InDel markers, primer pairs, kits and applications related to 2E, 6Z-nonadienal content in cucumber

Abstract

The present invention discloses an InDel marker, a primer pair, a kit and an application related to the 2E, 6Z-nonadienal content trait of cucumber, and belongs to the field of molecular marker-assisted breeding technology. The InDel marker provided by the present invention is an InDel9 marker or an InDel48 marker, and the InDel9 marker is amplified by a specific primer pair as shown in a sequence such as SEQ ID NO. 1~2; the InDel48 marker is amplified by a specific primer pair as shown in a sequence such as SEQ ID NO. 3~4. The InDel marker of the present invention can be used to quickly and more accurately judge the content of 2E, 6Z-nonadienal in cucumber fruit, which is helpful to assist in screening cucumber varieties with high flavor quality, and can be widely used in cucumber breeding work.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular marker-assisted breeding, and in particular to an InDel marker, a primer pair, a kit and applications related to the 2E, 6Z-nonadienal content trait of cucumber. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance the understanding of the overall background of the invention and should not be necessarily regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] With economic development and consumption upgrading, the flavor quality of cucumbers has attracted more and more attention from consumers. Flavor quality is a combination of smell and taste, among which volatile substances, especially aromatic substances that constitute the fragrance, are important factors in determining flavor. Cucumber fruits contain more than 70 kinds of aromatic substances, among which 2E, 6Z-nonadienal and 2E, 6Z-nonadienol are recognized as the characteristic aromatic substances of cucumbers, especially 2E, 6Z-nonadienal, which has an important influence on the flavor of cucumbers.

[0004] The key to improving cucumber flavor in production lies in the discovery, evaluation and screening of excellent germplasm resources. The existing evaluation methods mainly rely on traditional sensory evaluation and physical and chemical testing. Sensory evaluation is a simple characteristic description of cucumber flavor by breeders based on sensory perception, which has high requirements for the experience and training of relevant personnel, and the quantitative standards are not unified, and there is subjectivity and one-sidedness. The physical and chemical detection method uses physical or chemical methods to detect the content of flavor substances, which is objective, but has the defects of expensive detection instruments and reagents, cumbersome operation, and long cycle. Developing molecular markers linked to cucumber flavor quality traits for screening excellent germplasm resources or assisting the genetic improvement process can eliminate the drawbacks of existing evaluation methods and greatly improve the efficiency of flavor variety breeding. However, there are few reports on molecular markers linked to cucumber flavor quality traits in existing studies, so it is necessary to develop an InDel marker related to the cucumber 2E, 6Z-nonadienal content trait. Summary of the invention

[0005] In view of this, the present invention provides an InDel marker, a primer pair, a kit and an application related to the 2E, 6Z-nonadienal content trait of cucumber, wherein the marker can be used to assist in screening cucumber materials with high 2E, 6Z-nonadienal content.

[0006] In a first aspect, the present invention provides an InDel marker related to the 2E, 6Z-nonadienal content trait of cucumber, wherein the InDel marker is an InDel9 marker or an InDel48 marker, wherein the InDel9 marker is amplified by a specific primer pair whose sequences are shown in SEQ ID NOs. 1 to 2; and the InDel48 marker is amplified by a specific primer pair whose sequences are shown in SEQ ID NOs. 3 to 4.

[0007] The primer sequence of the InDel9 marker is:

[0008] Forward primer (upstream primer): 5′-GTAACCCTTTCTTCGTTCGAGA-3′ (SEQ ID NO. 1);

[0009] Reverse primer (downstream primer): 5′-ACTCTGGTTGAAAGAAGAATGACA-3′ (SEQ ID NO. 2);

[0010] The primer sequence of the InDel48 marker is:

[0011] Forward primer (upstream primer): 5′-ATTTGATGAACGAGGCATGCAT-3′ (SEQ ID NO. 3);

[0012] Reverse primer (downstream primer): 5'-TTGTTAGTGTCAACCTCGAAGC-3' (SEQ ID NO. 4).

[0013] The InDel marker of the present invention is located on chromosome 2 of the cucumber genome, and the above-mentioned InDel9 marker and InDel48 marker can be used alone or in combination.

[0014] In a second aspect, the present invention provides a specific primer pair for detecting the above-mentioned InDel marker related to the cucumber 2E, 6Z-nonadienal content trait, whose sequence is shown in SEQ ID NO. 1~2 or SEQ ID NO. 3~4.

