InDel marker primer combination of prunus serrulata l. germplasm resources and application thereof

Abstract

The application provides an InDel marker primer combination of a Prunus serrulata L. germplasm resource and application thereof, and belongs to the detection field of cherry breeding; the application identifies the core germplasm resource of Prunus serrulata L. by using an InDel primer group, including the Prunus serrulata L. germplasm of Rucheng County in Hunan, the Prunus serrulata L. germplasm of Yizhang County in Hunan, the Prunus serrulata L. germplasm of Xinning County in Hunan, the Prunus serrulata L. germplasm of Dongan County in Hunan, the Prunus serrulata L. germplasm of Longshan County in Hunan, the Prunus serrulata L. germplasm of Nanping City in Fujian and the Prunus serrulata L. germplasm of Taipei City in Taiwan; the InDel primer group includes an IDE2 marker primer group, an IDE4 marker primer group, an IDE7 marker primer group, an IDG1 marker primer group and an IDH5 marker primer group; the products amplified by PCR of the five primer groups are subjected to agarose gel electrophoresis, a DNA fingerprint map is established according to the size difference of molecules, and the DNA fingerprint map is used for identifying the Prunus serrulata L. germplasm resource, thereby laying a foundation for accurate identification and breeding utilization of the Prunus serrulata L. germplasm resource.

Description

Technical Field

[0001] This invention relates to the field of cherry blossom breeding detection, specifically to an InDel marker primer combination for the germplasm resources of *Prunus campanulata* and its application. Background Technology

[0002] Cherry blossoms belong to the genus *Prunus* in the family Rosaceae. Prunus Subgenus *Cercis*. Cerasus The term "Prunus" is a general term for plants in the genus *Prunus*, widely used in landscaping and urban beautification due to their excellent ornamental value. *Prunus* species are mainly distributed in temperate and subtropical regions of the Northern Hemisphere. There are over 150 species of *Prunus* worldwide, with 52 species and varieties found in China. More than one-third of *Prunus* species are native to China. my country's *Prunus* species are not only numerous but also exhibit a rich variety of variations, possessing broad prospects for development and utilization. *Prunus campanulata* (Bellflower Prunus) Prunus campanulata It is a deciduous tree, also known as bell-shaped cherry, Fujian cherry, and winter cherry. Its flowers are mainly scarlet, and it has an early flowering period, good heat resistance, strong resistance, and high ornamental value, making it very popular in the southern garden and flower market.

[0003] my country has abundant resources of *Prunus campanulata*, widely distributed in Fujian, Taiwan, Guangdong, Guangxi, Jiangxi, Hunan, Yunnan, and other provinces. *Prunus campanulata* possesses rich genetic diversity, making it a valuable material for the creation of superior cherry blossom germplasm and the breeding of new varieties. In recent years, Chinese breeders have successively selected a series of new varieties from wild *Prunus campanulata* resources through seedling cultivation, including the reported 'Xichun'. P. campanulata 'Xichun', 'Xiangsi Hong' ( P. campanulata 'Xiangsihong', 'Xiangyan' P. campanulata 'Xiangyan', 'Canxia' P. campanulata (e.g., 'Canxia'). In recent years, national-level cherry blossom germplasm resource banks (repositories) have been established in Wuhan, Changsha, and other places, collecting a large number of cherry blossom germplasm resources and conducting research on ornamental value evaluation and genetic diversity analysis; technical regulations for the selection of superior cherry blossom plants and seedling propagation have been formulated; and good progress has been made in the development and utilization of germplasm resources, but the protection of germplasm resources has been neglected to some extent. Constructing DNA fingerprint maps of core cherry blossom germplasm resources using modern molecular marker technology can provide technical support for the scientific protection of germplasm resources, while also promoting research related to the creation of new germplasm and the breeding of new varieties.

