A kasp molecular marker closely linked to citrus embryo color and application thereof

Abstract

The application discloses a KASP molecular marker closely linked with citrus embryo color and an application thereof, and belongs to the technical field of biotechnology. The application is located to a SNP site closely linked with the citrus embryo white / green color through BSA-seq, and the SNP site is located at the 8739586th position of the 5th chromosome of a sweet orange second-generation reference genome. The base type of the SNP site is T or G, the TT or TG genotype and the GG genotype correspond to the citrus embryo color of white and green. The application develops a KASP molecular marker by using the SNP site, and the SNP site can be directly used for embryo color and corresponding genotype identification by detection. The marker development and implementation can not only effectively identify the citrus embryo color, but also accurately predict the embryo color of seedless varieties, and is used for assisting the citrus cross breeding parent matching. The marker can also be used for rapidly identifying the sexual embryos of polyembryonic varieties, and improving the sexual cross breeding efficiency of the polyembryonic varieties as the female parent.

Description

Technical Field

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

[0002] Citrus (Citrus) is the most widely cultivated and important economic fruit tree in southern my country. my country boasts abundant citrus resources, including pomelo, grapefruit, orange, mandarin, tangerine, hybrid citrus, lemon, and kumquat. Sexual hybridization is highly susceptible to generating new recombination variants both within and between species. Due to the influence of nucellar polyembryony and the lack of rapid and accurate methods for identifying sexual embryos in polyembryonic varieties, breeders prefer monoembryonic varieties as maternal parents for hybridization. However, due to the limited number of monoembryonic varieties, long-term sexual hybridization using a few monoembryonic varieties as maternal parents has gradually reduced the genetic diversity of the offspring. This has led to problems in some currently cultivated hybrids, such as premature aging, difficulty in management, and decreased flavor quality. Citrus polyembryonic varieties are abundant and serve as excellent breeding parents. Hybrids derived from these varieties exhibit rich genetic variation, effectively addressing the issue of genetic homogeneity in hybrids. However, the lack of efficient and early identification methods for zygotic embryos makes it difficult to effectively isolate sexual embryos from nucellar embryos, resulting in low breeding efficiency and slow progress in citrus hybridization using polyembryonic varieties as maternal parents.

[0003] Citrus embryo color, which includes white and green, is an important morphological marker and evolutionary trait. The inventors previously developed a method for efficiently identifying sexual embryos in polyembryonic citrus varieties using embryo color. This method accurately identifies sexual embryos based on the number of white / green embryos in polyembryonic cultivars (green embryos in "mostly white and one green" and white embryos in "mostly green and one white" are sexual embryos). However, the existing problem is that embryo color can only identify sexual embryos that are different in color from the nucellar embryo, while sexual embryos with the same color as the nucellar embryo are difficult to identify. Therefore, there is an urgent need to develop molecular markers that are tightly linked to embryo color and can accurately and quickly identify all sexual embryos. Summary of the Invention

[0004] The inventors' previous research found that the white / green trait of citrus embryos is controlled by a single gene, and white is dominant over green. By sexually hybridizing varieties with different embryo white / green traits to create sexual populations, combined with BSA-seq analysis, the inventors located the key gene controlling the white / green trait of citrus embryos. Through co-segregation analysis, they discovered a SNP variation site that is closely linked to this trait. Using KASP (Kompetitive Allele Specific PCR) technology, they developed a KASP molecular marker that is closely linked to the color of citrus embryos.

[0005] Based on the above finding, the present application provides a SNP site closely linked to citrus embryo color, which is located at the 8739586th position of chromosome 5 of the sweet orange second-generation 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 white citrus embryo color, and the GG genotype corresponds to green citrus embryo color.

[0006] The present application also provides a DNA fragment containing the above-mentioned SNP site in the citrus genome, which 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.

[0007] The present application also provides the application of the above-mentioned SNP site or the above-mentioned DNA fragment as a KASP molecular marker for the white / green trait of citrus embryo.

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

[0009] In one 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 one specific embodiment, the 5' ends of the first forward primer and the second forward primer are respectively labeled with different types of fluorescent labels.

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

[0015] The present application also provides the application of the above-mentioned SNP site, DNA fragment, primer set or kit, which includes the application in early identification of citrus embryo color, application in sexual embryo hybrid identification of citrus polyembryonic varieties, and application in molecular marker assisted breeding of citrus.