[0015] In a third aspect, the present invention provides a kit containing the specific primer pair.

[0016] Preferably, the kit further comprises other reagents for PCR amplification, including but not limited to PCR reaction buffer, DNA polymerase, dNTP, ddH2O, etc.

[0017] In a fourth aspect, the present invention provides an application of the InDel marker or the specific primer pair or the kit, wherein the application is application in identifying the content of 2E, 6Z-nonadienal in cucumber.

[0018] Furthermore, the application method is:

[0019] The genomic DNA of the cucumber to be tested is used as a template, and a specific primer pair as shown in SEQ ID NO. 1-2 or SEQ ID NO. 3-4 is used to perform PCR amplification; the size of the PCR amplification product is detected, and the content of 2E, 6Z-nonadienal in the cucumber is judged according to the size of the PCR amplification product.

[0020] Preferably, the reaction procedure of the PCR amplification is: pre-denaturation at 95°C for 1 min; denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 30 s, for a total of 32 cycles; and final extension at 72°C for 10 min.

[0021] Preferably, in the PCR amplification reaction system, the concentration of the forward primer and the reverse primer in the specific primer pair is 0.4-0.6 μM.

[0022] Furthermore, the reaction system of the PCR amplification is as follows: 2 μL of 10 ng / μL template DNA, 1 μL of 10 pmol / μL forward primer, 1 μL of 10 pmol / μL reverse primer, 10 μL of 2X M5 HiPer plus Taq HiFi PCR mix, and 6 μL of ultrapure water.

[0023] Preferably, the method for detecting the size of the PCR amplification product is: performing electrophoresis detection on the PCR amplification product in 2-4wt% agarose gel.

[0024] Further, the specific method for judging the content of 2E, 6Z-nonadienal in cucumber according to the size of the PCR amplification product is as follows: when PCR amplification is performed with the InDel9 marker specific primer pair, if only a band with a length of 223 bp appears in the PCR amplification product, it is judged to be a cucumber variety with a low 2E, 6Z-nonadienal content; if only a band with a length of 255 bp appears in the PCR amplification product, it is judged to be a cucumber variety with a high 2E, 6Z-nonadienal content;

[0025] When PCR amplification was performed using the InDel48 marker-specific primer pair, if only a band of 267 bp appeared in the PCR amplification product, it was judged to be a cucumber variety with a low 2E, 6Z-nonadienal content; if only a band of 233 bp appeared in the PCR amplification product, it was judged to be a cucumber variety with a high 2E, 6Z-nonadienal content.

[0026] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0027] The present invention provides an InDel marker related to the 2E, 6Z-nonadienal content trait of cucumber, which can be used to quickly and relatively accurately determine the content of 2E, 6Z-nonadienal in cucumber fruits, and is helpful to assist in screening cucumber varieties with high flavor quality. The marker can be widely used in cucumber breeding work, providing a powerful molecular tool for cultivating new cucumber varieties with excellent flavor, and has important industrial practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The drawings constituting part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute an improper limitation of the present invention. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0029] Figure 1 is a comparison chart of 2E, 6Z-nonadienal content in cucumber inbred line fruits of different sources and ecological types in Example 1 of the present invention;

[0030] Figure 2 is a box plot of 2E, 6Z-nonadienal content in fruits in the genetic population of Example 1 of the present invention;

[0031] Figure 3 This is a BSA-seq analysis diagram of the 2E, 6Z-nonadienal content gene in cucumber fruit in Example 1 of the present invention;

[0032] Figure 4 This is a graph showing the electrophoresis results of the PCR amplification products of the InDel9 marker of the F2 population in Example 1 of the present invention;

[0033] Figure 5 This is a graph showing the electrophoresis results of the PCR amplification products of the InDel48 marker of the F2 population in Example 1 of the present invention;

[0034] Figure 6 2E, 6Z-nonadienal content box plot of different genotypes of cucumber fruits marked by InDel9 and InDel48 in the F2 population in Example 2 of the present invention, wherein (a) is marked by InDel9, and (b) is marked by InDel48; NS. indicates no significant difference; * indicates significant difference, P < 0.05; ** indicates extremely significant difference, P < 0.01. DETAILED DESCRIPTION

[0035] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0036] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0037] Example 1

[0038] This example provides the development of InDel markers related to the 2E, 6Z-nonadienal content trait in cucumber.