[0004] At present, the relatively mature and widely used DNA fingerprinting construction markers include RAPD, ISSR, InDel, etc. These technologies have their own advantages. For example, RAPD and ISSR molecular markers require less DNA, are simple to operate, and the most prominent advantage is that the genome sequence of the research object does not need to be known in advance. InDel refers to the insertion or deletion (Insertion-deletion) of different sizes of nucleotide fragments at the same site in the genome between closely related species or different individuals of the same species, which is a phenomenon of gap generated by homologous sequence alignment. The InDel variant sequence is usually flanked by a relatively conserved single copy sequence, which can be amplified by PCR on genomic DNA by designing specific primers, and the variation can be distinguished by detecting the length of the amplified product. InDel molecular markers have the characteristics of abundant quantity, high polymorphism, good repeatability, genetic co-dominance, stable band amplification, high precision, short detection time, mature technology, etc., and have broad application prospects in the construction of DNA fingerprinting of plant germplasm resources. SUMMARY

[0005] To solve the above technical problems, InDel markers are used to detect the core germplasm resources of Prunus subhirtella var. pendula, and the DNA fingerprinting of the core germplasm resources is constructed by PCR product electrophoresis analysis, so as to realize the rapid and accurate differentiation of the germplasm resources.

[0006] To achieve the above purpose, the present application first provides a Prunus subhirtella var. pendula InDel marker primer combination, which comprises an IDE2 marker primer group, an IDE4 marker primer group, an IDE7 marker primer group, an IDG1 marker primer group and an IDH5 marker primer group.

[0007] The nucleotide sequence of the IDE2 marker primer group is shown in SEQ ID NO. 1-SEQ ID NO. 2;

[0008] The nucleotide sequence of the IDE4 marker primer group is shown in SEQ ID NO. 3-SEQ ID NO. 4;

[0009] The nucleotide sequence of the IDE7 marker primer group is shown in SEQ ID NO. 5-SEQ ID NO. 6;

[0010] The nucleotide sequence of the IDG1 marker primer group is shown in SEQ ID NO. 7-SEQ ID NO. 8;

[0011] The nucleotide sequence of the IDH5 marker primer group is shown in SEQ ID NO. 9-SEQ ID NO. 10.

[0012] Based on a total invention concept, the scheme also provides an application of a clock flower cherry InDel marker primer combination in constructing a DNA fingerprint to identify clock flower cherry germplasm, including the following steps:

[0013] S1, using a modified CTAB method to extract total DNA of leaves of the clock flower cherry sample to be tested;

[0014] S2, using the total DNA extracted in step S1 as an amplification template, and performing PCR amplification with the five InDel marker primer groups respectively to obtain PCR products;

[0015] S3, performing agarose gel electrophoresis on the PCR products obtained in step S2, and recording the band with a larger molecular weight as number 1 and the band with a smaller molecular weight as number 2, and arranging the digital codes of the electrophoresis bands of the PCR products amplified by the five primer groups to obtain the DNA fingerprint of the clock flower cherry to be tested, and identifying the clock flower cherry germplasm to be tested according to the DNA fingerprint.

[0016] As a preferred, the germplasm of the clock flower cherry to be tested in step S1 includes seven kinds, which are clock flower cherry germplasm RC-1 in Rucheng County, Hunan, clock flower cherry germplasm YZ-3 in Yizhang County, Hunan, clock flower cherry germplasm XN-2 in Xinning County, Hunan, clock flower cherry germplasm DA-1 in Dong'an County, Hunan, clock flower cherry germplasm LS-2 in Longshan County, Hunan, clock flower cherry NP-4 in Nanping City, Fujian, and clock flower cherry germplasm TB-3 in Taipei City, Taiwan.

[0017] As a preferred, the reaction system of the PCR amplification in step S2 is: 2×Taq Master Mix 12.5 μL, 10 μmol / L of the upstream and downstream primers each 1 μL, ddH2O 9.5 μL, 50 ng / μL of the DNA of the material to be tested 1 μL, and the reaction system is 25 μL in total.

[0018] As a preferred, the reaction program of the PCR amplification in step S2 is: 98℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 54~57℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 3 min, 4℃ preservation; the annealing temperature of the IDE4 and IDG1 marker primer groups is 54℃, the annealing temperature of the IDE2 and IDE7 marker primer groups is 55℃, and the annealing temperature of the IDH5 marker primer group is 57℃.

