KASP molecular marker closely linked with citrus embryo color and application of KASP molecular marker

Abstract

The invention discloses a KASP molecular marker closely linked with the color of a citrus embryo and application of the KASP molecular marker, and belongs to the technical field of biology. The SNP locus closely linked with the white / green character of the citrus embryo is positioned through BSA-seq, the SNP locus is located at the 8739586 site of the fifth chromosome of a sweet orange second-generation reference genome, the base type of the locus is the genotype of T or G, TT or TG, and the color of the citrus embryo corresponding to the genotype of GG is white or green. The KASP molecular marker is developed by utilizing the SNP locus, the SNP locus can be directly used for identifying the embryo color and the corresponding genotype, and the development and implementation of the marker not only can effectively identify the citrus embryo color, but also can accurately predict the embryo color of a seedless variety, and is used for assisting the selection and matching of citrus crossbreeding parents; the marker can also be used for quickly identifying sexual embryos of the polyembryonic variety, and the sexual crossbreeding efficiency taking the polyembryonic variety as a female parent is improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a KASP molecular marker closely linked to citrus embryo color and an application thereof. Background Art

[0002] Citrus is the most widely cultivated and important economic fruit tree in southern my country. my country is rich in citrus resources, including pomelo, grapefruit, orange, mandarin, tangerine, hybrid citrus, lemon and kumquat. Both inter-species and intra-species sexual hybridization are very easy to produce new recombinant mutations. Due to the influence of nucellar polyembryony and the lack of rapid and accurate identification of polyembryonic varieties with sexual embryos, breeders prefer to select monoembryonic varieties as female parents for hybrid breeding. However, due to the limited number of monoembryonic varieties, long-term sexual hybridization with a few monoembryonic varieties as female parents has gradually reduced the genetic diversity of offspring, resulting in some hybrid varieties currently cultivated. Problems such as premature aging of the tree, difficulty in management and decreased flavor quality. Citrus polyembryonic varieties are rich in types and are excellent breeding parents. The hybrid offspring with them as female parents have rich genetic variation, which can effectively solve the problem of genetic homogeneity of hybrid offspring. However, there is a lack of early and efficient identification methods for zygotic embryos in breeding, making it difficult to effectively separate sexual embryos from nucellar embryos, resulting in low breeding efficiency, resulting in slow progress in citrus hybrid breeding with polyembryonic varieties as female parents.

[0003] Citrus seed embryo colors include white and green, which are important morphological markers and evolutionary traits. The inventors previously developed a method for efficiently identifying sexual embryos in citrus polyembryonic varieties using seed embryo colors, that is, sexual embryos can be accurately identified based on the number of white / green embryos in polyembryonic variety seeds (green embryos of "mostly white and 1 green" and white embryos of "mostly green and 1 white" are sexual embryos). However, the existing problem is that seed embryo color can only identify sexual embryos with different colors from nucellar embryos, while sexual embryos with the same color as nucellar embryos are difficult to identify. Therefore, it is urgent to develop molecular markers that are closely linked to seed embryo color and can accurately and quickly identify all sexual embryos. Summary of the invention

[0004] The inventors' previous studies found that the white / green trait of citrus embryos is controlled by a single gene, and white is a dominant trait over green; sexual populations were created by sexual hybridization of varieties with different white / green embryo traits, and combined with BSA-seq analysis, the key genes controlling the white / green trait of citrus embryos were located; through co-segregation analysis, a SNP variation site closely linked to the trait was discovered; and using KASP (Kompetitive Allele Specific PCR) technology, a KASP molecular marker closely linked to the color of citrus embryos was developed.

[0005] Based on the above findings, the present invention provides a SNP site closely linked to the color of citrus embryos, which is located at position 8739586 of chromosome 5 of the second-generation sweet orange reference genome, and the base type of the site is T or G. The TT or TG genotype of the base type of the site corresponds to a white citrus embryo color, and the GG genotype corresponds to a green citrus embryo color.

[0006] The present invention also provides a DNA fragment containing the above-mentioned SNP site in the citrus genome, which contains the nucleotide sequence shown in SEQ ID NO:1, and the SNP site is located at the 24th position of the nucleotide sequence shown in SEQ ID NO:1.

