An indel molecular marker primer pair, kit and method for identifying yichang orange

Abstract

The application provides an Indel molecular marker primer pair, a kit and a method for identifying Yichang orange, and belongs to the technical field of biochemistry. The Indel molecular marker primer pair comprises at least one of an InDel-2 primer pair and an InDel-3 primer pair. The Indel molecular marker primer pair or the kit can specifically identify Yichang orange, realizes efficient and accurate identification of Yichang orange and its hybrid offspring, and has a wide application prospect in aspects of citrus germplasm resource protection, variety right interest maintenance, breeding selection and market supervision. The method for identifying Yichang orange can accurately identify Yichang orange and its hybrid offspring, has high specificity, economy and efficiency, is an accurate, economical and efficient hybrid identification method, and can effectively identify Yichang orange varieties and hybrids, thereby providing strong technical support for scientific research and breeding work.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, and more specifically, relates to an Indel molecular marker primer pair, kit, and method for identifying Yichang oranges. Background Technology

[0002] Citrus fruits are the world's largest category of fruits, with immense economic value. Citrus is an important economic fruit tree, and the Yichang orange, named after its earliest discovery in Xingshan County, Yichang City, Hubei Province, is a wild citrus species endemic to my country, distributed in the northern and central subtropical regions. The Yichang orange is a rare monoembryonic citrus resource within the Citrus genus, possessing cold resistance, tolerance to poor soil, and shade tolerance, making it a valuable material for conventional hybridization breeding and cell fusion breeding of citrus. Hybridization breeding is a primary means of germplasm innovation. In citrus variety identification, a combination of morphological, cytological, and molecular marker methods is commonly used. However, existing morphological markers are limited, and the juvenile stage of citrus is relatively long. Furthermore, at the seedling stage, it is difficult to accurately distinguish between citrus varieties of the same type or those with similar parentage. With the continuous enrichment of citrus varieties and the diversification of market demands, there is an urgent need to solve the problem of accurate and rapid identification of citrus varieties, which is of great significance for germplasm resource protection, variety rights maintenance, and market supervision.

[0003] Because citrus and other fruit trees have a long juvenile period, relying solely on biochemical analysis and phenotypic evaluation methods is insufficient for achieving rapid progress in citrus resource development and breeding within a short period. With the development of molecular biology techniques, DNA molecular markers are widely used in plant variety identification. Among these, SSR, RAPD, and AFLP markers have been applied in citrus variety identification, but these methods suffer from drawbacks such as cumbersome operation, poor reproducibility, and high cost.

[0004] In recent years, with the rapid development of high-throughput sequencing technology, the development of molecular markers based on whole-genome resequencing data has become possible. Among them, Indel markers have shown great potential in plant variety identification due to their high polymorphism, co-dominance, and good stability. However, specific Indel markers for Yichang oranges have not yet been reported.

[0005] Therefore, it is of great significance to develop an Indel molecular marker based on resequencing data that can specifically identify Yichang oranges. Summary of the Invention

[0006] The purpose of this invention is to provide an Indel molecular marker primer pair, kit, and method for identifying Yichang oranges, so as to specifically identify Yichang oranges and achieve efficient and accurate identification of Yichang oranges and their hybrid offspring.

[0007] To achieve the above objectives, a first aspect of the present invention provides an InDel molecular marker primer pair for identifying Yichang oranges. The InDel molecular marker primer pair includes at least one of an InDel-2 primer pair and an InDel-3 primer pair. The sequences of the InDel-2 primer pair are shown in SEQ ID No. 1 and SEQ ID No. 2, and the sequences of the InDel-3 primer pair are shown in SEQ ID No. 3 and SEQ ID No. 4. Both the InDel-2 and InDel-3 primer pairs can effectively identify Yichang oranges; either of these two primer pairs can be selected for identification.

[0008] In a second aspect, the present invention provides a kit for identifying Yichang oranges, the kit comprising the Indel molecular marker primer pair described above.

[0009] Furthermore, the kit also includes PCR amplification reagents and double-distilled water.

[0010] A third aspect of the present invention provides the application of the Indel molecular marker primer pair or the kit described above in the identification of Yichang oranges.

