Molecular marker primer group for identifying or screening wax gourd spaceflight breeding materials and application of molecular marker primer group

Abstract

The invention discloses a molecular marker primer group for identifying or screening wax gourd spaceflight breeding materials and application of the molecular marker primer group. Six pairs of InDel marker primers capable of identifying the wax gourd aerospace material and the offspring thereof are developed and screened from 606959 InDel markers of the wax gourd aerospace breeding material, the primers can be used for identification in the seedling stage, the wax gourd aerospace material can be accurately bred according to the insertion / deletion size of the primers, the breeding period is shortened, and the breeding efficiency is improved. And the white gourd aerospace material and the offspring thereof can be bred quickly, accurately and efficiently. Meanwhile, when the InDel marker is applied to screening and offspring selection of aerospace breeding materials, the recognition and utilization efficiency of excellent variation in aerospace breeding can be remarkably improved, the offspring selection efficiency is optimized, and the breeding process of the aerospace breeding materials is accelerated. Compared with a traditional breeding identification method, the method is accurate and reliable, breeding target progenies can be screened in advance, the breeding cost is reduced, and more high-quality and efficient new white gourd varieties are provided for agricultural production.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological breeding. More specifically, it relates to a molecular marker primer set for identifying or screening Benincasa hispida space breeding materials and its application. Background Art

[0002] Benincasa hispida (Thunb.) Cogn. is an annual trailing or climbing herbaceous plant of the genus Benincasa in the gourd family, with yellow flowers and is usually monoecious; the fruit is cylindrical or nearly spherical, and its surface is covered with bristles and white frost. For some Benincasa hispida, a powdery wax appears on the fruit surface after maturity, which is called the powdery skin Benincasa hispida; while for others, due to less wax powder after maturity, the fruit skin presents black or green, which is called the green skin or black skin Benincasa hispida. Excellent Benincasa hispida varieties have the characteristics of strong disease resistance, vigorous growth, high yield, and good quality, and breeding excellent Benincasa hispida varieties has always been the goal of Benincasa hispida breeding.

[0003] Currently, the methods for Benincasa hispida breeding are as follows: (1) Conventional cross-breeding, that is, creating genetic variation through hybridization and then selecting new varieties with excellent phenotypes through phenotypic selection. (2) Chemical mutagenesis breeding, which is mainly divided into two categories: one is to induce gene mutations or chromosome breaks, and the other is to induce the generation of polyploids. However, the existing methods have the defects of a long breeding cycle, low mutagenesis success rate, and inability to select excellent varieties in advance. Space breeding is a technical means of mutating crop germplasm by using unique environmental factors such as cosmic ray radiation, high vacuum, heavy particles, and microgravity in space, and then directionally screening and cultivating excellent new varieties. Compared with traditional breeding, space breeding significantly shortens the breeding cycle, thus greatly accelerating the breeding process. At present, space breeding of Benincasa hispida has been achieved, and the selected excellent germplasm resources have the characteristics of delicious taste, high yield, and regular fruit shape. In order to create more Benincasa hispida space breeding materials, it is necessary to select and screen space breeding materials and their offspring, accurately select space materials, optimize the efficiency of offspring selection, improve the recognition and utilization efficiency of excellent variations in space breeding, and thus accelerate the breeding process of space breeding materials.

[0004] Molecular markers are an important technology that can directly reflect genetic differences at the DNA level and are widely used in the genetic analysis and identification of germplasm resources of various crops. InDel (insertion / deletion) molecular markers are based on the insertion or deletion of nucleotide fragments and can distinguish different genotypes through length differences by means of conventional PCR and gel electrophoresis techniques. This marker method has the advantages of simple operation, low cost, low requirements for instruments and equipment, and accurate results, and is an ideal tool for the rapid identification and genetic analysis of germplasm resources. In addition, the polymorphism of InDel markers in different germplasm resources can provide effective support for trait screening and gene mapping in the breeding process. In existing research, molecular markers have been used to distinguish different wax gourd varieties and genotypes for the study of wax gourd genetic diversity and interspecific genetic relationships, but few have been used for the identification and screening of space breeding materials. In order to accelerate the breeding process of space breeding materials, it is urgent to develop methods or means that can pre-select wax gourd space materials in advance, shorten the breeding cycle, improve the accuracy of screening excellent mutations, reduce the breeding cost, and provide more high-quality and efficient new wax gourd varieties for agricultural production. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies of the existing identification methods for wax gourd space breeding materials and provide a molecular marker primer set for identifying or screening wax gourd space breeding materials and its application.