[0007] The present invention also provides the use of the SNP site or the DNA fragment as a KASP molecular marker for the white / green trait of citrus embryos.

[0008] The present invention also provides a primer set for detecting KASP molecular markers of white / green traits of citrus embryos, which comprises primers for detecting the base type of the above-mentioned SNP site.

[0009] In a specific embodiment, the primers include a first forward primer, a second forward primer, and a reverse primer;

[0010] The sequence of the first forward primer is: 5'-AATTCTCAGGTAATCCTTTGATCT-3' (SEQ ID NO: 2);

[0011] The sequence of the second forward primer is: 5'-AATTCTCAGGTAATCCTTTGATCG-3' (SEQ ID NO: 3);

[0012] The sequence of the reverse primer is: 5'-CTGATTAGGCACCTGGATTTTG-3' (SEQ ID NO: 4).

[0013] In a specific embodiment, the 5' ends of the first forward primer and the second forward primer are respectively labeled with different types of fluorescent markers.

[0014] The present invention also provides a kit for detecting the KASP molecular marker of the white / green trait of citrus embryos, which comprises the above primer set.

[0015] The present invention also provides applications of the above-mentioned SNP sites, DNA fragments, primer sets or kits, including: applications in early identification of citrus embryo color, applications in identification of sexual embryo hybrids of citrus polyembryonic varieties, and applications in citrus molecular marker-assisted breeding.

[0016] The present invention also provides a method for early identification of citrus embryo color, which comprises the following steps: using the DNA of the citrus sample to be detected as a template, performing PCR amplification using the above primer set, and detecting the gene type of the above SNP site, the TT or TG genotype is white, and the GG gene type is green.

[0017] The present invention also provides a method for identifying sexual embryo hybrids of citrus polyembryonic varieties, the method comprising the following steps:

[0018] The DNA of the parents and the offspring to be identified as hybrids is used as a template, and PCR amplification is performed using the above primer set to detect the gene type of the above SNP site.

[0019] If the genotype TT is the mother parent (the nucellar embryo genotype is TT and the embryo color is white), when the hybrid combination is 'TT×GG', the genotype of the sexual embryo is TG; when the hybrid combination is 'TT×TG', the genotype of the sexual embryo is TT and TG; when the hybrid combination is 'TT×TT', the genotype of the sexual embryo is TT; this marker can identify sexual embryos with a genotype of TG, but sexual embryos with a genotype of TT cannot be identified by this marker because they have the same genotype as the nucellar embryo.

[0020] If the genotype TG is the mother parent (the nucellar embryo genotype is TG, and the embryo color is white), when the hybrid combination is 'TG×TT', the genotypes of the sexual embryos are TT and TG; when the hybrid combination is 'TG×TG', the genotypes of the sexual embryos are TT, TG and GG; when the hybrid combination is 'TG×GG', the genotypes of the sexual embryos are TT and TG; this marker can identify sexual embryos with genotypes of TT and GG. Since the sexual embryos with genotype of TG are consistent with the genotype of the nucellar embryo, this marker cannot be used for hybrid identification.

[0021] If the genotype GG is the female parent (the nucellar embryo genotype is GG, and the embryo color is green), when the hybrid combination is 'GG×TT', the genotype of the sexual embryo is TG; when the hybrid combination is 'GG×TG', the genotype of the sexual embryo is TG and GG; when the hybrid combination is 'GG×GG', the genotype of the sexual embryo is GG; this marker can identify sexual embryos with a genotype of TG, and sexual embryos with a genotype of GG cannot be used for hybrid identification because they have the same genotype as the nucellar embryo.

[0022] The beneficial effects of the present invention are as follows: the present invention locates a SNP site closely linked to the white / green trait of citrus embryos through BSA-seq, and develops a KASP molecular marker using the SNP site. By detecting the SNP site, it can be directly used for embryo color and corresponding genotype identification. The development and implementation of the marker can not only effectively identify the color of citrus embryos, but also accurately predict the embryo color of seedless varieties, which is used to assist in the selection of parents for citrus hybrid breeding; at the same time, the marker can be used to quickly identify the sexual embryos of polyembryo varieties, thereby improving the efficiency of sexual hybrid breeding with polyembryo varieties as maternal parents. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The color of the embryos of the hybrid parents and sexual hybrid offspring of the combination of 'Kiyomi Mandarin Orange (white) × Ponkan (green)'. The embryos of the hybrid offspring have a 1:1 segregation of white and green traits.