[0011] A fourth aspect of the present invention provides a method for identifying Yichang oranges, comprising the following steps:

[0012] Extract genomic DNA from the sample to be tested;

[0013] Using the extracted genomic DNA of the sample to be tested as a template, a PCR amplification system was established using the Indel molecular marker primer pair to perform PCR amplification and obtain amplification products.

[0014] The amplification products were subjected to agarose gel electrophoresis to obtain electrophoresis images;

[0015] The variety of the sample to be tested is identified based on the band information on the electrophoresis image.

[0016] Furthermore, the PCR amplification system comprises the following components: 10 μl 2 x Taq plus master mix, 0.5 μl positive primer, 0.5 μl reverse primer, 150 ng genomic DNA template of the sample to be tested, and ddH2O to bring the total volume to 20 μl.

[0017] Furthermore, the PCR amplification conditions are as follows: first, pre-denaturation at 95 ℃ for 5 min; then denaturation at 95 ℃ for 30 s, annealing at 56 ℃ for 30 s, extension at 72 ℃ for 50 s, for 35 cycles; and finally, extension at 72 ℃ for 5 min.

[0018] Furthermore, the agarose gel electrophoresis uses 2.0% to 4.0% by mass agarose gel, a voltage of 100 to 150 V, a current of 300 to 500 mA, and an electrophoresis time of 10 to 30 min.

[0019] Furthermore, the step of identifying the species of the sample to be tested based on the band information on the electrophoresis image includes the following steps:

[0020] When a 330bp or 400bp band is displayed on the agarose gel, the sample to be tested is of another variety;

[0021] When an agarose gel shows a band less than 330 bp or a band less than 400 bp, the sample to be tested is Yichang orange.

[0022] Compared with the prior art, the present invention has the following technical effects:

[0023] The Indel molecular marker primer pair or kit of the present invention for identifying Yichang oranges can specifically identify Yichang oranges, realize efficient and accurate identification of Yichang oranges and their hybrid offspring, and has broad application prospects in citrus germplasm resource protection, variety rights protection, breeding selection and market supervision.

[0024] The present invention provides a method for identifying Yichang oranges using Indel molecular marker primer pairs. This method can accurately identify Yichang oranges and their hybrid offspring, exhibiting high specificity, economy, and efficiency. It is an accurate, economical, and efficient hybrid identification method that can effectively identify Yichang orange varieties and their hybrids, providing strong technical support for scientific research and breeding work. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a gel image showing the characteristic bands of 64 citrus varietal samples detected by electrophoresis using Indel-2, as provided in Example 2 of this invention; M represents the molecular weight standard.

[0027] Figure 2 This is a gel image of characteristic bands from 64 citrus cultivar samples detected by electrophoresis using Indel-3, as provided in Example 2 of this invention.

[0028] Figure 3These are characteristic band gel images of Ziyang oranges obtained by electrophoresis detection using Indel-2 and Indel-3 provided in Embodiment 3 of the present invention. Figure 3 (a) shows the identification results of Indel-2. Figure 3 (b) shows the identification results of Indel-3; YCC1-YCC4 represent four Yichang orange varieties, and ZYXC represents the Ziyang sweet orange variety;

[0029] Figure 4 The image shows the characteristic bands of Indel-2 and Indel-3 in 44 hybrids of Sanhong Honey Pomelo and Yichang Orange provided in Example 4 of this invention, detected by electrophoresis. Lanes 1 and 2, excluding the marker, represent the hybrid parents. In the image, Indel02 is Indel-2, Indel03 is Indel-3, YCC represents the Yichang Orange variety, SH represents the Sanhong Honey Pomelo variety, and the other 44 samples are hybrid offspring of Sanhong Honey Pomelo and Yichang Orange. Detailed Implementation

[0030] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0031] The terminology used in the embodiments of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. The singular forms “a,” “the,” and “the” used in the embodiments of this invention are also intended to include the plural forms unless the context clearly indicates otherwise.

[0032] This invention provides an InDel molecular marker primer pair based on resequencing data of Yichang oranges and various citrus varieties from a public database. This InDel molecular marker primer pair can accurately identify Yichang oranges and their hybrid offspring, exhibiting high specificity, economy, and efficiency. It is an accurate, economical, and efficient method for hybrid identification, effectively identifying Yichang orange varieties and their hybrids, and providing strong technical support for scientific research and breeding work.

