Set of InDel molecular markers for identifying wide-peel citrus variety and application of InDel molecular markers

Abstract

The invention relates to the technical field of molecular marker identification, and discloses a set of InDel molecular markers for identifying a wide-peel citrus variety and application of the InDel molecular markers, the InDel molecular markers comprise 42 InDel sites, each site is in two states, and each marker represents a chromosome site; 42 pairs of InDel molecular marker primers are correspondingly arranged on the 42 InDel sites, and the nucleotide sequences of the primers are shown as SEQ ID NO. 1 to SEQ ID NO. 84 in a sequence table. The 42 citrus InDel loci provided by the invention are stable in typing, have space-time crossing performance and can be used for constructing a citrus InDel fingerprint database. The method is beneficial to citrus variety identification, classification, germplasm resource protection and genetic diversity research.

Description

Technical Field

[0001] This invention relates to the field of molecular marker identification technology, specifically to a set of InDel molecular markers for identifying loose-skinned citrus varieties and their applications. Background Technology

[0002] Citrus fruits are an important fruit crop in my country and globally. Common cultivated varieties include mandarin oranges (…). Citrus reticulata Blanco), sweet orange ( Citrus sinensis (L.) Osbeck), grapefruit Citrus grandis (L.)Osbeck), grapefruit Citrus paradise Macfad, lemon ( Citrus limon (L.) Burm.f.), kumquat ( Fortunella (spp.), etc. Among them, loose-skinned mandarins have loose peels that are easy to peel, are juicy and delicious, and are very popular with consumers, accounting for more than two-thirds of the total citrus production. There are many varieties of loose-skinned mandarins, and dozens of common varieties in my country, including the popular Wogan, Chun Jian ("Pa Pa Gan"), and Ehime 28 ("Red Beauty", "Jelly Orange"), etc. Accurate and efficient variety identification is crucial for citrus breeding, new variety rights protection, and seedling quality assurance.

[0003] Citrus variety identification primarily relies on morphological or molecular markers. Morphological identification identifies varieties by observing differences in the external morphology of the plant, leaves, flowers, and fruit. Specificity, uniformity, and stability (DUS) testing based on morphological observations is an internationally recognized and legally grounded method. However, this method is time- and land-intensive and easily affected by ecological environment and cultivation management. Molecular markers are genetic markers based on nucleic acid sequence variations. Molecular marker detection allows for rapid, accurate, objective, and environmentally unaffected variety identification. SSR and SNP markers are common molecular markers suitable for variety identification. SSR markers have the advantages of high polymorphism and co-dominance, but their locus richness is insufficient, and their detection generally relies on polyacrylamide gel electrophoresis or capillary electrophoresis, which is cumbersome and has low stability. SNP markers have high density, high stability, wide distribution, and are suitable for automated and high-throughput detection, but genotyping is more difficult and requires sophisticated experimental equipment and skilled personnel.

[0004] InDels (Insertion / Deletion) are variant sites in the genome formed by the insertion or deletion of base sequences (typically 1-50 bp in length). In plant genomes, InDels are widely distributed, dense, and genetically stable. For InDels longer than 30 bp, typing results can be obtained by ordinary agarose gel electrophoresis, which is economical and convenient.

[0005] Therefore, developing a set of InDel molecular markers for identifying loose-skinned citrus varieties can effectively make up for the shortcomings of existing technologies and is of great significance for the accurate and rapid identification of loose-skinned citrus varieties. It will also facilitate further work on citrus breeding, new variety rights protection, seedling quality assurance, and other related tasks. Summary of the Invention

[0006] The present invention aims to provide a set of InDel molecular markers for identifying mandarin orange varieties and their applications, in order to solve the technical problem that existing technologies for identifying mandarin orange varieties cannot simultaneously achieve both accuracy and convenience.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: a set of InDel molecular markers for identifying loose-skinned citrus varieties, including 42 InDel sites distributed on all 9 chromosomes of citrus, all of which exhibit a dimorphism; the 42 InDel markers correspond to 42 pairs of primers for PCR-based typing, and the nucleotide sequences of each primer are shown in SEQ_ID_NO.1 to SEQ_ID_NO.84 in the sequence listing.

[0008] Preferably, as an improvement, this scheme also provides a primer combination of InDel molecular markers for identifying mandarin orange varieties and its application in mandarin orange variety identification, InDel fingerprinting and database construction, germplasm resource genetic diversity evaluation and phylogenetic analysis.