[0024] Figure 2 This is a fine-mapping of candidate genes closely linked to citrus embryo color; Figure A is a preliminary BSA-seq mapping of embryo color trait genes. Figure B is a fine-mapping of embryo color trait genes.

[0025] Figure 3 The KASP marker of the SNP closely linked to the citrus embryo color developed by the present invention was used to perform genotyping on the sexual hybrid population of 'Clementine × HB pomelo'; the blue ones are HB pomelo embryo samples, the green ones are Clementine embryo samples, the red ones are white embryo samples, and the gray ones are blank controls (H2O).

[0026] Figure 4 This is a genotyping diagram of the 'Qingjian×Wogan' population using the KASP marker developed by the present invention that is tightly linked to the SNP of citrus embryo color; the blue one is the Qingjian sample, the green one is the Wogan sample, and the gray one is the blank control (H2O);

[0027] Figure 5 This is the genotyping diagram of the 'Qingjian×Cocktail Grapefruit' population using the KASP marker developed by the present invention, which is a SNP tightly linked to the color of citrus embryos; the blue one is the Qingjian sample, the green one is the Cocktail sample, and the gray one is the blank control (H2O);

[0028] Figure 6 This is a genotyping diagram of a natural population of 55 citrus white / green embryos using the KASP marker developed by the present invention, which is a SNP tightly linked to the color of citrus embryos. DETAILED DESCRIPTION

[0029] In order to clearly and completely describe the purpose, technical solutions and advantages of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. The described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Example 1

[0031] Mapping of tightly linked genes for citrus embryo color and development of KASP markers

[0032] This example is based on BSA-seq analysis of a sexual hybrid population with 1:1 segregation of white / green embryo traits of 'Kiyomi Mandarin Orange × Ponkan', and locates the key gene for citrus embryo color and develops a SNP site that is closely linked to the white / green trait of the embryo. KASP primers are designed for the SNP site, and specifically include the following steps:

[0033] 1. Genetic population construction and phenotypic statistics

[0034] The white / green trait of the embryo color of the hybrid seed was analyzed by sexual hybridization of the Kiyomizu orange (white embryo) as the female parent and the Ponkan (green embryo) as the male parent. A total of 946 seeds were obtained from this hybrid combination. The embryo color phenotype was investigated and statistically analyzed. The white / green trait of the embryo color of the sexual offspring was separated ( Figure 1 ), white embryo: green embryo = 471:475, consistent with the Mendelian segregation ratio of 1:1 (χ 2 0.05 =0.017<3.84), indicating that the white / green trait of citrus embryo is a quality trait, among which white is a dominant trait over green.

[0035] 2. Localization of citrus embryo color genes

[0036] From the 946-strain F1 sexually segregating population of 'Kiyomi Jucheng × Ponkan', 30 white embryo seedlings and 30 green embryo seedlings were randomly selected to construct white embryo pools and green embryo pools for mixed pool sequencing (BSA-seq). Through bioinformatics analysis, the gene controlling the white / green trait of citrus embryos was preliminarily located in the 7.37Mb-12.10Mb interval of chromosome 5. Using the parental resequencing data, polymorphic SNP molecular markers between parents were developed in the initial positioning interval, and the 946-strain F1 sexually segregating population of 'Kiyomi Jucheng × Ponkan' was used as the material for fine positioning of the white / green trait of the embryo, further narrowing the positioning interval, and finally the gene controlling the white / green trait of the citrus embryo was located to the 103kb interval ( Figure 2). Linkage analysis was performed on all variant sites and traits within this interval, and a SNP molecular marker (T / G) was identified at position 8739586 of chromosome 5, which was closely linked to the white / green trait of citrus embryos. The color of citrus embryos with base types of TT or TG was white, and the color of citrus embryos with base types of GG was green.

[0037] 3. Design of KASP primers and their application

[0038] According to the chr5_8739586 site and the DNA base sequence information on both sides, KASP primers were designed using Snapgene software. The amplification sequence and primer sequences are as follows:

[0039] Amplified sequence: 5′-AATTCTCAGGTAATCCTTTGATCNACAAAATCCAGGTGCCTAATCAG-3′ (SEQ ID NO: 1, N represents G or T).