[0006] The object of the present invention is to provide an InDel molecular marker primer set.

[0007] Another object of the present invention is to provide the application of the InDel molecular marker primer set.

[0008] Another object of the present invention is to provide a kit.

[0009] Another object of the present invention is to provide the application of the kit.

[0010] Another object of the present invention is to provide a method for identifying or screening wax gourd space breeding materials and their offspring.

[0011] The above objects of the present invention are achieved by the following technical solutions:

[0012] An InDel molecular marker primer set of the present invention includes one or more of InDel1, InDel2, InDel3, InDel4, InDel5, and InDel6 primers: The specific primer sequences are as follows:

[0013] InDel1:

[0014] F: 5’-ACTAAGGCCCCATTTGGT-3’;

[0015] R: 5’-AGACTCCGTTTCATAACT-3’;

[0016] InDel2:

[0017] F: 5’-TGAAGAGAGAGGAGAGAATGAT-3’;

[0018] R: 5’-ACTCCCTCCTCCTTTTTCTCT-3’;

[0019] InDel3:

[0020] F: 5’-TGAACCGCAGTCCAATAGAT-3’;

[0021] R: 5’-TGCAAGTGTTGAACGCAAA-3’;

[0022] InDel4:

[0023] F: 5’-TGGGGTTGTTTGGGACA-3’;

[0024] R: 5’-ACCTGTTGTTATTTCGCTAAATA-3’;

[0025] InDel5:

[0026] F: 5’-TAGAGCCTTAAATAGGACTAGG-3’;

[0027] R: 5’-TGTGATGTGGCAATCTTGTC-3’;

[0028] InDel6:

[0029] F: 5’-AAGTGTGTAGTATCTGTAGCG-3’;

[0030] R: 5’-TGGGATTGATGTCCTAAATCTTGTG-3’.

[0031] Through whole-genome resequencing, the present invention conducted genetic variation analysis on spaceflight materials and non-spaceflight materials of wax gourd, and developed 6 pairs of InDel molecular marker primers InDel1-6 for identifying and screening spaceflight materials of wax gourd among 606,959 InDel markers. These primers can identify wax gourd materials at the seedling stage, without waiting for the appearance of material traits for identification, greatly reducing the breeding cycle, and enabling rapid, accurate, and efficient identification of spaceflight materials of wax gourd and their offspring. The present invention applies InDel molecular markers to the screening of spaceflight breeding materials and the selection of offspring, which can significantly improve the recognition and utilization efficiency of excellent variations in spaceflight breeding, can more accurately identify those mutations with important agricultural traits, thereby optimizing the efficiency of offspring selection and accelerating the breeding process of spaceflight breeding materials. Compared with the traditional breeding phenotype identification method, the use of InDel molecular marker primers for identification is accurate and reliable, can screen breeding target offspring in advance, improve the screening accuracy of excellent mutations, reduce breeding costs, and provide more high-quality and efficient new wax gourd varieties for agricultural production.

[0032] Therefore, the present invention provides the application of the InDel molecular marker primer set in identifying or screening spaceflight breeding materials of wax gourd and their offspring.

[0033] The present invention provides the application of the InDel molecular marker primer set in the preparation of a kit for identifying or screening spaceflight breeding materials of wax gourd and their offspring.

[0034] The present invention provides a kit containing the InDel molecular marker primer set.

[0035] Preferably, it contains reagents for extracting sample DNA.

[0036] More preferably, it also contains reagents required for PCR amplification.

[0037] The present invention provides the application of the above kit in identifying or screening spaceflight breeding materials of wax gourd and their offspring.