[0009] Preferably, as an improvement, this solution also provides a method for identifying mandarin orange varieties based on InDel molecular markers, including the following steps: S1. Take samples of the loose-skinned citrus varieties to be identified and extract genomic DNA; S2. Using the DNA from step S1 as a template, PCR amplification was performed using the above 42 pairs of InDel molecular marker primers to construct the amplification system and obtain PCR products. S3. Perform electrophoresis on an agarose gel containing nucleic acid dyes on the PCR products obtained in S2, and take a picture to obtain the electrophoresis image; S4. Based on the electrophoresis images obtained in S3, the tested varieties are classified.

[0010] It also includes S5, comparing the typing of the above 42 InDel sites in the two test samples. If the typing of two or more sites is different, the two test samples are determined to be different; if all typing is the same, the two test samples are determined to be the same variety.

[0011] Preferably, as an improvement, the amplification system is 20 μL, containing 10 μL of 2×Es Taq MasterMix, 1 μL of 10 μM upstream primer, 1 μL of 10 μM downstream primer, 1 μL of 10 ng / μL DNA, and 7 μL of ddH2O.

[0012] Preferably, as an improvement, the PCR amplification program is as follows: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 35 s, 58℃ annealing for 35 s, 72℃ extension for 35 s, for a total of 32 cycles; and finally 72℃ extension for 2 min.

[0013] Preferably, as an improvement, this scheme also provides a method for constructing a citrus fingerprint database based on InDel molecular markers, including the following: typing of the aforementioned 42 InDel loci in multiple citrus varieties, and combining them to form an InDel fingerprint database for citrus. Specifically, after typing the sample of each citrus variety, the samples are sequentially arranged based on genomic location to form the InDel fingerprint of that sample; the genomic and typing primer sequences of the 42 InDel loci are shown in Table 1.

[0014] Preferably, as an improvement, this solution also provides a method for determining the variety of a sample to be tested based on a citrus fingerprint database, including the following steps: first, determine the genotyping of the sample to be tested using the aforementioned 42 InDel loci, arrange them according to genomic location to form the InDel fingerprint map of the sample; then, compare the InDel fingerprint map of the sample to be tested with the aforementioned citrus fingerprint database. If the genotyping of all loci of one or more varieties in the database is the same, then the sample to be tested is the same as that variety; if there is no variety in the database that is the same as the genotyping of all loci of the sample to be tested, then the sample to be tested is a variety outside the scope of the database.

[0015] The principles and beneficial effects of this solution: 1. This invention provides 42 citrus InDel markers and their PCR-based typing primers, which provide clear typing results and are easy to use. By comparing the typing results with standard varieties, varieties can be directly identified, especially suitable for loose-skinned citrus, including currently cultivated varieties such as Wogan, Chunjian (“PaPa Gan”), Ehime 28 (“Hong Meiren”, “Guo Dong Cheng”), Jinqiu Sugar Orange (“Zhonggansuo No. 5”), Sugar Orange, Wenzhou Mandarin, Sunshine No. 1, Asuka, and Ganping.

[0016] 2. This invention provides 42 stable InDel loci for citrus, exhibiting spatiotemporal cross-spatial characteristics, which can be used to construct an InDel fingerprint database for citrus. This is helpful for citrus variety identification, classification, germplasm resource conservation, and genetic diversity research. Attached Figure Description

[0017] Figure 1 These are agarose gel electrophoresis images and typing results of some markers and varieties in embodiments of the present invention.

[0018] Figure 2 This represents the number of differential sites among 63 citrus varieties at 42 InDel sites in this embodiment of the invention.

[0019] Figure 3 This refers to the genetic distance among 63 citrus varieties based on the average number of state similarities (IBS), obtained through genotyping at 42 InDel loci in this embodiment of the invention.

[0020] Figure 4 This is a phylogenetic tree of 63 citrus varieties based on genetic distance, constructed using the UPGMA (unweighted group average method) in an embodiment of the present invention. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0022] Overview of the Plan This scheme provides a set of InDel molecular markers for identifying loose-skinned citrus varieties, including 42 InDel loci, each of which is dimorphic, and each marker represents a chromosome locus; the genomic locations of the 42 InDel loci and the genotyping primer sequences are shown in Table 1.

