amino acids (SEQ ID No. 6), including a GARP-DNA binding domain (176-243 amino acids) and a C-terminal domain GCTbox (342-439 amino acids).

[0034] The coding sequence of the AcGLK gene is shown in SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 2.

[0035] SEQ ID NO. 1:

[0036]

[0037] SEQ ID NO.2:

[0038] MLAVSPLRSSHGGERGCDGDVGGSGFVDDFSIIGDGGDDMLDDIDFSELFVGLEDGDILPDLEVDPAEILAELSACEPSSVATRTSAEEGLVREEEYNKGGGGGGEEEEEDKEVVTMAEMRRREGDVSTDGNTVAADARSSSSLSSSSEEGDNRRRKSPSTAAATAKNSQGKRKVKVDWTPELHRRFVQAVEQLGVDKAVPSRILELMGIDCLTRHNVASHLQKYRSHRKHLLAREAEAAQWSQRRQMYGVGGPSSGVKRDVNPWLAPTIGFPPPPPVPHFRPLHVWGHPTVDPPLVHMWARHLAPRSTSPPWPPQAPVPPPPPPDPSYWHHHYQRGAHSLTQGTPCFPQALPVMRFPASAVPAVIPHPMYRPIPPPTTAKNASSQVPLDPHPSKESVDAAIGDVLAKPWLPLPLGLKAPSTESVMMELQKQGISKVPPTCGSSCGNYSVLTTTAS.

[0039] 6. Haplotype analysis

[0040] Haplotypes of genes in 'HZZ' and 'LZZ' were analyzed using existing genotype data. Haplotypes were constructed based on SNPs in untranslated regions (UTRs) and exonic regions by Haploview software. Through haplotype analysis of AcGLK, two haplotypes were identified in the pale green leaf color germplasm: Hapl (AcGLK without insertion) and Hap2 (AcGLK with an 8515 bp insertion in intron 5), while only Hapl existed in the green leaf trait germplasm (Hap2 was not found in the green leaf trait germplasm) Figure 3 ), and thus AcGLK is a candidate gene associated with the pineapple green leaf trait. Further analysis of the characteristics of the insertion sequence showed that AcGLK in the Hap2 haplotype has an 8515 bp insertion in intron 5, and the sequence of 2858 bp near exon 5 is complementary to the sequence between introns 1-5, forming a hairpin structure Figure 4 ).

[0041] Therefore, primers AcGLK-F and AcGLK-R were designed before and after the insertion sequence, and primer AcGLK-H was designed on the insertion fragment to identify the two haplotypes of AcGLK. The primer sequences of AcGLK are shown in Table 1.

[0042] Table 1 AcGLK gene amplification primer and amplification results

[0043]

[0044] Note: "√" represents amplification, "X" represents no amplification.

[0045] Example 2

[0046] Development of AcGLK haplotype molecular markers and application in F1 segregating population

[0047] Two haplotypes of AcGLK were designed using Primer Premier 5 software. The F1 different green leaf single plants were amplified with the primer combination of AcGLK gene, the amplification system is shown in Table 2, the amplification program is shown in Table 3, and electrophoresis detection was performed. If only the 1402bp target band is amplified, the leaf of the plant is dark green, and the synthesis of chlorophyll in the pericarp causes the color of anthocyanin to be affected, resulting in the red pericarp being covered. If the 1402bp and 1063bp target bands are amplified at the same time, the leaf of the plant is light green, and the pericarp does not synthesize chlorophyll, and the normal color of anthocyanin causes the pericarp to be red.

[0048] Table 2 Amplification system

[0049]

[0050] Table 3 Amplification program

[0051]

[0052] The results showed that the chlorophyll index of each single plant found that the results of the primer in distinguishing the leaf color of 10 F1 single plants were 100% (Table 4); primer combination P1 had 1402bp target band amplification in all F1 leaf color single plants, and primer P2 only had 1063bp target band amplification in F1 light green leaf color single plants. Figure 5 It is indicated that the AcGLK gene marker primer combination P1 and P2 can distinguish the two haplotypes of AcGLK, and can also efficiently and accurately distinguish the green leaf color and light green leaf color and pineapple color.

[0053] Table 4 Chlorophyll index of samples and primer amplification

[0054]

[0055] Note: "√" represents amplification, "X" represents no amplification.

[0056] Finally, it is to be understood that the above description is intended to be illustrative and not restrictive. Many embodiments of the application will be apparent to those of skill in the art upon reviewing the above description. The scope of the application should, therefore, be determined not with reference to the above description, but instead with reference to the appended claims, along with their full scope of equivalents.

Claims

1. A molecular marker for identifying the color of pineapple peel, characterized in that, The molecular marker is the AcGLK gene, and the nucleotide sequence of the coding region of the AcGLK gene is shown in SEQ ID No.

1.

2. A molecular marker for assessing chlorophyll content in pineapple, characterized in that, The molecular marker is the AcGLK gene, and the nucleotide sequence of the coding region of the AcGLK gene is shown in SEQ ID No.

1.

3. The molecular marker for identifying the color of pineapple peel according to claim 1 or the molecular marker for assessing the chlorophyll content of pineapple according to claim 2, characterized in that, The AcGLK gene includes haplotypes Hap1 and Hap2; the AcGLK gene of haplotype Hap2 has an 8515bp insertion in intron 5, and the 2858bp near exon 5 is inversely complementary to the sequence between introns 1-5, forming a hairpin structure.

4. The molecular marker for identifying the color of pineapple peel according to claim 1 or the molecular marker for assessing the chlorophyll content of pineapple according to claim 2, characterized in that, The protein encoded by the AcGLK gene contains the amino acid sequence shown in SEQ ID No.

2.

5. The molecular marker according to claim 4, characterized in that, The amino acid sequence includes a GARP-DNA binding domain and a C-terminal GCT box, wherein the GARP-DNA binding domain is amino acid 176 to 243 of the amino acid sequence; and the C-terminal GCT box is amino acid 342 to 439 of the amino acid sequence.

6. A primer set for detecting the molecular marker for identifying the color of pineapple peel as described in claim 1 or the molecular marker for assessing the chlorophyll content of pineapple as described in claim 2, characterized in that, The primer set includes primers with nucleotide sequences as shown in SEQ ID No. 3-6.

7. A kit for detecting the molecular marker for identifying pineapple peel coloration as described in claim 1 or the molecular marker for assessing pineapple chlorophyll content as described in claim 2, characterized in that, Includes the primer set as described in claim 6.

8. The reagent kit according to claim 7, characterized in that, The hybrid parents of the pineapple are rock sugar red pineapple and glossy pineapple.

9. The application of the primer set of claim 6 or the kit of claim 7 in the preparation of reagents for identifying or assisting in the identification of pineapple peel coloration or for evaluating or assisting in the evaluation of pineapple chlorophyll content or for pineapple screening and breeding.

10. The application of the molecular marker for identifying pineapple peel coloration as described in claim 1, or the molecular marker for assessing pineapple chlorophyll content as described in claim 2, or the primer set as described in claim 6, or the kit as described in claim 7, in pineapple screening breeding.

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