Ssr marker primer and application thereof in identification of elephant grass hybrid

Abstract

The application discloses an SSR marker primer and application thereof in elephant grass hybrid identification, and an SSR marker primer pair used for real identification of elephant grass hybrid, wherein the primer pair is a primer Pphyb-g1; and the SSR marker primer combination further comprises a primer Pphyb-w1. By using the SSR molecular marker technology, the application can accurately distinguish the true and false of the elephant grass hybrid, and in particular, when the primer Pphyb-w1 and the primer Pphyb-g1 are combined for identification, the effective identification rate of the real hybrid can reach 64.8% or more, and the identification accuracy is significantly improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant molecular breeding, and relates to a zebra grass hybrid true identification primer, an identification method thereof and application. BACKGROUND

[0002] Zebra grass (Pennisetum purpureum) is a perennial tropical herb, which is originally from African grassland and belongs to the genus Pennisetum of Poaceae. Due to its strong tillering ability, rich nutrition, high yield and strong disease and pest resistance, zebra grass has important value in grassland animal husbandry in tropical and subtropical regions.

[0003] With the rapid development of grassland animal husbandry, the demand for forage grasses and feeds is increasing, and research departments and breeding experts in China have also carried out a large amount of research and application work in the fields of zebra grass introduction and domestication, selection, hybridization and radiation breeding. By 2024, the National Forage Variety Approval Committee has registered a total of 7 zebra grasses, including 3 introduced varieties, 2 bred varieties and 2 local varieties.

[0004] Although breeding work has made some progress, zebra grass as a cross-pollinated plant, its hybrid offspring is prone to false hybridization, which seriously affects the development process of hybrid breeding. Traditional zebra grass breeding mainly relies on hybridization, but this method has the disadvantages of low efficiency, long cycle and great environmental influence. Therefore, it is very important to establish a fast and effective identification technology system that is not affected by the environment and can accurately distinguish different genotypes.

[0005] The commonly used methods for variety identification at present mainly include morphological identification, cytological identification, biochemical marker identification and molecular marker identification.

[0006] SSR (Simple Sequence Repeats) marker, also known as microsatellite DNA molecular marker, is a molecular marker technology based on specific primer PCR. SSR marker technology has been widely used in genetic research field due to its high polymorphism, genetic stability and wide distribution.

[0007] However, there are few reports on the application of SSR molecular marker technology in zebra grass variety or germplasm resource identification at present, and a mature and standardized identification system has not yet been formed. SUMMARY

[0008] In view of this, one of the purposes of the application is to provide a pair of SSR marker primers for zebra grass hybrid true identification.

[0009] The second purpose of the application is to provide a combination of SSR marker primers for zebra grass hybrid true identification.

[0010] The third object of the present application is to provide an application of the SSR marker in authenticity identification of elephant grass hybrid.

[0011] The fourth object of the present application is to provide a method for authenticity identification of elephant grass hybrid using the SSR marker primer combination.

[0012] The inventors, through long-term exploration and trial, and multiple experiments and efforts, and continuous reform and innovation, provide a technical solution to solve the above technical problems, which is a SSR marker primer pair for authenticity identification of elephant grass hybrid, the primer pair is primer Pphyb-g1:

[0013] The upstream primer g1F is 5'-CCAAGCGCTCCTTCTTCTGA-3';

[0014] The downstream primer g1R is 5'-CACGGCACTGATCACTCTGT-3'.

[0015] The present application also provides a SSR marker primer combination for authenticity identification of elephant grass hybrid, which comprises:

[0016] A) the primer Pphyb-g1 in claim 1,

[0017] B) primer Pphyb-w1:

[0018] The upstream primer w1F is 5'-TGTGTTTTTCATCCTCAAGTGTTGT-3';

[0019] The downstream primer w1R is 5'-CAAATGTAAGATGATAAATGCAGCG-3'.

[0020] The present application also provides a use of the SSR marker primer pair or the SSR marker primer combination, which is used for identifying the authenticity of elephant grass hybrid through PCR amplification.

[0021] The present application also provides a method for authenticity identification of elephant grass hybrid using the SSR marker primer combination, which comprises the following steps:

[0022] a. DNA extraction: collecting fresh leaves free of pests and diseases from seedling plants of hybrid combinations prepared from elephant grass female parent and elephant grass male parent, and extracting DNA;

[0023] b. PCR amplification: performing PCR amplification on the extracted DNA using the SSR marker primer combination;

[0024] c. Electrophoresis detection: performing polyacrylamide gel electrophoresis on the PCR amplification product to detect the amplified fragments.

[0025] According to one embodiment of the method, the PCR amplification uses a 15 μL system, including 2x Taq PCR Master Mix, template DNA, upstream and downstream primers, and sterilized water.