[0038] Meanwhile, the present invention also provides a method for identifying or screening spaceflight breeding materials of wax gourd and their offspring, including the following steps:

[0039] S1. Respectively extract the genomic DNA of the wax gourd sample to be tested;

[0040] S2. Using the genomic DNA extracted in S1 as a template, perform PCR amplification with the InDel molecular marker primer set, and then conduct agarose gel electrophoresis detection;

[0041] S3. Analyze the detection results of S1: When using the InDel1 primer for detection, if the amplification result shows a 39bp deletion, it is a space breeding material or its offspring; when using the InDel2 primer for detection, if the amplification result shows a 52bp insertion, it is a space breeding material or its offspring; when using the InDel3 primer for detection, if the amplification result shows a 32bp insertion, it is a space breeding material or its offspring; when using the InDel4 primer for detection, if the amplification result shows a 39bp insertion, it is a space breeding material or its offspring; when using the InDel5 primer for detection, if the amplification result shows a 41bp deletion, it is a space breeding material or its offspring; when using the InDel6 primer for detection, if the amplification result shows a 75bp deletion, it is a space breeding material or its offspring.

[0042] Preferably, in S2, one or more of the primers InDel1, InDel2, InDel3, InDel4, and InDel5 are used for detection.

[0043] Furthermore, the PCR amplification program is as follows: First, pre-denature at 94°C for 3 minutes, and then perform amplification cycles: denature at 94°C for 30 seconds, anneal at 55°C for 30 seconds, extend at 72°C for 30 seconds, for 38 cycles, and then extend at 72°C for 2 minutes.

[0044] The present invention has the following beneficial effects:

[0045] Through the re-sequencing data of wax gourd space breeding materials and genetic variation analysis with non-space materials, the present invention develops and screens out 6 pairs of InDel molecular marker primers that can identify wax gourd space materials and their offspring from 606959 InDel markers. These primers can identify wax gourd materials at the seedling stage, and can accurately select wax gourd space materials according to the size of their insertions / deletions, greatly reducing the breeding cycle and enabling the rapid, accurate, and efficient selection of wax gourd space materials and their offspring. At the same time, the present invention provides a method for identifying or screening wax gourd space breeding materials and their offspring. Applying InDel molecular markers to the screening of space breeding materials and the selection of offspring can significantly improve the recognition and utilization efficiency of excellent variations in space breeding, can more accurately identify those mutations with important agricultural traits, thereby optimizing the efficiency of offspring selection and accelerating the breeding process of space breeding materials. Compared with the traditional breeding phenotype identification method, using InDel molecular marker primers for identification is accurate and reliable, can screen breeding target offspring earlier, improve the screening accuracy of excellent mutations, reduce breeding costs, and provide more high-quality and efficient wax gourd new varieties for agricultural production. Description of the Drawings

[0046] Figure 1 are different wax gourd materials.

[0047] Figure 2Genetic variation analysis of space breeding materials (on the left in the figure is the statistical analysis of molecular marker types, and on the right is the PCA analysis of genomic variation).

[0048] Figure 3 Statistical results of variant marker regions and GO enrichment analysis.

[0049] Figure 4 Analysis results of the distribution of InDel markers on different chromosomes.

[0050] Figure 5 PCR detection results of some InDel molecular marker primers (in the figure, M is DNA Maker, H is spaceflight material, and FH is non-spaceflight material).

[0051] Figure 6 Accuracy verification results of InDel molecular marker primer InDel1-3 (in the figure, M is DNA Maker, H is spaceflight material, and FH is non-spaceflight material).