[0023] Forty-two pairs of InDel molecular marker primers were designed for each of the 42 InDel sites, and the nucleotide sequences of each primer are shown in SEQ_ID_NO.1 to SEQ_ID_NO.84 in the sequence listing. The 42 pairs of InDel molecular marker primers in this scheme were designed based on the genome sequence of Clementine mandarin (a type of loose-skinned citrus).

[0024] This solution also provides a primer set for identifying InDel molecular markers of mandarin orange varieties and its application in mandarin orange variety identification, InDel fingerprinting and database construction, germplasm resource genetic diversity evaluation and phylogenetic analysis.

[0025] This solution also provides a method for identifying loose-skinned citrus varieties based on InDel molecular markers, including the following steps: S1. Take samples of the loose-skinned citrus varieties to be identified and extract genomic DNA; S2. Using the DNA from step S1 as a template, PCR amplification was performed using the above 42 pairs of InDel molecular marker primers to construct the amplification system and obtain PCR products. As a reference, the amplification system was 20 μL, containing 10 μL of 2×Es Taq MasterMix (purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd.), 1 μL of 10 μM upstream primer, 1 μL of 10 μM downstream primer, 1 μL of 10 ng / μL DNA, and 7 μL of ddH2O.

[0026] The PCR amplification program was as follows: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 35 s, 58℃ annealing for 35 s, 72℃ extension for 35 s, for a total of 32 cycles; and finally 72℃ extension for 2 min.

[0027] S3. Perform electrophoresis on an agarose gel containing nucleic acid dye (GelRed optional) and take a picture to obtain the electrophoresis image; S4. Based on the electrophoresis images obtained in S3, classify the tested varieties, such as... Figure 1 As shown, the electrophoresis results of some markers and varieties are displayed. The typing results can be directly read based on the number and position of the bands.

[0028] S5. Compare the genotypes of the above 42 InDel sites in the two test samples. If the genotypes of two or more sites are different, the two test samples are determined to be different; if all genotypes are the same, the two test samples are determined to be of the same variety.

[0029] This solution also provides a method for constructing a citrus fingerprint database based on InDel molecular markers, including the following: Step 1: After performing genotyping on the above 42 InDel loci for each citrus variety sample, arrange the 42 InDel loci in genomic order as shown in Table 1 to form the InDel fingerprint map of the sample.

[0030] Step 2: Perform genotyping detection on the above 42 InDel sites on multiple citrus varieties and combine them to form an InDel fingerprint database of citrus.

[0031] This solution also provides a method for determining the variety of a sample to be tested based on a citrus fingerprint database, including the following steps: First, the sample to be tested was subjected to genotyping of the above 42 InDel loci. The 42 InDel loci were arranged in genomic order as shown in Table 1 to form the InDel fingerprint of the sample.

[0032] Next, the InDel fingerprint spectrum of the sample to be tested is compared with the above-mentioned citrus fingerprint spectrum database. If the genotypes of all loci of one or more varieties in the database are the same, then the sample to be tested is the same as that variety; if there is no variety in the database that is the same as the genotypes of all loci of the sample to be tested, then the sample to be tested is a variety outside the database range.

[0033] Table 1. Genomic locations and typing primer sequences of the 42 InDel sites in this scheme.

[0034] Example 1: Establishment of an InDel fingerprint database for 63 citrus varieties.

[0035] The experimental materials consisted of 63 varieties from six types: mandarin orange, sweet orange, pomelo, grapefruit, lemon, and kumquat, sourced from the National Citrus Germplasm Resource Center (Chongqing), as shown in Table 2.

[0036] According to the above scheme, the 63 varieties were genotyped at 42 InDel loci. The genotypes of each marker were arranged according to the physical order of chromosomes to form an InDel fingerprint database of 63 citrus varieties, as detailed in Table 2.

[0037] Table 2. Sources of the 63 citrus varieties used in this experiment and their InDel fingerprint database.

[0038]

[0039] Genetic diversity of 42 InDel markers in 63 varieties was statistically analyzed. The calculation methods were as follows: allele A frequency was the ratio of allele A to the total number of alleles at that locus (p); allele A frequency was the ratio of allele A to the total number of alleles at that locus (q); minimum gene frequency was the smaller allele frequency, i.e., the smaller values ​​of p and q; expected heterozygosity (He) was the expected frequency of heterozygotes in randomly selected individuals under Hardy-Weinberg equilibrium. Observed heterozygosity (Ho) is the frequency of heterozygotes measured in actual observations, calculated by the proportion of experimental heterozygote frequencies; Shannon diversity index. The results are shown in Table 3.