[0033] The InDel molecular marker primer pairs of this invention include at least one of the InDel-2 primer pair and the InDel-3 primer pair; the sequences of the InDel-2 primer pair are shown in SEQ ID No. 1 and SEQ ID No. 2, and the sequences of the InDel-3 primer pair are shown in SEQ ID No. 3 and SEQ ID No. 4.

[0034] This invention also provides a kit for identifying Yichang oranges, which includes the aforementioned InDel molecular marker primer pair. Furthermore, the kit includes PCR amplification reagents and double-distilled water (ddH2O). Specifically, the PCR amplification reagents are 2 x Taq plus master mix.

[0035] The Indel molecular marker primer pair of this invention is a Yichang orange-specific Indel molecular marker primer pair developed based on whole-genome resequencing data. Specifically, it is a primer pair of relevant InDel markers designed through whole-genome resequencing of Yichang oranges and various citrus varieties. Both the Indel molecular marker primer pair and the kit of this invention can be used to identify whether a sample is a Yichang orange, and can also be used to determine whether hybrid offspring have Yichang orange lineage. The Indel molecular marker primer pair and kit of this invention have broad application prospects in citrus germplasm resource protection, variety rights maintenance, breeding selection, and market supervision.

[0036] This invention also provides a method for identifying Yichang oranges, comprising the following steps:

[0037] (1) Extract genomic DNA from the sample to be tested;

[0038] (2) Using the extracted genomic DNA of the sample to be tested as a template, a PCR amplification system was established with Indel molecular marker primers to perform PCR amplification and obtain amplification products;

[0039] (3) Perform agarose gel electrophoresis on the amplification products to obtain electrophoresis images;

[0040] (4) Identify the species of the sample to be tested based on the band information on the electrophoresis image.

[0041] In step (1) above, the genomic DNA of the sample to be tested can be extracted using existing conventional methods.

[0042] In step (2) above, the PCR amplification system of this embodiment includes the following components: 10 μl 2 x Taq plusmaster mix, 0.5 μl positive primer, 0.5 μl reverse primer, 150 ng genomic DNA template of the sample to be tested, and ddH2O to make up to 20 μl. The positive and reverse primers are the positive and reverse primers of the InDel-2 primer pair and / or the positive and reverse primers of the InDel-3 primer pair.

[0043] The PCR amplification conditions in this embodiment are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 s, annealing at 56 ℃ for 30 s, extension at 72 ℃ for 50 s, for 35 cycles; and finally extension at 72 ℃ for 5 min.

[0044] In step (3) above, agarose gel electrophoresis is performed using 2.0% to 4.0% agarose gel by mass, with a voltage of 100 to 150V, a current of 300 to 500 mA, and an electrophoresis time of 10 to 30 min.

[0045] In step (4) above, the variety identification of the sample to be tested is based on the band information on the electrophoresis image, including the following steps: when a band of 330bp and / or a band of 400bp is displayed on the agarose gel, the sample to be tested is another variety; when a band less than 330bp and / or a band less than 400bp is displayed on the agarose gel, the sample to be tested is Yichang orange.

[0046] The following specific embodiments illustrate an Indel molecular marker primer pair for identifying Yichang oranges and its application method according to an embodiment of the present invention.

[0047] Example 1

[0048] Example 1 of this invention provides an Indel molecular marker primer pair for identifying Yichang oranges and its design method, comprising the following steps:

[0049] (1) By selecting whole genome resequencing data of Yichang orange and citrus pomelo and combining it with multiple publicly available whole genome resequencing data of tangerine, orange and pomelo, and using sweet orange as the reference genome to generate the bam file required for analysis, it was found that at positions 6954900 and 26137400 on the first chromosome, Yichang orange is missing a fragment of about 100bp compared to other varieties. Based on this, relevant specific high-quality InDel molecular marker primer pairs were screened and designed.

[0050] (2) Then, primers were synthesized for the designed InDel molecular marker primer pair. The synthesized primers were dissolved in sterile ultrapure water (ddH2O) and finally stored at a concentration of 10 uM.