[0040] Primer sequences:

[0041] F1: 5'-FAM-gaaggtgaccaagttcatgct-AATTCTCAGGTAATCCTTTGATCT-3' (SEQ ID NO: 5, the lowercase letter part is the specific fluorescent tag sequence FAM);

[0042] F2: 5'-HEX-gaaggtcggagtcaacggatt-AATTCTCAGGTAATCCTTTGATCG-3' (SEQ ID NO: 6, the lowercase letter part is the specific fluorescent tag sequence HEX);

[0043] R: 5'-CTGATTAGGCACCTGGATTTTG-3' (SEQ ID NO: 4).

[0044] The detection method is as follows:

[0045] 1) Extracting genomic DNA from citrus leaves;

[0046] 2) Fluorescence quantitative PCR detection was performed with the following reaction system: 2.5 μL of genomic DNA (DNA content: 5-50 ng), 2.5 μL of 2×KASP reaction mixture (purchased from LGC, UK) and 0.07 μL of primer mixture (mixed with equal volumes of F1 (concentration: 36 μM), F2 (concentration: 36 μM) and R (concentration: 90 μM)).

[0047] The reaction procedure was as follows: pre-denaturation at 94°C for 15 min; the first stage amplification cycle, denaturation at 94°C for 20 sec, annealing / extension at 61-55°C for 60 sec, decreasing by 0.6°C per cycle, for a total of 10 cycles; the second stage amplification cycle, denaturation at 94°C for 20 sec, annealing / extension at 55°C for 60 sec, for a total of 26 cycles; fluorescence reading at 37°C for 60 sec to obtain the PCR amplification product.

[0048] The result graph was automatically generated using LightCycler R480 SW1.5.1 software, and cluster analysis of PCR amplification products was performed according to the fluorescence type.

[0049] Example 2

[0050] The embryo color of Clementine orange is green, the embryo color of HB pomelo is white, and the embryo color of the hybrid seed of 'Clementine orange × HB pomelo' is white. 42 plants formed by germination of white seeds of the combination of 'Clementine orange × HB pomelo' were randomly selected, and the genomic DNA of the above 42 plants and parents was extracted respectively by the improved CTAB method. The KASP primers were used to amplify the parents and the hybrid plants. The method was as described in Example 1. The genotyping results showed that the Clementine orange was GG, the HB pomelo was TT, and the genotypes of the 42 plants formed by germination of white embryos at this site were all TG, indicating that the marker can accurately distinguish between white embryos and green embryos of citrus ( Figure 3 ).

[0051] Example 3

[0052] The embryos of Qingjian orange and Wogan are white, and the embryos of 'Qingjian orange × Wogan' have separated in color, with white embryo:green embryo=274:72, which is consistent with the Mendelian separation ratio of 3:1. 32 seedlings formed by green embryo germination and 27 seedlings formed by white embryo germination were randomly selected, and the genomic DNA of the above 59 individual plants and the hybrid parents were extracted by the improved CTAB method. The KASP primers developed above were used to perform genotyping on the parents and the hybrid individual plants. The method was as described in Example 1. The genotyping results showed that the genotype of Qingjian orange was TG, the genotype of Wogan was also TG, the genotype of 32 green embryo seedlings was GG, the genotype of 15 of the 27 white embryo seedlings was TG, and the genotype of 12 was TT ( Figure 4 ), indicating that the marker can accurately distinguish between white and green embryos of citrus.

[0053] Example 4

[0054] The embryos of the Qingjian orange and the cocktail grapefruit are white. The sexual embryos obtained by the hybridization of 'Qingjian orange × cocktail grapefruit' have color separation, with white embryo: green embryo = 154:58, which is consistent with the Mendelian segregation ratio of 3:1 32 seedlings formed by green embryo germination and 27 seedlings formed by white embryo germination were randomly selected, and the genomic DNA of the above 59 individual plants and parents was extracted by the improved CTAB method. The KASP primers developed above were used to perform genotyping on the parents and hybrid individual plants. The method was as described in Example 1. The genotyping results showed that the genotype of Kiyomi mandarin orange was TG, the genotype of cocktail grapefruit was TG, the genotype of 32 green embryo seedlings was GG, and the genotype of 15 of the 27 white embryo seedlings was TG, and the genotype of 12 was TT ( Figure 5 ), indicating that the marker can accurately distinguish between white and green embryos of citrus.