[0039] (1) Analysis and screening of 2E, 6Z-nonadienal content in cucumber fruit inbred lines:

[0040] Twenty-seven cucumber inbred line materials from different sources and ecological types were selected, including 13 North China types (including XY1, AZ-1, N26-5-1, DRT276, DJ04, F11-15, 228, D1503, M1XT, 18a03, DRTJY-2, 170-1-2 and MC7-2-1), 5 South China types (including A72, XB23, TM011, Cuiyu and M1-2-h-10), 8 European and American greenhouse types (including stoke, Tony, SJ11, Jenny, Shoushui1, Shoushui12, HL7-921h and HL1128) and 1 European and American processing type material (Mini). The contents of 2E, 6Z-nonadienal in the fruits of these materials were detected by GC-MS.

[0041] Detection method: Insert the extraction head into the injection port of gas chromatography-mass spectrometry (GC-MS), age at 250℃ for 40 min, take 30 g of the pulp from the middle part of the cucumber, squeeze it into a homogenate with a juicer, weigh 10 g of the homogenate and put it into a clean 25 mL conical flask, and quickly add 10 μL of the internal standard substance octanal (concentration of 1 mg / mL) and 1 g of analytical grade NaCl, quickly seal it with aluminum foil, mix it on a magnetic stirrer, insert the aged extraction head into the headspace of the conical flask and push out the fiber head, heat and extract at 40℃ for 30 min, then insert it into the GC-MS injection port, and analyze it at 250℃ for 5 min.

[0042] Chromatographic conditions: The chromatographic column was a DB-1 capillary column, the initial temperature was 36°C, maintained for 3 min, then heated to 210°C at 6°C / min and maintained for 7 min, the injection port temperature was 230°C, the flow rate was 1.0 mL / min, the carrier gas was helium, the injection method was splitless injection, and the injection time was 0.5 min.

[0043] Mass spectrometry conditions were as follows: ion source temperature was 200 °C, interface temperature was 200 °C, solvent delay time was 1 min, and detector voltage was 0.8 kV.

[0044] Test results such as Figure 1 As shown, the results showed that the content of 2E, 6Z-nonadienal in the fruits of different inbred lines varied greatly. Among them, SJ11 was the material with the highest content, with a 2E, 6Z-nonadienal content of about 18 μg / g, and Mini was the material with the lowest content, with a 2E, 6Z-nonadienal content of 4 μg / g. The content of SJ11 was about 4 times that of Mini.

[0045] (2) Construction of population and phenotypic detection of 2E, 6Z-nonadienal content in cucumber fruit:

[0046] According to the above results, the material SJ11 with the highest 2E, 6Z-nonadienal content was selected as the female parent (P1), and the material Mini with the lowest content was selected as the male parent (P2) to construct the F1 combination, and further self-pollination was performed to obtain the F2 population. A total of 288 individual plants of the F2 population were planted in the greenhouse, and 256 individual plants of commercial melons were obtained during the harvest period and tested for 2E, 6Z-nonadienal content.

[0047] The test results showed that the average content of 2E, 6Z-nonadienal in P1 commercial melons was 13.7 μg / g, the average content of 2E, 6Z-nonadienal in P2 commercial melons was 7.36 μg / g, the average content of 2E, 6Z-nonadienal in F1 commercial melons was 16.2 μg / g, the average content of 2E, 6Z-nonadienal in F2 commercial melons was 14.2 μg / g, and the content range of F2 population was 3.03~56.71 μg / g. Figure 2 shown.

[0048] (3) Genetic analysis of 2E, 6Z-nonadienal content in cucumber fruit:

[0049] Genetic analysis was performed on the F2 population test results using a major gene + polygene mixed genetic model. The results showed that the 2E, 6Z-nonadienal content in this population was controlled by two pairs of major effect genes, which was consistent with the additive-dominant genetic model controlled by two pairs of major effect genes, and the heritability of the major effect genes reached 85%.

[0050] (4) BSA-seq (cluster segregation analysis sequencing) analysis of 2E, 6Z-nonadienal content genes in cucumber fruits:

[0051] 50 plants with extremely high and extremely low 2E, 6Z-nonadienal contents were selected from the F2 population and mixed to construct two extreme pools. Whole genome resequencing was performed on both parents and the two extreme pools, with a sequencing depth of 50X. Differential SNP (single nucleotide polymorphism) sites and InDel (insertion / deletion polymorphism) sites were detected, and the median+3SD (median value + three standard deviations) of the fitted values ​​of all sites were taken as the association threshold for analysis.