[0040] Table 3. Genetic diversity statistics of 42 InDel markers in this scheme across 63 varieties.

[0041] Data show that the 42 InDel loci in this protocol exhibit genetic diversity across 63 varieties. Specifically, the minimum gene frequencies of the 42 InDel loci in this protocol range from 0.175 to 0.500, the expected heterozygosity ranges from 0.288 to 0.500, the observed heterozygosity ranges from 0.286 to 0.661, and the Shannon Diversity Index (SHDI) ranges from 0.700 to 1.093 across the 63 varieties.

[0042] For 47 varieties of mandarin oranges, 4 varieties of sweet oranges, 3 varieties of grapefruits, Guanxi honey pomelo, and Beijing lemons, all 42 InDel loci can be genotyped; for Shatin pomelo, Dongshi early pomelo, Eureka lemon, Fino lemon, crisp honey kumquat, smooth-skinned kumquat, and golden bullet, 39, 40, 39, 40, 34, 34, and 34 loci can be genotyped, respectively.

[0043] The number of InDel differential sites among any of the above 63 varieties is as follows: Figure 2 As shown, apart from a few indifferential loci among varieties, there are varying numbers of differentially expressed loci among other varieties.

[0044] Furthermore, there were 0 different loci among the three types of kumquat (crisp honey kumquat, smooth kumquat, and golden bullet kumquat), among the four types of sweet orange (Newhall navel orange, rock sugar orange, Beibei 447, and Tarocco blood orange), and among Eureka lemon and Fino lemon (except for Eureka lemon, which could not be effectively genotyped at the I54Q4BK locus).

[0045] For loose-skinned citrus, there were 0 different loci among the four types of Satsuma mandarin (Miyagawa, Okitsu, Owari, and Yura), between Daiya and Chunjian, between Wogan and 091 seedless Wogan, between Yangxiao 2-6 and seedless Kishu mandarin, and between Ponkan and Lugan. Different Satsuma mandarin varieties originated from bud mutations; Daiya and Chunjian are phenotypic hybrid sister lines; 091 seedless Wogan originated from radiation-induced mutation of Wogan; Yangxiao 2-6 is a superior strain of Nanfeng mandarin, while the seedless Kishu mandarin originated from a variety formed in Japan from Nanfeng mandarin; Lugan is a local variety of Ponkan formed in Fujian. The genetic backgrounds within these groups of varieties are extremely similar.

[0046] There are only 2-3 differentiating sites among the three types of pomelo. Therefore, this invention is not recommended for identifying pomelo varieties.

[0047] Furthermore, there are at least nine differentiating sites among other varieties. Therefore, this invention can identify varieties of mandarin oranges and grapefruits, but cannot distinguish some varieties with very similar genetic backgrounds; it can identify the tested varieties as belonging to the categories of kumquat, sweet orange, pomelo, and lemon, but cannot determine the specific variety.

[0048] The genetic distance between any two individuals was calculated by averaging the number of identical states (IBS) at 42 loci, and a genetic distance matrix of 623 varieties was further constructed, as shown below. Figure 3 Varieties with high genetic similarity have small genetic distances.

[0049] The method for calculating genetic distance is as follows: Where L is the number of InDel loci used to calculate genetic distance between individuals (43); when individuals i and j have the same genotype at locus k (AA|AA or BB|BB), d kij =0; when individuals i and j share one allele at locus k (AB|AA or AB|BB), d kij =0.5; when individuals i and j have completely different genotypes at locus k (AA|BB), d kij =1.

[0050] Based on the above genetic distance matrix, a phylogenetic tree was constructed using the UPGMA (unweighted group average) method, as follows: Figure 4 Varieties with high genetic similarity tend to cluster within the smaller branches of the tree.

[0051] The InDel fingerprint obtained by this invention has cross-temporal and spatiotemporal characteristics and is cumulative. As the number of tested varieties increases, the InDel fingerprint database of citrus fruits can be continuously updated and enriched.

