InDel marker related to orange flesh character of cucumber, primer pair, kit and application

Abstract

The invention discloses an InDel marker related to orange flesh traits of cucumbers, a primer pair, a kit and application, and belongs to the technical field of molecular marker-assisted breeding. The polymorphism of the InDel marker provided by the invention is a deletion / insertion sequence, and the InDel marker is obtained by amplifying a specific primer pair as shown in SEQ ID NO.1-2 or SEQ ID NO.3-4. The InDel marker can be used for distinguishing and identifying white green pulp cucumbers and orange pulp cucumbers, has higher accuracy, can be used for efficiently screening orange pulp cucumber materials, and can be used for identifying the cucumbers with the white green pulp and the orange pulp cucumbers. The method can be applied to 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 orange flesh 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] Flesh color is one of the important breeding traits of cucumbers. It not only affects the appearance quality of the fruit, but also is an indicator of some nutrients. Currently, cucumbers on the market mainly have white-green flesh and low β-carotene content; while orange flesh (orange flesh) is an apparent characteristic rich in β-carotene. At present, there are relatively few orange-fleshed cucumber germplasm resources on the market. The development of molecular markers that are closely linked to the orange flesh trait is of great significance for expanding the orange-fleshed cucumber germplasm resources and accelerating the breeding of high-quality orange-fleshed cucumber varieties. The existing molecular markers have the problem of a long linkage distance between the marker and the orange flesh trait, a low accuracy rate in the early identification of hybrid offspring, and complex marker types and cumbersome identification steps. Therefore, it is necessary to develop molecular markers with high accuracy and convenient operation. Summary of the invention

[0004] In view of this, the present invention provides an InDel marker, a primer pair, a kit and an application related to the orange flesh trait of cucumber, wherein the marker has high accuracy in identifying the cucumber flesh (orange / non-orange) trait.

[0005] In a first aspect, the present invention provides an InDel marker related to the orange flesh trait of cucumber, wherein the polymorphism of the InDel marker is an insertion / deletion sequence, and the InDel marker is amplified by a specific primer pair such as a sequence shown in SEQ ID NO. 1~2 or SEQ ID NO.3~4.

[0006] Further, the InDel marker includes an Indel47 marker or an OIndel5 marker, wherein the primer sequence of the Indel47 marker is: Forward primer (upstream primer): 5′-TGATGGCGTTATGGTGTAGGA-3′ (SEQ ID NO. 1); Reverse primer (downstream primer): 5′-GGCTGGGACCGATACTTTTAG-3′ (SEQ ID NO. 2); The primer sequences for the OIndel5 marker are: Forward primer (upstream primer): 5′-TCACATGTTCTTTTGGTAAGAGGA-3′ (SEQ ID NO. 3); Reverse primer (downstream primer): 5'-TGTGTTGTGAAAAGGACTAGATGG-3' (SEQ ID NO. 4).

[0007] The InDel marker of the present invention is located on chromosome 4 of the cucumber genome, and the Indel47 marker and OIndel5 marker can be used alone or in combination.

[0008] In a second aspect, the present invention provides a specific primer pair for detecting the above-mentioned InDel marker related to the orange flesh trait of cucumber, and its sequence is shown in SEQ ID NO. 1~2 or SEQ ID NO. 3~4.

[0009] In a third aspect, the present invention provides a kit containing the specific primer pair.

[0010] Preferably, the kit further comprises other reagents for PCR amplification, including but not limited to PCR reaction buffer, DNA polymerase, dNTP, ddH2O, etc.

[0011] 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 an application in the identification or auxiliary identification of the orange flesh color trait of cucumber.

[0012] Furthermore, the application method is: 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 for PCR amplification; the size of the PCR amplification product is detected to determine the genotype of the cucumber to be tested, and whether the cucumber fruit has orange flesh is inferred based on the genotype.

[0013] 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.

[0014] 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.

[0015] 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 upstream primer, 1 μL of 10 pmol / μL downstream primer, 10 μL of 2X M5 HiPer plus Taq HiFi PCR mix, and 6 μL of ultrapure water.