[0051] (3) Extract genomic DNA from the sample to be tested;

[0052] (4) Using the extracted genomic DNA of the samples to be tested as templates, PCR amplification was performed on 64 citrus varietal samples using all synthesized InDel molecular marker primer pairs. The PCR amplification system was 20 μl. Due to the high quality and specificity of the primer design, the relatively inexpensive 2 x Taq plus master mix could be used for amplification (each mix is ​​1 ml, which can identify 100 samples). The PCR amplification system included the following components: 10 μl 2 x Taq plus master mix, 0.5 μl positive primer, 0.5 μl reverse primer, 150 ng genomic DNA template of the samples to be tested, and ddH2O was added to make up to 20 μl. Among them, the positive and reverse primers are the positive and reverse primers of the InDel-2 primer pair and / or the positive and reverse primers of the InDel-3 primer pair.

[0053] The PCR amplification conditions were as follows: pre-denaturation at 95 ℃ for 5 min; followed by denaturation at 95 ℃ for 30 s, annealing at 56 ℃ for 30 s, extension at 72 ℃ for 50 s, for 35 cycles; and finally extension at 72 ℃ for 5 min.

[0054] (5) The amplified products were detected by 2% agarose gel electrophoresis to identify the amplified bands. Specifically, the agarose gel electrophoresis was performed using a 2.0% (w / w) agarose gel at 130V, 400 mA, and for 18 min. Based on the amplification results, InDel molecular marker primer pairs that could amplify characteristic bands were selected as candidate effective identification primers. The information of the finally screened effective InDel molecular marker primer pairs is shown in Table 1 below. These effective InDel molecular marker primer pairs can distinguish Yichang oranges from other varieties.

[0055]

[0056] Example 2

[0057] To verify the effectiveness of the InDel molecular marker primer pairs InDel-2 and InDel-3 screened in Example 1, Example 2 of this invention provides a method for identifying Yichang oranges, comprising the following steps:

[0058] (1) Sixty-four citrus varieties were selected, including four Yichang orange varieties and sixty other various citrus cultivars, wild resources and distant relatives, to verify the effectiveness of the InDel molecular marker primer pair developed in Example 1.

[0059] (2) Genomic DNA was extracted from these 64 citrus cultivar samples;

[0060] (3) Using genomic DNA extracted from 64 citrus cultivar samples as templates, 20 μl PCR amplification systems were established using Indel molecular marker primer pairs: 10 μl 2 x Taq plus master mix, 0.5 μl forward primer, 0.5 μl reverse primer, 150 ng DNA template, and ddH2O was added to bring the total volume to 20 μl. The amplification conditions were: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 s, annealing at 56 ℃ for 30 s, extension at 72 ℃ for 50 s, for 35 cycles; and final extension at 72 ℃ for 5 min.

[0061] (4) After PCR amplification as described above, take 7 μl of the PCR amplification product and detect it by 2.0% agarose gel electrophoresis (voltage 130 V, current 400 mA, electrophoresis time 18 min).

[0062] (5) Based on the test results, two effective primer pairs were finally identified that yielded differential bands: When other varieties were used as templates for PCR amplification, the amplification products showed a 330bp (Indel-2) bright band and a 400bp (Indel-3) bright band on agarose gel electrophoresis. However, the bands obtained from the amplification products of Yichang orange were significantly smaller than 330bp (Indel-2) and 400bp (Indel-3), indicating that primer pairs Indel-2 and Indel-3 could distinguish Yichang orange from other varieties. The electrophoresis results are as follows: Figure 1 , Figure 2 As shown.

[0063] Example 3

[0064] To verify the effectiveness of the Indel-2 and Indel-3 molecular marker primer pairs developed in this invention in identifying Yichang oranges and varieties with Yichang orange lineage, Ziyang fragrant oranges (Yichang oranges are the direct parents of Ziyang fragrant oranges) were selected. Amplification and electrophoresis were performed according to the PCR steps in Example 2. The electrophoresis results are as follows: Figure 3 As shown. Figure 3 The results indicate that there are different bands between the Yichang orange variety and the tested varieties. The amplification products of the Yichang orange material have band sizes of less than 330bp (Indel-2) and less than 400bp (Indel-3). Ziyang Xiangcheng orange also has the same short bands as Yichang orange. After amplification, it was proved that the Indel-2 and Indel-3 molecular marker primer pairs are effective in identifying varieties with Yichang orange lineage.