[0019] As preferred, the method for identifying the to-be-tested clock cherry germplasm of the S3 step is: if the DNA fingerprint is 11112, it is the clock cherry germplasm RC-1 in Rucheng County, Hunan; if the DNA fingerprint is 12221, it is the clock cherry germplasm YZ-3 in Yizhang County, Hunan; if the DNA fingerprint is 21211, it is the clock cherry germplasm XN-2 in Xinning County, Hunan; if the DNA fingerprint is 21112, it is the clock cherry germplasm DA-1 in Dong'an County, Hunan; if the DNA fingerprint is 21212, it is the clock cherry germplasm LS-2 in Longshan County, Hunan; if the DNA fingerprint is 22121, it is the clock cherry germplasm NP-4 in Nanping City, Fujian; and if the DNA fingerprint is 21122, it is the clock cherry germplasm TB-3 in Taipei City, Taiwan.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] (1) The present application is based on the clock cherry genome resequencing technology, and InDel sites are first excavated and used for constructing the DNA fingerprint of the clock cherry core germplasm resource. The present application not only can provide technical support for the scientific protection of the species, but also can promote the related researches on new germplasm creation and new variety breeding. The InDel molecular marker has the characteristics of rich quantity, high polymorphism, good repeatability, genetic co-dominance, stable band amplification, high precision, short detection time, mature technology, etc., and has a broad application prospect in the construction of the DNA fingerprint of plant germplasm resources.

[0022] (2) The amplification products of the five InDel markers designed in the present application are easy to distinguish, do not need to be detected by polyacrylamide gel or capillary electrophoresis, and can be detected by ordinary agarose gel electrophoresis, and have high resolution. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0024] Figure 1 The detection results of 7 clock cherry germplasm resources by using the IDE2 marker in Experimental Example 1 are shown in Table 1.

[0025] Figure 2 The detection results of 7 clock cherry germplasm resources by using the IDE4 marker in Experimental Example 1 are shown in Table 2.

[0026] Figure 3 The detection results of 7 clock cherry germplasm resources by using the IDE7 marker in Experimental Example 1 are shown in Table 3.

[0027] Figure 4 The detection results of 7 Prunus serrulata germplasm resources using IDG1 marker for Experimental Example 1 are shown in Table 1.

[0028] Figure 5 The detection results of 7 Prunus serrulata germplasm resources using IDH5 marker for Experimental Example 1 are shown in Table 2. DETAILED DESCRIPTION

[0029] To make the technical problems, technical solutions and advantages of the present application clearer, the following will be described in detail with reference to the drawings and specific examples.

[0030] The following examples are used to illustrate the present application, but are not used to limit the scope of the present application. Modifications or replacements of the methods, steps or conditions of the present application, without departing from the spirit and essence of the present application, all belong to the scope of the present application.

[0031] If not specifically indicated, the technical means used in the examples is the conventional means known to those skilled in the art; if not specifically indicated, the reagents used in the examples are commercially available.

[0032] Example 1: Design and screening of InDel primers

[0033] 1. Genome resequencing analysis of Prunus serrulata germplasm resources

[0034] Seventeen Prunus serrulata tender leaf samples from Hunan, Fujian, Taiwan and other places were collected, and the total DNA was extracted by using the improved CTAB method. After the DNA detection was qualified, the DNA fragments were fragmented by mechanical disruption (ultrasonic wave), and then the fragmented DNA was subjected to fragment purification, end repair, 3' end A addition, ligation of sequencing adapters, and then subjected to fragment size selection by agarose gel electrophoresis, PCR amplification to form a sequencing library. The library was first subjected to library quality inspection, and the qualified library was subjected to sequencing by using the Illumina NovaSeq6000 platform. The sequencing depth was more than 30 times (data more than 10 Gb). The obtained sequencing reads were repositioned to the Prunus serrulata reference genome, the positions of the Clean reads on the reference genome were aligned and positioned, and the sequencing depth and genome coverage of each sample were counted, so as to facilitate the next step of variation detection.

[0035] 2. Development of Prunus serrulata InDel markers

[0036] According to the positioning results of the clean reads of the Prunus subhirtella genome in the Prunus subhirtella reference genome, the GATK v4.1.4.1 software is used to detect insertion / deletion mutations (InDels), and then the obtained variant sites are filtered to screen high-reliability variant results, to obtain the final InDel site set, and the InDel marker statistics are performed.

[0037] 3. Identification of Prunus subhirtella polymorphic InDel markers

[0038] From the statistical InDel site set, InDel markers that can distinguish different Prunus subhirtella germplasm resources, i.e. polymorphic InDel markers, are selected, to obtain a batch of candidate markers that can be used for the construction of DNA fingerprint of germplasm resources, for subsequent experimental verification.