[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 tested as a template, performing PCR amplification using the above-mentioned primer set, and detecting the gene type of the above-mentioned SNP site, where the TT or TG genotype is white, and the GG genotype 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 hybridized was used as a template and the above primer set was used for PCR amplification to detect the gene type of the above SNP site.

[0019] If the genotype TT is the female 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 used for hybrid identification because they have the same genotype as the nucellar embryo.

[0020] If the genotype TG is the female 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 tightly 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 this 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 parent selection in citrus hybrid breeding; at the same time, the marker can be used to quickly identify sexual embryos of polyembryonic varieties, thereby improving the efficiency of sexual hybrid breeding with polyembryonic varieties as maternal parents. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The color of the embryos of the hybridized parents and sexual hybrid offspring of the combination of 'Kiyomi Mandarin Orange (white) × Ponkan (green)' is shown. The offspring embryos 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 shows the preliminary BSA-seq mapping of embryo color genes. Figure B shows the fine-mapping of embryo color genes.

[0025] Figure 3 Genotyping of a 'Clementine × HB pomelo' sexual hybrid population using the KASP markers developed in this invention, which are closely linked to citrus embryo color. Blue represents HB pomelo embryo samples, green represents Clementine embryo samples, red represents white embryo samples, and gray represents a blank control (H2O).

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

[0027] Figure 5 This is a genotyping diagram of the 'Kiyomi × Cocktail Grapefruit' population using the KASP marker developed by the present invention, which is tightly linked to the SNP of citrus embryo color. The blue color represents the Kiyomi sample, the green color represents the Cocktail sample, and the gray color represents 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 with reference to the accompanying drawings and embodiments. The described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making 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 of 'Kiyomi Mandarin Orange × Ponkan' segregating 1:1 for white / green embryos. Key genes for citrus embryo color were located and a single nucleotide polymorphism (SNP) site tightly linked to the white / green embryo trait was discovered. KASP primers were designed targeting this SNP site. The specific steps involved:

[0033] 1. Genetic population construction and phenotypic statistics

[0034] A sexual hybrid was conducted using Kiyomizu orange (white embryo) as the female parent and Ponkan (green embryo) as the male parent, and the white / green trait segregation of the embryo color of the hybrid seeds was statistically analyzed. A total of 946 seeds were obtained from this hybrid combination, and the embryo color phenotype was investigated and statistically analyzed. The white / green trait of the embryo color of the sexual offspring was segregated ( 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 embryos is a quality trait, in which white is dominant to green.

[0035] 2. Localization of citrus embryo color genes

[0036] From the 946-strain F1 sexually segregating population of 'Kiyomi Citrus × Ponkan', 30 white 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 within the 7.37Mb-12.10Mb interval of chromosome 5. Using the parental resequencing data, polymorphic SNP molecular markers between the parents were developed within the initial positioning interval. The 946-strain F1 sexually segregating population of 'Kiyomi Citrus × Ponkan' was used as the material for fine positioning of the white / green trait of the embryo, further narrowing the positioning interval. Finally, the gene controlling the white / green trait of the citrus embryo was located within the 103kb interval ( Figure 2). Linkage analysis was performed on all the variation sites and traits in the interval, and a SNP molecular marker (T / G) at position 8739586 on chromosome 5 was identified to be closely linked to the citrus embryo white / green trait. Citrus embryos with base type TT or TG were white in color, and those with base type GG were green in color.

[0037] 3. Design of KASP primer and its application

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

[0039] Amplification sequence: 5'-AATTCTCAGGTAATCCTTTGATCNACAAAATCCAGGTGCCTAATCAG-3'(SEQ ID NO: 1, N represents G or T).

[0040] Primer sequence:

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

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

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

[0044] The detection method is as follows:

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

[0046] 2) Perform fluorescent quantitative PCR detection, and the reaction system is as follows: genomic DNA 2.5 μL (DNA content is 5-50 ng), 2×KASP reaction mixture (purchased from LGC company in the United Kingdom) 2.5 μL and 0.07 μL primer mixture (mixed by F1 (concentration is 36 μM), F2 (concentration is 36 μM), R (concentration is 90 μM) and the like in equal volume).