[0026] According to one embodiment of the method, the SSR-PCR amplification procedure includes 5 minutes of pre-denaturation at 94℃, 30 seconds of denaturation at 94℃, 45 seconds of annealing at 58-60℃, 1 minute of extension at 72℃, a total of 35 cycles, and 7 minutes of extension at 72℃.

[0027] According to one embodiment of the method, the polyacrylamide gel electrophoresis detection includes 30 minutes of pre-electrophoresis at 200 V, 1.3 hours of electrophoresis at 300 V, and silver staining using 0.1% AgNO3 solution and color development using NaOH solution.

[0028] According to one embodiment of the method, the method identifies true hybrids with parent complementary or paternal specific bands.

[0029] Compared with the prior art, one of the above technical solutions has the following advantages:

[0030] a) By using the SSR molecular marker technology, the present application can accurately distinguish between true and false elephant grass hybrids, especially when using primers Pphyb-w1 and Pphyb-g1 for combined identification, the effective identification rate of true hybrids can reach more than 64.8%, significantly improving the accuracy of identification.

[0031] b) The method of the present application greatly shortens the identification time of true elephant grass hybrids. From DNA extraction to the final electrophoresis detection, the entire process can be completed in a short time, greatly improving the efficiency of breeding work.

[0032] c) The identification steps provided by the present application are simple and easy to operate, do not require complex pretreatment or professional skill knowledge, and are convenient for rapid identification in the laboratory and on site.

[0033] d) Compared with identification methods that rely on external morphological characteristics, the molecular marker technology of the present application is not affected by environmental changes, and can maintain stable identification effects even in poor environmental conditions.

[0034] e) The present application provides important technical support for elephant grass molecular breeding, which helps to accelerate the breeding process of new varieties and promote the development of grassland animal husbandry.

[0035] f) Through the identification method of the present application, the genetic background of elephant grass hybrids can be better understood, providing a scientific basis for further genetic improvement and protection of germplasm resources. Attached Figure Description

[0036] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0037] Figure 1 This is a diagram showing the results of amplification of target grasses and hybrids using primer Pphyb-g1.

[0038] Figure 2 This is a diagram showing the results of amplification of target grasses and hybrids using primer Pphyb-w1.

[0039] Figure 3 This is a graph showing the results of polyacrylamide electrophoresis amplification of the target grass parent and 287 F1 hybrids labeled with Pphyb-g1.

[0040] Figure 4 This is a graph showing the results of polyacrylamide electrophoresis amplification of the target grass parent and 287 F1 hybrids labeled with Pphyb-w1. Detailed Implementation

[0041] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; and the materials and reagents used, unless otherwise specified, are commercially available.

[0042] Based on transcriptome data, the inventors developed 132 pairs of SSR primers and screened out SSR primers that showed specific band amplification in the parental lines of the hybrid combination. After screening and analysis, only the markers Pphyb-g1 and Pphyb-w1 could effectively distinguish between the parental lines and the hybrid. The primer sequences of Pphyb-g1 are shown in Seq ID NO.1-2 of the sequence listing, and the primer sequences of Pphyb-w1 are shown in Seq ID NO.3-4 of the sequence listing.

[0043] Using the Pphyb-g1 marker, over 43.9% of true elephant grass hybrids can be identified, while using the Pphyb-w1 marker can identify over 38.68%. Using both Pphyb-g1 and Pphyb-w1 markers further improves accuracy, identifying over 64.80% of true elephant grass hybrids. Therefore, both the Pphyb-g1 and / or Pphyb-w1 markers can be used for rapid identification of the authenticity of elephant grass hybrids.

[0044] The specific steps of rapid identification of the authenticity of the elephant grass hybrid in this embodiment are as follows.

[0045] (1) DNA extraction: In the present application, elephant grass 'GM1' is used as the female parent and elephant grass 'F-B' is used as the male parent to prepare a hybrid combination. The seedling plants of the parents and their intergeneric hybrid F1 generation are used as materials. Fresh leaves of 2-3 pieces without diseases and insect pests are randomly collected from each hybrid single plant of the parents and the leaves are wiped clean with absolute ethanol. After wiping with non-woven cloth, the leaves are stored in a self-sealing bag with silica gel and taken back to the laboratory. The high-efficiency plant genomic DNA extraction kit (Tiangen Biotech Co., Ltd., model: DP350-03) is used to extract the DNA of the parents and their hybrids. The purity and integrity of the total DNA of each variety are detected by 1% agarose gel electrophoresis. The absorbance values of each sample total DNA at 260 nm and 280 nm are measured by ultraviolet spectrophotometry to determine the purity and concentration of the DNA. A small amount of original DNA solution of each variety is diluted to 20 ng / uL and stored in a -20℃ refrigerator for standby use.