[0052] Figure 3 The results showed that one locus controlling the 2E, 6Z-nonadienal content in cucumber fruit was detected on chromosome 2 (Chr2), chromosome 3 (Chr3) and chromosome 5 (Chr5), with physical ranges of 2.5 Mb, 2.2 Mb and 1.1 Mb, respectively, and they were named FNC2.1, FNC3.1 and FNC5.1, respectively.

[0053] (5) Development of marker for 2E, 6Z-nonadienal content in cucumber fruit:

[0054] Based on the sequencing data of BSA-seq and the sequence information published by the cucumber reference genome, a total of 72 pairs of InDel markers were developed in the preliminary gene positioning segment, including 38 pairs on chromosome 2, 27 pairs on chromosome 3, and 7 pairs on chromosome 5. PCR amplification was performed using 3 Mini (low 2E, 6Z-nonadienal content), 3 SJ11 (high 2E, 6Z-nonadienal content) and two genomic DNAs of F1 of the two materials as templates. Primers were screened by agarose gel electrophoresis detection. Finally, primers that showed polymorphism between the parents and the two mixed pools, with clear bands and easy statistics were selected for subsequent experiments. The selected markers were InDel9 and InDel48, located on chromosome 2 of the cucumber genome. The primer sequence of the InDel9 marker was:

[0055] Forward primer: 5′-GTAACCCTTTCTTCGTTCGAGA-3′ (SEQ ID NO. 1);

[0056] Reverse primer: 5′-ACTCTGGTTGAAAGAAGAATGACA-3′ (SEQ ID NO. 2);

[0057] The primer sequence for InDel48 marker is:

[0058] Forward primer: 5′-ATTTGATGAACGAGGCATGCAT-3′ (SEQ ID NO. 3);

[0059] Reverse primer: 5'-TTGTTAGTGTCAACCTCGAAGC-3' (SEQ ID NO. 4).

[0060] Example 2

[0061] This example provides the application of the InDel marker provided in Example 1 in the identification of 2E, 6Z-nonadienal content.

[0062] A total of 256 F2 population plants were planted in the field, and DNA was extracted from leaves at the seedling stage, followed by PCR amplification and molecular marker detection and analysis. The 2E, 6Z-nonadienal content of the corresponding cucumber fruit was measured at the fruiting stage. The specific steps are as follows:

[0063] (1) DNA extraction:

[0064] ①Take 1cm 2 Large and small cucumber leaves;

[0065] ② Add steel beads and 500 μL extraction buffer (12.1 g Tris, 28.1 g NaCl, 18.6 g EDTA, add water to 1 L), grind the sample for 3 min, and centrifuge at 12,000 rpm for 10 min;

[0066] ③ Pipette 400 μL of supernatant into a 1.5 mL centrifuge tube, add an equal volume of isopropanol for precipitation, place at -20°C for about 30 minutes, and centrifuge for 5 minutes;

[0067] ④ Pour off the ethanol, add 500μL 75% ethanol, soak and wash for 10 minutes, then pour off the 75% ethanol, and repeat the wash once;

[0068] ⑤ After drying, add 100 μL ddH2O and dissolve for about 1 hour to obtain the cucumber sample DNA solution.

[0069] (2) PCR amplification:

[0070] The cucumber sample DNA solution prepared above was taken and PCR amplified using the InDel9 marker specific primers and the InDel48 molecular marker specific primers designed in Example 1. The PCR amplification system is shown in Table 1.

[0071] Table 1 PCR amplification system (20 μL)

[0072]

[0073] The PCR amplification program was as follows: pre-denaturation at 95°C for 1 min; then denaturation at 95°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 30 s, for a total of 32 cycles; and finally final extension at 72°C for 10 min.

[0074] (3) Detection of product amplification results:

[0075] The PCR amplified product was loaded onto a 3 wt % agarose gel containing 0.01 wt % GelRed nucleic acid dye, and electrophoresed at a constant voltage of 180 V for 30 min. The gel imaging system was used to take photos, archive, and analyze the labeling results.