[0052] Figure 7 Accuracy verification results of InDel molecular marker primer InDel4-6 (in the figure, M is DNA Maker, H is spaceflight material, and FH is non-spaceflight material). Specific implementation manners

[0053] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0054] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0055] Example 1 Development of InDel molecular markers

[0056] 1. Test materials

[0057] The test materials are 11 strains of wax gourd of space breeding materials carried by Shenzhou XVI. The wax gourd materials were inbred line DG29 before being carried (male parent: Taro-scented small wax gourd; female parent: Japanese Huazhen small wax gourd, a line obtained by self-crossing and purification; DG29 is an excellent inbred line obtained by separating and purifying the hybrid offspring of the two). The inbred line DG29 has neat and stable traits and typical wax gourd characteristics. Three strains of wax gourd inbred line DG29 without spaceflight treatment were used as controls, and different wax gourd materials are as Figure 1 shown. All experimental materials were planted in the Wanjiang Base of Dongguan Agricultural Science Research Center to ensure the same planting environment for comparative analysis of the differences between space breeding and conventional breeding materials.

[0058] 2. Extraction of Genomic DNA, Library Construction, and High-throughput Sequencing

[0059] Whole-genome resequencing with a high depth (30x) was performed on 14 wax gourd materials. To ensure the accuracy of the data, strict quality control and filtering were carried out on the generated raw sequencing data to remove low-quality data and adapter sequences, eliminating any possible human bias. The specific quality control steps included the following key filtering criteria: (1) removing sequencing reads with more than 10% of the total number of nucleotides being uncertain; (2) removing sequencing reads with more than 50% of the total number of bases being low-quality bases (quality value ≤ 5); (3) identifying and removing sequencing reads containing adapter sequences; (4) removing duplicate sequences that might be generated by PCR amplification. After these steps, the high-quality data obtained was further used to generate a quality report of the sequencing data through FastQC (version 0.12.1).

[0060] To accurately align the filtered paired-end sequencing data to the high-quality wax gourd reference genome, the read alignment operation was performed using the default parameters of the BWA-MEM (version 0.7.17) tool, and a Sam format file was generated. After the alignment was completed, Samtools (version 1.15) was used to convert and index the alignment results to generate a bam format file. To improve the alignment accuracy, the MarkDuplicates function of Picard (version 2.25.7) was further used to mark and filter the duplicate sequences generated during the PCR amplification process.

[0061] After the alignment and duplicate removal processes were completed, the SNPs (single nucleotide polymorphisms) and InDels (insertion / deletion variations) in the wax gourd population were jointly called using the default parameters of GATK HaplotypeCaller and Variant Filter (version 4.2.6.1) according to the recommended hard filtering criteria. Finally, SnpEff (version 5.1) was used to perform functional annotation on the identified variations to reveal the possible biological effects of these variations, providing an important basis for subsequent trait research and functional analysis.

[0062] 3. Analysis of the Genomic Characteristics of Space-Bred Wax Gourd Materials

[0063] Based on the SNP and InDel marker information in the wax gourd materials obtained above, the number of locus variations in different materials was counted, and principal component analysis (PCA) was performed using Plink software to view the distribution characteristics of different materials.

[0064] The results are as Figure 2As shown, genetic variation analysis was carried out on spaceflight materials and non-spaceflight materials through whole-genome resequencing. A total of 3,485,348 high-quality SNPs and 606,959 InDels were detected in 14 samples. The sample statistics results show that spaceflight breeding materials generally contain more genetic variations and exhibit richer genetic diversity ( Figure 2 left). In addition, the results of PCA analysis show that there are significant differentiations between spaceflight breeding materials and the control group at the genomic level ( Figure 2 right). This indicates that under the action of space radiation, the genomic characteristics of spaceflight materials have changed significantly, which may endow them with more favorable biological characteristics, so that they can be better applied to crop breeding and provide an important genetic basis for the rapid breeding of new varieties.

[0065] At the same time, 1,142,743 newly generated SNPs and 115,311 InDels were found in spaceflight breeding materials. As Figure 3 shown, these variations are mainly located in the upstream and downstream and intergenic regions of genes ( Figure 3 left). The enrichment analysis results show that the variations of these genes may significantly affect the material transport, cytoskeleton regulation, metabolic regulation, external stimulus response and development-related genes of plants, indicating that the space environment may have a wide range of effects on the growth and environmental adaptability of wax gourd materials ( Figure 3 right).