[0016] The size of the PCR amplification product can be detected by conventional techniques in the art, such as electrophoresis detection, DNA sequencing, etc. In the present invention, agarose gel electrophoresis is preferably used for detection.

[0017] Preferably, the specific method for inferring whether the cucumber fruit has orange flesh based on the genotype is: when PCR amplification is performed with specific primers marked with Indel47, if only a band with a length of 242 bp appears in the PCR amplification product, the cucumber to be tested is identified as orange flesh; if a band with a length of 321 bp appears in the PCR amplification product, the cucumber to be tested is identified as non-orange flesh; when PCR amplification is performed with specific primers marked with OIndel5, if only a band with a length of 153 bp appears in the PCR amplification product, the cucumber to be tested is identified as orange flesh; if a band with a length of 127 bp appears in the PCR amplification product, the cucumber to be tested is identified as non-orange flesh.

[0018] In a fifth aspect, the present invention provides an application of the InDel marker or the specific primer pair or the kit, wherein the application is an application in breeding orange-fleshed cucumbers.

[0019] In a sixth 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 the detection of orange pulp gene in cucumber.

[0020] Compared with the prior art, the present invention has achieved the following beneficial effects: The present invention provides an InDel marker related to the orange flesh trait of cucumber, which can be used to distinguish and identify white-green flesh cucumbers and orange flesh cucumbers with high accuracy, can be used to efficiently screen orange flesh cucumber materials, can be widely used in cucumber breeding work, and provides a powerful molecular tool for cultivating new cucumber varieties with excellent nutritional quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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.

[0022] Figure 1 This is a diagram showing the changes in flesh color during the fruit development of the wild type and mutant materials of Example 1 of the present invention; Figure 2 is a graph showing changes in the content of β-carotene in the wild type and mutant materials of Example 1 of the present invention at different fruit development stages; Figure 3 is a distribution diagram of the Euclidean distance (ED) association value of the cucumber flesh color on the chromosome in Example 1 of the present invention; Figure 4 This is a graph showing the electrophoresis results of PCR amplification products of 8 polymorphic InDel markers screened in Example 1 of the present invention; Figure 5 This is a graph showing the electrophoresis results of PCR amplification products of some Indel47 markers of the F2 population in Example 2 of the present invention; Figure 6 This is a graph showing the electrophoresis results of PCR amplification products of some OIndel5 markers of the F2 population in Example 2 of the present invention. DETAILED DESCRIPTION

[0023] 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.

[0024] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0025] Example 1 This example provides the development of InDel markers associated with the orange flesh trait of cucumber.

[0026] (1) Identification of the color of cucumber flesh during the development period: The preserved wild-type (WT) materials and mutant materials were sown at the Vegetable Research Institute of Shandong Academy of Agricultural Sciences in Jinan, Shandong Province in mid-July 2023. When the seedlings grew to three leaves and one heart, they were planted in the cucumber arch shed at the Vegetable Research Institute of Shandong Academy of Agricultural Sciences. The plants were marked at different growth stages (0-55 days), and the fruits were harvested on the same day. The color changes of the flesh at different stages were observed and photographed.

[0027] The results are as follows Figure 1 As shown in the figure, the wild-type material has green flesh color from 0 to 25 days, and the flesh color gradually changes from green to white from 25 to 55 days; the mutant material has green flesh color from 0 to 25 days, and the flesh color gradually changes from green to orange from 25 to 55 days. Around 25 days, the mutant material begins to differ from the wild-type material, and finally the mature flesh of the wild-type material is white, while the mature flesh of the mutant material is orange.

[0028] Based on the liquid chromatography-mass spectrometry (LC-MS / MS) detection platform, the content of carotenoid metabolites in the pulp of wild-type and mutants at different developmental stages was measured, and a total of 68 carotenoids were detected. According to the cluster analysis of carotenoid metabolites, the contents of 16 major carotenoid substances were detected to be significantly different between the wild type and the mutant, including zeaxanthin, α-carotene, phytoene, lutein, neoxanthin, violaxanthin, violaxanthin dilaurate, violaxanthin myristate, β-carotene, β-cryptoxanthin, luteinmyristate, lutein palmitate, violaxanthin-myristate-caprate, violaxanthin-myristate-laurate, lutein dilaurate, and lutein ester. dilaurate and lutein dimyristate.