[0039] 4. Design of Prunus subhirtella polymorphic InDel marker PCR primers

[0040] The Prunus subhirtella genome sequence is used as a template, and the Primer Premier 6.0 software is used to design PCR amplification primers of the polymorphic InDel site. The main parameters of primer design are: length 18-26 bp; GC content 40%-60%, melting temperature (Tm) 57-61℃, amplification product size 100-400 bp; the primer itself cannot have consecutive 4 bases complementary to avoid forming a hairpin structure; the primers cannot have consecutive 4 bases complementary to avoid forming primer dimers; and the specificity and high amplification efficiency of the primers are ensured.

[0041] Example 1: Construction of DNA fingerprint of Prunus subhirtella core germplasm resources

[0042] 1. Experimental materials

[0043] The tender leaves of 7 Prunus subhirtella core germplasm resources were collected respectively, as shown in Table 1 below, for InDel marker screening and specificity verification.

[0044]

[0045] 2. InDel marker detection

[0046] 2.1 DNA extraction

[0047] The total DNA of the above-mentioned Prunus subhirtella core germplasm resources samples was extracted by the improved CTAB method.

[0048] 2.2 PCR amplification

[0049] The DNA of the core germplasm resource of Prunus triloba extracted in step 2.1 was used as an amplification template, and the specific primers of the five InDel markers were used for PCR amplification, and the primer information is shown in Table 2.

[0050] Table 2 InDel marker and its specific primer information

[0051]

[0052] 2.3 PCR reaction system

[0053] The PCR reaction system was 25 μL, including 2×Taq Master Mix 12.5 μL, 1 μL of upstream and downstream primers (10 μmol / L) each, 9.5 μL of ddH2O, 1 μL of 50 ng / μL of DNA of the material to be tested.

[0054] 2.4 PCR reaction program

[0055] The PCR reaction program was 98℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 54℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 3 min, 4℃ storage. The annealing temperature of the primer pair of IDE4, IDG1 marker was 54℃, the annealing temperature of the primer pair of IDE2, IDE7 marker was 55℃, and the annealing temperature of the primer pair of IDH5 marker was 57℃.

[0056] 2.5 Electrophoretic detection of PCR amplification products

[0057] The PCR amplification products were electrophoresed on a 2% agarose gel (5 μL of GelRed 10000×stock solution was added to each 50 mL agarose solution), and the electrophoresis buffer was 1×TAE buffer, the voltage was set to 110 V, and the electrophoresis time was 40 min. The electrophoresis results were photographed in the gel imaging system, if the PCR product size was consistent with the expected and there was no non-specific band, it was considered that the target nucleic acid fragment amplification was successful, the larger band was marked as number 1, and the smaller band was marked as number 2, the electrophoretic band number coding arrangement of the PCR products amplified by the five primers was the DNA fingerprint of the Prunus triloba to be tested, and the Prunus triloba germplasm to be tested could be judged according to the DNA fingerprint.

[0058] 3. Specific InDel markers of Prunus triloba core germplasm resource and detection analysis

[0059] 3.1 Identification results of IDE2 marker

[0060] The identification results of IDE2 marker are shown in Figure 1As shown in the figure, the PCR product electrophoresis bands of the core germplasm resources 1 and 2 are larger, and are marked as 1; the PCR product electrophoresis bands of the core germplasm resources 3-7 are smaller, and are marked as 2.

[0061] 3.2 Identification results of the IDE4 marker

[0062] The identification results of the IDE4 marker are shown in the figure. Figure 2 As shown in the figure, the PCR product electrophoresis bands of the 7 core germplasm resources are encoded as 1, 2, 1, 1, 1, 2 and 1, respectively.

[0063] 3.3 Identification results of the IDE7 marker

[0064] The identification results of the IDE7 marker are shown in the figure. Figure 3 As shown in the figure, the PCR product electrophoresis bands of the 7 core germplasm resources are encoded as 1, 2, 2, 1, 2, 1 and 1, respectively.

[0065] 3.4 Identification results of the IDG1 marker

[0066] The identification results of the IDG1 marker are shown in the figure. Figure 4 As shown in the figure, the PCR product electrophoresis bands of the 7 core germplasm resources are encoded as 1, 2, 1, 1, 1, 2 and 2, respectively.

[0067] 3.5 Identification results of the IDH5 marker

[0068] The identification results of the IDH5 marker are shown in the figure. Figure 5 As shown in the figure, the PCR product electrophoresis bands of the 7 core germplasm resources are encoded as 2, 1, 1, 2, 2, 1 and 2, respectively.