[0052] Example 2: Identification of a suspected new citrus variety – Kawazu No. 5 Kawazu No. 5 is a high-quality, early-maturing citrus variety that has already obtained plant variety rights and is currently under protection. A company in Yunnan is breeding and selling seedlings that appear to be Kawazu No. 5. This invention will be used to identify whether it is indeed Kawazu No. 5.

[0053] According to the above scheme, three unknown varieties (c1, c2, c3) suspected to be Kawazu 5 were genotyped at 42 InDel sites, while Kawazu 5 was set as a control, which came from the inventor of the variety.

[0054] The typing of the test samples (c1, c2, c3) and Kawazu No. 5 at 42 InDel sites is shown in Table 4, and the four are completely identical.

[0055] Table 4 shows the InDel typing results of three suspected Kawazu 5 samples (c1, c2, c3) and the Kawazu 5 from the breeder.

[0056] The results showed that the InDel fingerprints of the tested samples (c1, c2, c3) and Kawajin No. 5 (breeder) were all “BBABAAAAABABAAAAAAAAABABAAABABBBABAAAAAAAAB BBBABBBABABBBBBABABABABAAABABABABAAABABAB”, which were completely consistent with the InDel fingerprint database (Table 2) provided by this invention, and inconsistent with other varieties in the database.

[0057] In summary, using the method provided in this embodiment, the tested samples (c1, c2, c3) were confirmed to be of the variety Kawazu 5 or varieties with a very similar genetic background, such as bud mutations or inductions. (However, given that Kawazu 5 is a protected new variety, no such variety currently exists, so the tested samples can only be Kawazu 5.) The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A set of InDel molecular markers for identifying varieties of mandarin oranges, characterized by: It includes 42 InDel sites, each of which is dimorphic, and each marker represents a chromosomal site; there are 42 pairs of InDel molecular marker primers for the 42 InDel sites, and the nucleotide sequences of each primer are shown in SEQ_ID_NO.1~SEQ_ID_NO.84 in the sequence listing.

2. The application of the primer combination of InDel molecular markers for identifying mandarin orange varieties as described in claim 1 in the identification of mandarin orange varieties, the construction of InDel fingerprint maps and databases, the evaluation of genetic diversity of germplasm resources, and the analysis of kinship.

3. A method for identifying mandarin orange varieties based on InDel molecular markers, characterized in that: Includes the following steps: S1. Take samples of the loose-skinned citrus varieties to be identified and extract genomic DNA; S2. Using the DNA from step S1 as a template, PCR amplification is performed using the 42 pairs of InDel molecular marker primers described in claim 1 to construct an amplification system and obtain PCR products. S3. The PCR products obtained in S2 were electrophoresed on an agarose gel containing GelRed nucleic acid dye, and the electrophoresis images were taken. S4. Based on the electrophoresis images obtained in S3, the tested varieties are classified.

4. The method for identifying mandarin orange varieties based on InDel molecular markers according to claim 3, characterized in that: It also includes S5, typing determination: the typing of the above 42 InDel sites of two test samples is detected. If the typing of two or more sites is different, the two test samples are determined to be different; if all typing is the same, the two test samples are determined to be the same variety.

5. A method for constructing a citrus fingerprint database based on InDel molecular markers, characterized in that: It includes the following: performing genotyping detection on multiple citrus varieties using the 42 InDel sites described in claim 1, and combining them to form an InDel fingerprint database of citrus.

6. The set of InDel molecular markers for identifying loose-skinned citrus varieties as described in claim 5 and their applications, characterized in that: After typing the samples of each citrus variety, the samples are also arranged according to chromosome order to form the InDel fingerprint of the sample.

7. The set of InDel molecular markers for identifying loose-skinned citrus varieties as described in claim 6 and their applications, characterized in that: The genomic order of the 42 InDel sites is shown in Table 1.

8. A method for determining the variety of a sample to be tested based on a citrus fingerprint database, characterized in that: The process includes the following steps: First, the sample to be tested is subjected to genotyping of the 42 InDel loci as described in claim 1, and the loci are arranged according to chromosome order to form the InDel fingerprint map of the sample; then, the InDel fingerprint map of the sample to be tested is compared with the citrus fingerprint database as described in claim 7. If the genotyping of all loci of one or more varieties in the database is the same, then the sample to be tested is the same as that variety; if there is no variety in the database that is the same as the genotyping of all loci of the sample to be tested, then the sample to be tested is a variety outside the scope of the database.

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