[0029] Figure 2 The content of β-carotene in wild type and mutants changes from 10 to 50 days. It can be seen that the content of β-carotene in wild type and mutants is high in the early stage of fruit development. The content of β-carotene in wild type and mutants shows a downward trend from 10 to 30 days, and there is a difference after 30 days. The content of β-carotene in wild type continues to decline, while the content of β-carotene in mutants begins to rise. Combined with the changes in the flesh color of mutants at different developmental stages and the content and color of these substances, it is speculated that the difference in the content of β-carotene in the flesh of mutants is the main factor leading to the change in its flesh color.

[0030] (2) Analysis of genetic rules of flesh color mutation: The residual heterozygous line (RHL) was established to construct near-isogenic lines to analyze the genetic law of flesh color mutation. The flesh color phenotypes of the residual heterozygous line F6 and F7 populations obtained after multiple generations of self-pollination were observed. The results showed that there were 133 plants with white-green flesh and 44 plants with orange flesh in the F6 population. The chi-square test showed that the segregation ratio of white-green flesh to orange flesh was 3:1 (chi-square value χ 2 = 0.001883, P = 0.97). There were 229 plants with white-green flesh and 84 plants with orange flesh in the F7 population. The chi-square test showed that the separation ratio of white-green flesh to orange flesh was 3:1 (χ 2 =0.004357, P = 0.95). The results showed that orange flesh in cucumber was controlled by a recessive single gene, which was named CsOre2.

[0031] (3) Development of cucumber pulp color marking: The two extreme pools in the remaining heterozygous population were resequenced, and a total of 15,254 SNPs and 11,835 InDel sites were obtained between the mixed pools. BSA-seq analysis was performed based on the differential sites, and the intersection of the SNP (single nucleotide polymorphism) and InDel (insertion / deletion polymorphism) associated regions was taken. Figure 3 As shown in the figure, there is an obvious association signal in the interval of 4,850,000 to 9,470,000 bp and a total length of 4.62 Mb on chromosome 4 (Chr4) of the cucumber genome, and the Euclidean distance (ED value) obviously exceeds the significant association threshold. Therefore, the CsOre2 gene that controls the orange flesh trait of cucumber is preliminarily located in this interval. 45 pairs of primers were designed in this interval. These primers were screened using DNA from 4 plants of the orange flesh parent, the white flesh parent and the F1, and a total of 8 polymorphic InDel markers were obtained, as shown in the figure. Figure 4 As shown. Among them, the length of the amplified fragment of Indel47 in the orange-fleshed parent plant is 242bp, and the length of the amplified fragment in the white-fleshed parent plant is 321bp. In the heterozygous F1 plant, the above two fragments can be amplified simultaneously. The length of the amplified fragment of OIndel5 in the orange-fleshed parent plant is 153bp, and the length of the amplified fragment in the white-fleshed parent plant is 127bp. In the heterozygous F1 plant, the above two fragments can be amplified simultaneously.

[0032] The specific primer sequences for the Indel47 marker are: Forward primer: 5′-TGATGGCGTTATGGTGTAGGA-3′ (SEQ ID NO. 1); Reverse primer: 5'-GGCTGGGACCGATACTTTTAG-3' (SEQ ID NO. 2).

[0033] The specific primer sequences for the OIndel5 marker are: Forward primer: 5′-TCACATGTTCTTTTGGTAAGAGGA-3′ (SEQ ID NO. 3); Reverse primer: 5'-TGTGTTGTGAAAAGGACTAGATGG-3' (SEQ ID NO. 4).

[0034] Example 2 This example provides the application of the InDel marker provided in Example 1 in cucumber pulp color identification.

[0035] A total of 600 F2 population plants were planted in the field, and leaves were taken for DNA extraction at the seedling stage, followed by PCR amplification and molecular marker detection and analysis, and the sex type of each material was investigated during the flowering period. The specific steps are as follows: (1) DNA extraction: ①Take 1cm 2 Large and small cucumber leaves; 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; 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; 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; ⑤ After drying, add 100 μL ddH2O and dissolve for about 1 hour to obtain the cucumber sample DNA solution.