[0066] 4. Development of specific InDel markers

[0067] In order to screen out specific InDel markers, taking the non-spaceflight materials of the control group and the reference genome as references, the newly mutated sites specifically generated in spaceflight breeding materials were extracted. Further, the Plink software was used to calculate the heterozygosity of these sites, and only the homozygous variant sites were retained for subsequent analysis. These specific InDel markers will help to better understand the genetic variations induced in the spaceflight breeding process and provide strong support for the selection in the breeding process.

[0068] By filtering out heterozygous variant sites and screening InDel markers that show consistent changes in all spaceflight materials, the study identified 76 InDel sites. Among them, two regions on Chr6 and Chr8 are mutation hotspots, as Figure 4 shown. In addition, 11 markers show specific variant sites relative to the reference genome and the control group. As shown in Table 1, these markers can be used for subsequent molecular marker development to distinguish spaceflight and non-spaceflight materials.

[0069] Table 1 Details of 11 molecular markers

[0070]

[0071] Screening for the Development of Specific InDel Markers in Example 2

[0072] According to the analysis results of Example 1, polymorphic InDel sites with an insertion / deletion sequence length greater than 20 bp were selected, and 250 bp of sequences on both sides of the InDel sites were retrieved. Specific primers were designed using Primer5. The upstream and downstream primers were located on both sides of the InDel site, the annealing temperature was set at 53 - 57 °C, the amplification fragment length was 100 - 400 bp, and multiple InDel marker primers were designed and synthesized. Some of the primers are shown in Table 2. Using the samples of Example 1, PCR amplification was carried out.

[0073] Table 2 Some InDel Marker Primers

[0074]

[0075] The PCR amplification system was 10 μL, including 1 μL of genomic DNA (150 ng / μL), 5 μL of 2×MIX, 0.2 μL each of the upstream and downstream primers (10 μmol / L), and 3.6 μL of ddH2O. The PCR amplification program was: pre-denaturation at 94 °C for 3 minutes, followed by amplification cycles (denaturation at 94 °C for 30 seconds, annealing at 55 °C for 30 seconds, extension at 72 °C for 30 seconds), a total of 38 cycles, and then extension at 72 °C for 2 minutes. Take 3 μL of the PCR product for detection by 3% agarose gel electrophoresis (190 V), and use a gel imager to take pictures to confirm the amplification effect of the target fragment.

[0076] The detection results are as Figure 5 shown. The results show that only the InDel1 (6 - 1), InDel2 (6 - 2), InDel3 (6 - 3), InDel4 (6 - 4), InDel5 (8 - 1), and InDel6 (8 - 4) markers can well distinguish between space - grown and non - space - grown wax gourd materials. Among them, the molecular marker InDel1 (6 - 1) can amplify a 339 - bp fragment in ordinary wax gourd materials, while in the genome of space - grown materials, due to a 39 - bp mutation and deletion, the amplified fragment is 300 bp; the molecular marker InDel6 (8 - 4) can amplify a 348 - bp fragment in ordinary wax gourd materials, while in the genome of space - grown materials, due to a 75 - bp mutation and deletion, the amplified fragment is 273 bp. Therefore, these InDel markers can accurately distinguish between space - grown and non - space - grown materials, avoiding confusion between materials. During the screening process of space - grown materials for wax gourd breeding hybrid offspring, these markers have important application value and also provide a reference basis for further studying the mechanism of space radiation - induced mutations.

[0077] Example 3 A Method for Identifying Wax Gourd Space - Breeding Materials Using InDel Molecular Markers

[0078] Use the 6 pairs of molecular marker primers identified in Example 2: InDel1 - InDel6. Randomly select 38 spaceflight wax gourd materials planted in the Wanjiang Base of Dongguan Agricultural Science Research Center and 10 non - spaceflight wax gourd materials as controls in combination with breeding phenotypes. Verify the accuracy of these 6 molecular marker primers in different spaceflight materials. Extract the genomic DNA of the wax gourd materials to be tested respectively, and perform detection with reference to the detection method conditions in Example 2.