[0076] When PCR amplification was performed with the InDel9 marker-specific primers, the fragment size of the PCR amplification product was determined. If it contained only a 223 bp band, its genotype marker was FNC2.1 FNC2.1 (maternal genotype), the cucumber to be tested was identified as having a low 2E, 6Z-nonadienal content; if it contained only a 255 bp band, its genotype marker was fnc2.1 fnc2.1 (paternal genotype), the cucumber to be tested was identified as having a high 2E, 6Z-nonadienal content. The gel electrophoresis results of the identification results of some plants are shown in the figure below. Figure 4 shown.

[0077] When PCR amplification is performed with specific primers labeled with InDel48, the fragment size of the PCR amplification product is determined. If it contains only a band of 267 bp in length, its genotype marker is FNC2.1 FNC2.1 (female genotype), and the cucumber to be tested is identified as having a low 2E, 6Z-nonadienal content; if it contains only a band of 233 bp in length, its genotype marker is fnc2.1 fnc2.1 (male genotype), and the cucumber to be tested is identified as having a high 2E, 6Z-nonadienal content. The gel electrophoresis results of the identification results of some plants are shown in the figure below. Figure 5 shown.

[0078] The above identification results were compared with the results of 2E, 6Z-nonadienal content determination in cucumber fruits during the fruiting period. The box plot of 2E, 6Z-nonadienal content in different genotypes of cucumber fruits marked by InDel9 in the F2 population is shown in the figure. Figure 6 As shown in (a), the box plot of 2E, 6Z-nonadienal content of different genotypes of cucumber fruit marked by InDel48 in the F2 population is shown in Figure 6 As shown in (b) in the figure, it can be seen that the average content of 2E, 6Z-nonadienal in the fruits of the plants identified as having high 2E, 6Z-nonadienal content (genotype fnc2.1 fnc2.1) is significantly higher than that of the plants identified as having low 2E, 6Z-nonadienal content (genotype FNC2.1 FNC2.1), indicating that the above markers can accurately and quickly distinguish between materials with high 2E, 6Z-nonadienal content and materials with low 2E, 6Z-nonadienal content at the cucumber seedling stage.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a specific primer pair for detecting the trait related to the content of 2E, 6Z-nonadienal in cucumber in the identification of the content of 2E, 6Z-nonadienal in cucumber, characterized in that: The specific primer pair is shown in SEQ ID NO. 1-2 or SEQ ID NO. 3-4; The method of application is: Using the genomic DNA of the cucumber to be tested as a template, PCR amplification is performed using the specific primer pairs shown in SEQ ID NOs. 1-2 or SEQ ID NOs. 3-4; detecting the size of the PCR amplification product, and judging the content of 2E,6Z-nonadienal in the cucumber according to the size of the PCR amplification product; The specific method for judging the content of 2E, 6Z-nonadienal in cucumber according to the size of the PCR amplification product is as follows: when PCR amplification is performed using a specific primer pair with a sequence as shown in SEQ ID NO. 1-2, if only a band with a length of 223 bp appears in the PCR amplification product, it is judged to be a cucumber variety with a low 2E, 6Z-nonadienal content; if only a band with a length of 255 bp appears in the PCR amplification product, it is judged to be a cucumber variety with a high 2E, 6Z-nonadienal content; When PCR amplification is performed using a specific primer pair with sequences shown in SEQ ID NOs. 3-4, if only a band with a length of 267 bp appears in the PCR amplification product, it is judged to be a cucumber variety with a low 2E, 6Z-nonadienal content; if only a band with a length of 233 bp appears in the PCR amplification product, it is judged to be a cucumber variety with a high 2E, 6Z-nonadienal content.

2. The use according to claim 1, characterized in that In the PCR amplification reaction system, the concentrations of the forward primer and the reverse primer in the specific primer pair are 0.4-0.6 μM.

3. The use according to claim 2, characterized in that The reaction system of the PCR amplification is as follows: 2 μL of 10 ng / μL template DNA, 1 μL of 10 pmol / μL forward primer, 1 μL of 10 pmol / μL reverse primer, 10 μL of 2X M5 HiPer plus Taq HiFiPCR mix, and 6 μL of ultrapure water.

4. The use according to claim 1, characterized in that The reaction procedure of the PCR amplification is: pre-denaturation at 95°C for 1 min; denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 30 s, for a total of 32 cycles; and final extension at 72°C for 10 min.

5. The use according to claim 1, characterized in that The method for detecting the size of the PCR amplification product is: performing electrophoresis detection on the PCR amplification product in 2-4wt% agarose gel.

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