[0055] Example 5

[0056] 55 citrus germplasm resources with different embryo colors were randomly selected, including 18 green embryo resources and 37 white embryo resources. The genomic DNA of the above 55 resources was extracted by the improved CTAB method. The 55 citrus germplasm resources with different embryo colors were genotyped using the developed KASP primers. The method was as described in Example 1. The genotyping results showed that the genotypes of the 18 green embryo resources were all GG, and the genotypes of the locus of 21 of the 37 white embryo resources were TG, and the genotypes of 16 were TT ( Figure 6 ), this molecular marker can accurately distinguish the white and green traits of the embryo.

Claims

1. A SNP locus closely linked to citrus embryo color, characterized by: The SNP site is located at position 8739586 of chromosome 5 of the citrus genome, and its base type is T or G; the color of citrus embryos with the genotype of the SNP site being TT or TG is white, and the color of citrus embryos with the genotype of the SNP site being GG is green.

2. A DNA fragment in a citrus genome comprising the SNP site of claim 1, characterized in that: It comprises the nucleotide sequence shown in SEQ ID NO:1, and the SNP site is located at the 24th position of the nucleotide sequence shown in SEQ ID NO:

1.

3. Use of the SNP site described in claim 1 or the DNA fragment described in claim 2 as a KASP molecular marker for the white / green trait of citrus embryos.

4. A primer set for detecting KASP molecular markers of white / green trait of citrus embryos, characterized in that: Contains primers for detecting the base type of the SNP site described in claim 1.

5. The primer set according to claim 4, characterized in that: The primers for detecting the base type of the SNP site described in claim 1 include a first forward primer, a second forward primer and a reverse primer; The sequence of the first forward primer is: 5'-AATTCTCAGGTAATCCTTTGATCT-3'; The sequence of the second forward primer is: 5'-AATTCTCAGGTAATCCTTTGATCG-3'; The sequence of the reverse primer is: 5'-CTGATTAGGCACCTGGATTTTG-3'.

6. The primer set according to claim 5, characterized in that: The 5' ends of the first forward primer and the second forward primer are respectively labeled with different types of fluorescent markers.

7. A kit for detecting KASP molecular markers of white / green trait of citrus embryos, characterized in that: A primer set comprising any one of claims 4 to 6.

8. Use of the SNP site according to claim 1, the DNA fragment according to claim 2, the primer set according to any one of claims 4 to 6, or the kit according to claim 7, characterized in that: include: Its application in early identification of citrus embryo color, in identification of sexual embryo hybrids of citrus polyembryonic varieties, and in molecular marker-assisted breeding of citrus.

9. A method for early identification of citrus embryo color, characterized in that: The method comprises the following steps: using the DNA of the citrus sample to be detected as a template, performing PCR amplification using the primer set described in any one of claims 4 to 6, and detecting the gene type of the SNP site described in claim 1, wherein the TT or TG genotype is white, and the GG genotype is green.

10. A method for identifying sexual embryo hybrids of citrus polyembryonic varieties, characterized in that: The following steps are involved: Using DNA of the parents and the offspring to be identified as hybrids as templates, PCR amplification is performed using the primer set described in any one of claims 4 to 6 to detect the gene type of the SNP site described in claim 1; If the genotype TT is the female parent, when the hybrid combination is 'TT×GG', the genotype of the sexual embryo is TG; when the hybrid combination is 'TT×TG', the genotype of the sexual embryo is TT and TG; when the hybrid combination is 'TT×TT', the genotype of the sexual embryo is TT; If the genotype TG is the female parent, when the hybrid combination is 'TG×TT', the genotypes of the sexual embryos are TT and TG; when the hybrid combination is 'TG×TG', the genotypes of the sexual embryos are TT, TG and GG; when the hybrid combination is 'TG×GG', the genotypes of the sexual embryos are TT and TG; If the genotype GG is the female parent, when the hybrid combination is 'GG×TT', the genotype of the sexual embryo is TG; when the hybrid combination is 'GG×TG', the genotype of the sexual embryo is TG and GG; when the hybrid combination is 'GG×GG', the genotype of the sexual embryo is GG.

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