[0036] (2) PCR amplification: The cucumber sample DNA solution prepared above was taken and PCR amplified using the Indel47 marker specific primers and the OIndel5 molecular marker specific primers designed in Example 1. The PCR amplification system is shown in Table 1.

[0037] Table 1 PCR amplification system (20 μL)

[0038] 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.

[0039] (3) Detection of product amplification results: The product after PCR amplification was loaded on a 3wt% agarose gel containing 0.01wt% GelRed nucleic acid dye, and electrophoresed at a constant voltage of 180V for 30min. The gel imaging system was used to take pictures, archive and analyze the labeling results. When PCR amplification was performed with the Indel47-labeled specific primer, the fragment length of the PCR amplification product was judged. If only a band of 242bp appeared, the cucumber to be tested was identified as orange flesh; if a band of 321bp appeared, the cucumber to be tested was identified as white-green flesh. When PCR amplification was performed with the OIndel5-labeled specific primer, the fragment length of the PCR amplification product was judged. If only a band of 153bp appeared, the cucumber to be tested was identified as orange flesh; if a band of 127bp appeared, the cucumber to be tested was identified as white-green flesh. The identification results were compared with the field phenotypes during the flowering period to determine the accuracy of the identification.

[0040] The electrophoresis results of some Indel47-labeled PCR amplification products are as follows Figure 5 As shown, the electrophoresis results of some OIndel5-labeled PCR amplification products are as follows Figure 6 As shown, the results showed that the detection results of 600 individual plants detected by Indel47 marker were completely consistent with the field phenotype, with an accuracy rate of 100%. There were 3 detection results inconsistent with the field phenotype of 600 individual plants detected by OIndel5 marker, with an accuracy rate of 99.5%. It can be seen that the InDel molecular marker of the present invention can accurately and quickly distinguish the white-green flesh color and the orange flesh color materials in the cucumber seedling stage.

[0041] 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. An InDel marker related to the orange flesh trait of cucumber, characterized in that: The polymorphism of the InDel marker is an insertion / deletion sequence, and the InDel marker is amplified by a specific primer pair such as the sequence shown in SEQ ID NO. 1~2 or SEQ ID NO. 3~4.

2. A specific primer pair for detecting the InDel marker associated with the orange flesh trait of cucumber according to claim 1, characterized in that: Its sequence is shown in SEQ ID NO. 1~2 or SEQ ID NO. 3~4.

3. A kit comprising the specific primer pair according to claim 2.

4. Use of the InDel marker according to claim 1, the specific primer pair according to claim 2, or the kit according to claim 3, characterized in that: The application is: application in identification or auxiliary identification of orange flesh color traits of cucumber.

5. The use according to claim 4, characterized in that The method of application is: 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 for PCR amplification; the size of the PCR amplification product is detected to determine the genotype of the cucumber to be tested, and whether the cucumber fruit has orange flesh is inferred based on the genotype.

6. The use according to claim 5, 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.

7. The use according to claim 5, 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.

8. The use according to claim 5, characterized in that The specific method for inferring whether the cucumber fruit has orange flesh according to the genotype is as follows: when PCR amplification is performed with the Indel47 marker-specific primers, if only a band with a length of 242 bp appears in the PCR amplification product, the cucumber to be tested is identified as having orange flesh; If a band with a length of 321 bp appears in the PCR amplification product, the cucumber to be tested is identified as non-orange flesh; when PCR amplification is performed with specific primers marked with OIndel5, if only a band with a length of 153 bp appears in the PCR amplification product, the cucumber to be tested is identified as orange flesh; if a band with a length of 127 bp appears in the PCR amplification product, the cucumber to be tested is identified as non-orange flesh.

9. Use of the InDel marker according to claim 1, the specific primer pair according to claim 2, or the kit according to claim 3, characterized in that: The application is: application in breeding orange flesh cucumbers.

10. Use of the InDel marker according to claim 1, the specific primer pair according to claim 2, or the kit according to claim 3, characterized in that: The application is: application in gene detection of orange flesh of cucumber.

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