[0065] Example 4

[0066] The Indel-2 and Indel-3 molecular marker primer pairs provided in this embodiment of the invention can be used to distinguish the Yichang orange variety and its hybrids in the early stage of seedling cultivation. Forty-four randomly selected hybrids of Sanhong honey pomelo and Yichang orange were used for identification. PCR amplification and electrophoresis were performed according to the steps in Example 2. The electrophoresis results are as follows: Figure 4 As shown. Figure 4 This indicates that the tested variety has the zoning pattern of both parents (Yichang orange and Sanhong honey pomelo), proving the effectiveness of the Indel-2 and Indel-3 molecular marker primer pairs in early hybrid identification.

[0067] This invention provides, for the first time, high-quality, highly specific InDel molecular marker primer pairs designed based on resequencing data of Yichang orange and various citrus varieties. Two effective primer pairs (Indel-2 and Indel-3) were ultimately selected for effective identification of Yichang orange varieties and their hybrid offspring. The InDel molecular marker primer pairs developed in this invention achieve efficient and accurate identification of Yichang orange and its hybrid offspring, providing strong scientific and technological support for citrus research, breeding, and industrial development. The above embodiments only illustrate several implementation methods of this invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of this invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of this invention, and these all fall within the protection scope of this invention.

Claims

1. An Indel molecular marker primer pair for identifying Yichang oranges, characterized in that, The Indel molecular marker primer pair includes at least one of the InDel-2 primer pair and the InDel-3 primer pair; the sequences of the InDel-2 primer pair are shown in SEQ ID No. 1 and SEQ ID No. 2, and the sequences of the InDel-3 primer pair are shown in SEQ ID No. 3 and SEQ ID No.

4.

2. A reagent kit for identifying Yichang oranges, characterized in that, The kit includes the Indel molecular marker primer pair as described in claim 1.

3. The reagent kit for identifying Yichang oranges as described in claim 2, characterized in that, The kit also includes PCR amplification reagents and double-distilled water.

4. The application of the Indel molecular marker primer pair as described in claim 1 or the kit as described in claim 2 or 3 in the identification of Yichang oranges.

5. A method for identifying Yichang oranges, characterized in that, Includes the following steps: Extract genomic DNA from the sample to be tested; Using the extracted genomic DNA of the sample to be tested as a template, a PCR amplification system was established using the Indel molecular marker primer pair described in claim 1, and PCR amplification was performed to obtain amplification products. The amplification products were subjected to agarose gel electrophoresis to obtain electrophoresis images; The variety of the sample to be tested is identified based on the band information on the electrophoresis image.

6. The method for identifying Yichang oranges as described in claim 5, characterized in that, The PCR amplification system comprises the following components: 10 μl 2 x Taq plus master mix, 0.5 μl positive primer, 0.5 μl reverse primer, 150 ng genomic DNA template of the sample to be tested, and ddH2O to bring the total volume to 20 μl.

7. The method for identifying Yichang oranges as described in claim 5, characterized in that, The PCR amplification conditions were as follows: pre-denaturation at 95℃ for 5 min; followed by denaturation at 95℃ for 30 s, annealing at 56℃ for 30 s, extension at 72℃ for 50 s, for 35 cycles; and finally extension at 72℃ for 5 min.

8. The method for identifying Yichang oranges as described in claim 5, characterized in that, The agarose gel electrophoresis was performed using 2.0%–4.0% agarose gel by mass, with a voltage of 100–150V, a current of 300–500 mA, and an electrophoresis time of 10–30 min.

9. The method for identifying Yichang oranges as described in claim 5, characterized in that, The process of identifying the species of the sample to be tested based on the band information on the electrophoresis image includes the following steps: When the Indel molecular marker primer pair is the InDel-2 primer pair, if a 330bp band is displayed on the agarose gel, the sample to be tested is another variety; if a band less than 330bp is displayed on the agarose gel, the sample to be tested is Yichang orange. When the Indel molecular marker primer pair is the InDel-3 primer pair, if a 400bp band is displayed on the agarose gel, the sample to be tested is another variety; if a band less than 400bp is displayed on the agarose gel, the sample to be tested is Yichang orange.

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