[0069] According to the detection results of the five InDel markers, the digital coding arrangement of the electrophoresis bands of the 7 core germplasm resources, i.e., the DNA fingerprint maps, are 11112, 12221, 21211, 21112, 21212, 22121 and 21122, respectively, as shown in the following table 3:

[0070]

[0071] Therefore, the five InDel markers designed in the scheme can distinguish the core germplasm resources of the Ching Hwa cherry, the DNA fingerprint maps of the core germplasm resources are constructed through the PCR product electrophoresis analysis, and the rapid and accurate distinction of the germplasm resources is realized.

[0072] The above only describes the preferred embodiments of the present application, and the protection scope of the present application is not limited to the above. Improvements and changes made by those skilled in the art without departing from the technical concept of the present application should also be considered as the protection scope of the present application.

Claims

1. A clock flower cherry InDel marker primer combination, characterized in that, The InDel marker primer combination comprises an IDE2 marker primer group, an IDE4 marker primer group, an IDE7 marker primer group, an IDG1 marker primer group and an IDH5 marker primer group; The nucleotide sequences of the IDE2 marker primer group are shown in SEQ ID NO. 1-2; The nucleotide sequences of the IDE4 marker primer group are shown in SEQ ID NO. 3-4; The nucleotide sequences of the IDE7 marker primer group are shown in SEQ ID NO. 5-6; The nucleotide sequences of the IDG1 marker primer group are shown in SEQ ID NO. 7-8; The nucleotide sequences of the IDH5 marker primer group are shown in SEQ ID NO. 9-10.

2. The application of a clock flower cherry InDel marker primer combination in constructing DNA fingerprint to identify clock flower cherry germplasm according to claim 1, characterized in that, The method comprises the following steps: S1, total DNA of leaves of the to-be-tested C. japonica sample is extracted by using a modified CTAB method; S2, total DNA extracted in the step S1 is used as an amplification template, and the five InDel marker primer groups in claim 1 are used for PCR amplification respectively to obtain PCR products; S3, the PCR products obtained in the step S2 are subjected to agarose gel electrophoresis, a band with a larger molecular weight is marked as a number 1, and a band with a smaller molecular weight is marked as a number 2, and the number coding arrangement of the electrophoresis bands of the PCR products amplified by the five primer groups is a DNA fingerprint of the to-be-tested C. japonica, and the to-be-tested C. japonica germplasm is identified according to the DNA fingerprint; The to-be-tested C. japonica germplasm in the step S1 comprises seven kinds, which are C. japonica germplasm RC-1 in Rucheng County, Hunan, C. japonica germplasm YZ-3 in Yizhang County, Hunan, C. japonica germplasm XN-2 in Xinning County, Hunan, C. japonica germplasm DA-1 in Dong'an County, Hunan, C. japonica germplasm LS-2 in Longshan County, Hunan, C. japonica germplasm NP-4 in Nanping City, Fujian, and C. japonica germplasm TB-3 in Taipei City, Taiwan; The method for identifying the to-be-tested C. japonica germplasm in the step S3 is that if the DNA fingerprint is 11112, it is the C. japonica germplasm RC-1 in Rucheng County, Hunan; if the DNA fingerprint is 12221, it is the C. japonica germplasm YZ-3 in Yizhang County, Hunan; if the DNA fingerprint is 21211, it is the C. japonica germplasm XN-2 in Xinning County, Hunan; if the DNA fingerprint is 21112, it is the C. japonica germplasm DA-1 in Dong'an County, Hunan; if the DNA fingerprint is 21212, it is the C. japonica germplasm LS-2 in Longshan County, Hunan; if the DNA fingerprint is 22121, it is the C. japonica germplasm NP-4 in Nanping City, Fujian; and if the DNA fingerprint is 21122, it is the C. japonica germplasm TB-3 in Taipei City, Taiwan.

3. Use according to claim 2, characterized in that, The reaction system of the PCR amplification in the step S2 is that 2×Taq Master Mix is 12.5 μL, 10 μmol / L of the upper and lower primers are each 1 μL, ddH2O is 9.5 μL, 50 ng / μL of the DNA of the to-be-tested material is 1 μL, and the reaction system is 25 μL.

4. Use according to claim 2, characterized in that, The reaction procedure of PCR amplification of the S2 step is: 98℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 54-57℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 3 min, 4℃ storage; the annealing temperature of the IDE4, IDG1 marker primer group is 54℃, the annealing temperature of the IDE2, IDE7 marker primer group is 55℃, and the annealing temperature of the IDH5 marker primer group is 57℃.

Images