[0079] The detection results are as Figure 6 and Figure 7 shown. After inspection, it shows that the accuracy rates of the molecular marker primers InDel1, InDel2, InDel3, InDel4, and InDel5 are 100%, and the accuracy rate of InDel6 is 81%. It shows that these 6 pairs of primers can all be used as molecular markers for identifying spaceflight and non - spaceflight wax gourd materials, can distinguish spaceflight materials and non - spaceflight materials, have high accuracy, and avoid confusion between materials. Among them, the molecular marker primer pairs InDel1, InDel2, InDel3, InDel4, and InDel5 have higher accuracy. In summary, using InDel molecular markers in the screening and identification of spaceflight breeding materials has many advantages such as shortening the breeding cycle, improving the accuracy of screening excellent mutations, and reducing breeding costs, which will provide more high - quality and efficient new varieties for agricultural production.

[0080] The above - mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above - mentioned embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. An InDel molecular marker primer set, characterized in that, Comprising one or more of the primers InDel1, InDel2, InDel3, InDel4, InDel5, InDel6: The specific primer sequences are as follows: InDel1: F: 5’-ACTAAGGCCCCATTTGGT-3’; R: 5’-AGACTCCGTTTCATAACT-3’; InDel2: F: 5’-TGAAGAGAGAGGAGAGAATGAT-3’; R: 5’-ACTCCCTCCTCCTTTTTCTCT-3’; InDel3: F: 5’-TGAACCGCAGTCCAATAGAT-3’; R: 5’-TGCAAGTGTTGAACGCAAA-3’; InDel4: F: 5’-TGGGGTTGTTTGGGACA-3’; R: 5’-ACCTGTTGTTATTTCGCTAAATA-3’; InDel5: F: 5’-TAGAGCCTTAAATAGGACTAGG-3’; R: 5’-TGTGATGTGGCAATCTTGTC-3’; InDel6: F: 5’-AAGTGTGTAGTATCTGTAGCG-3’; R: 5’-TGGGATTGATGTCCTAAATCTTGTG-3’.

2. Use of the primer set according to claim 1 in identifying or screening spaceflight breeding materials of wax gourd and their offspring.

3. Use of the primer set according to claim 1 in preparing a kit for identifying or screening spaceflight breeding materials of wax gourd and their offspring.

4. A kit, characterized in that, Containing the primer set according to claim 1.

5. The kit according to claim 4, wherein Containing reagents for extracting sample DNA.

6. The kit according to claim 5, characterized in that, Also containing reagents required for PCR amplification.

7. Use of the kit according to any one of claims 4 - 6 in identifying or screening spaceflight breeding materials of wax gourd and their offspring.

8. A method for identifying or screening space breeding materials of wax gourd and their offspring, characterized in that, Including the following steps: S1. Respectively extract the genomic DNA of the wax gourd sample to be tested; S2. Using the genomic DNA extracted in S1 as a template, perform PCR amplification with the primer set according to claim 1, and then perform agarose gel electrophoresis detection; S3. Analyze the detection results of S1: When using the InDel1 primer for detection, those with an amplified result showing a 39bp deletion are spaceflight breeding materials or offspring; when using the InDel2 primer for detection, those with an amplified result showing a 52bp insertion are spaceflight breeding materials or offspring; when using the InDel3 primer for detection, those with an amplified result showing a 32bp insertion are spaceflight breeding materials or offspring; when using the InDel4 primer for detection, those with an amplified result showing a 39bp insertion are spaceflight breeding materials or offspring; when using the InDel5 primer for detection, those with an amplified result showing a 41bp deletion are spaceflight breeding materials or offspring; when using the InDel6 primer for detection, those with an amplified result showing a 75bp deletion are spaceflight breeding materials or offspring.

9. The method according to claim 8, wherein In S2, one or more of the primers InDel1, InDel2, InDel3, InDel4, InDel5 are used for detection.

10. The method according to claim 8, characterized in that The PCR amplification program was as follows: pre-denaturation at 94°C for 3 minutes, followed by amplification cycles: denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds, for 38 cycles, and then extension at 72°C for 2 minutes.

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