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

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

[0049] Example 2

[0050] The embryos of Clementine mandarin are green, the embryos of HB pomelo are white, and the embryos of the hybrid seeds of 'Clementine mandarin × HB pomelo' are all white. Forty-two plants germinated from white seeds of the 'Clementine mandarin × HB pomelo' combination were randomly selected. Genomic DNA of the 42 plants and their parents was extracted using a modified CTAB method. The KASP primers were used to amplify the parental and hybrid plants using the method described in Example 1. Genotyping results showed that the Clementine mandarin was GG, the HB pomelo was TT, and the 42 plants germinated from white embryos were all TG at this locus, indicating that this marker can accurately distinguish between white and green citrus embryos ( Figure 3 ).

[0051] Example 3

[0052] The embryos of Qingjian Tangerine are white, and the embryos of Wogan are also white. The embryos of 'Qingjian Tangerine × Wogan' have undergone color separation, with white embryos: green embryos = 274:72, which conforms to the Mendelian segregation ratio of 3:1. Thirty-two seedlings germinated from green embryos and 27 seedlings germinated from white embryos were randomly selected. Genomic DNA of the 59 individual plants and the hybrid parents was extracted using a modified CTAB method. Genotyping of the parents and the hybrid plants was performed using the KASP primers developed above, as described in Example 1. The genotyping results showed that the genotype of the Qingjian orange was TG, the genotype of the Wogan was also TG, the genotype of the 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 white and green embryos in citrus.

[0053] Example 4

[0054] The embryos of the Kiyomizu orange and the Cocktail grapefruit are white. The sexual embryos obtained from the hybridization of Kiyomizu orange and Cocktail grapefruit exhibited color segregation, with a white embryo:green embryo ratio of 154:58, which conforms to the Mendelian segregation ratio of 3:1. Thirty-two seedlings germinated from green embryos and 27 seedlings germinated from white embryos were randomly selected. Genomic DNA of the 59 individual plants and their parents was extracted using a modified CTAB method. Genotyping of the parents and hybrid plants was performed using the KASP primers developed above, as described in Example 1. The genotyping results showed that the genotype of the Kiyomi mandarin orange was TG, the genotype of the cocktail grapefruit was TG, the genotype of the 32 green embryo seedlings was GG, and the genotype of the 27 white embryo seedlings was TG, and the genotype of 15 was TG and the genotype of 12 was TT ( Figure 5 ), indicating that the marker can accurately distinguish white and green embryos in citrus.

[0055] Example 5

[0056] Fifty-five citrus germplasm resources with different embryo colors were randomly selected, including 18 green embryo resources and 37 white embryo resources. Genomic DNA of the 55 resources was extracted using the modified CTAB method. Genotyping of the 55 citrus germplasm resources with different embryo colors was performed using the developed KASP primers. The method was as described in Example 1. The genotyping results showed that the genotype of the 18 green embryo resources was GG. Among the 37 white embryo resources, the genotype of this site was TG for 21 resources and TT for 16 resources. Figure 6 ), this molecular marker can accurately distinguish the white and green traits of the embryo.

Claims

1. A primer set for detecting the KASP molecular marker for the white / green trait in citrus embryos, characterized by: The invention comprises primers for detecting the base type of the SNP site at position 24 of the nucleotide sequence shown in SEQ ID NO: 1, wherein the base type of the SNP site is T or G; the color of a citrus embryo with a genotype of TT or TG at the SNP site is white, and the color of a citrus embryo with a genotype of GG at the SNP site is green; The primers 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'.

2. The primer set according to claim 1, wherein: The 5' ends of the first forward primer and the second forward primer are respectively labeled with different types of fluorescent markers.

3. A kit for detecting KASP molecular markers for the white / green trait of citrus embryos, characterized by: Comprising the primer set according to claim 1 or 2.

4. Use of the primer set according to claim 1 or 2 or the kit according to claim 3 in early identification of white / green citrus embryos.

5. 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 tested as a template, performing PCR amplification using the primer set described in claim 1 or 2, and detecting the gene type of the 24th SNP site of the nucleotide sequence shown in SEQ ID NO: 1, wherein the TT or TG genotype is white, and the GG genotype is green.

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