[0046] (2) Using SSR marker primers Pphyb-g1 and Pphyb-w1 for hybrid identification

[0047] Table 1 Primer sequences of SSR marker primers Pphyb-g1 and Pphyb-w1 of the present application

[0048] .

[0049] (3) The DNA separated from the leaves of the above-mentioned elephant grass parents and their hybrids is amplified and the detection method of the products is as follows:

[0050] The DNA solution diluted to 20 ng / uL is used for PCR amplification

[0051] Among them, the SSR-PCR adopts a 15 uL system, and the PCR reaction system is: 2 x Taq PCR Master Mix (Taq DNA polymerase, dNTPs, MgCl2, reaction buffer, PCR reaction enhancer and optimization agent, and stabilizer) 7.5 uL, template DNA 1.5 uL (20 ng / uL), upper and lower primers are 0.6 uL, and sterilized water is supplemented to 15 uL;

[0052] Among them, the SSR-PCR amplification program is: the first step is pre-denaturation at 94℃ for 5 min; the second step is denaturation at 94℃ for 30 s, annealing at 58-60℃ for 45 s, and extension at 72℃ for 1 min, for a total of 35 cycles; the third step is extension at 72℃ for 7 min, and finally stored in a 4℃ refrigerator.

[0053] PCR amplification products were detected by 8% polyacrylamide gel electrophoresis; 2uL of each sample was loaded, 2000bp DNA Ladder was used as molecular weight marker, pre-electrophoresis at 200V for 30min, then electrophoresis at 300V for 1.3h until the indicator band was 4-5cm from the bottom of the gel, silver staining in 0.1% AgNO3 solution, color development in NaOH solution, camera shooting for preservation for analysis, see Figs. 1 and 2 for the polyacrylamide gel electrophoresis detection of some hybridization of 2 pairs of SSR primer markers. Figure 1 and Figure 2 The results of amplification of the subject grasses and hybrids by primer Pphyb-g1 are shown in Fig. 3, and the results of amplification of the subject grasses and hybrids by primer Pphyb-w1 are shown in Fig. 4. Figure 1 Figure 2

[0054] Figure 1 In Fig. 1, lane F6 is paternal type; lanes F3 and F19 are maternal type; lanes F10, F11, F12, F13, F14, F15, F16, F17, F18, F20, F21, F22, F23, F24, F25, F27, F28, F29, F30 are parent complementary type; lanes F1, F2, F4, F5, F7, F8, F9, F26, F31, F32, F33 are other type.

[0055] Figure 2 In Fig. 2, lanes F2, F3, F11, F13, F15, F24, F29, F32 are paternal type; lanes F5, F7, F8, F9, F10, F12, F16, F18, F19, F20, F21, F22, F25, F26, F28, F30, F31 are maternal type; lanes F1, F4, F6, F17, F23, F27, F33 are parent complementary type; lane F14 is other type.

[0056] The method of the present embodiment can be used to know the genetic relationship between the parent material of elephant grass and its hybrid, and to identify the true and false of the elephant grass hybrid. The test results are arranged and analyzed by Excel, and in the identification results of the authenticity of the offspring, there are four types: parent complementary type with specific bands of both parents; paternal type with only specific bands of the paternal parent; maternal type with only specific bands of the maternal parent; other type without specific bands of both parents.

[0057] The parent complementary type and the paternal type can be identified as true hybrid.

[0058] The identification results of the authenticity of 287 F1 hybrids by Pphyb-g1 marker are shown in Table 2 below; the identification results of the authenticity of 287 F1 hybrids by Pphyb-w1 marker are shown in Table 3 below. ​​

[0059] As shown in Table 2, the Pphyb-g1 marker identified 126 true hybrids, with an effective identification rate of 43.9%. The polyacrylamide gel electrophoresis amplification results of the subject grass parent and 287 F1 hybrids using the Pphyb-g1 marker are shown in Figure 3 .

[0060] Table 2 Identification results of 287 F1 hybrids using the Pphyb-g1 marker

[0061]

[0062] As shown in Table 3, the Pphyb-w1 marker identified 111 true hybrids, with an effective identification rate of 38.68%. The polyacrylamide gel electrophoresis amplification results of the subject grass parent and 287 F1 hybrids using the Pphyb-w1 marker are shown in Figure 4 .

[0063] Table 3 Identification results of 287 F1 hybrids using the Pphyb-w1 marker

[0064]

[0065] As shown in Table 2 and Table 3, the two markers Pphyb-g1 and Pphyb-w1 identified a total of 186 true hybrids from 287 materials, with a true hybrid identification rate of more than 64.80%.

[0066] The combined use of the two markers Pphyb-g1 and Pphyb-w1 can complement each other, cover a wider genetic range, and provide more comprehensive genetic information, ensuring the comprehensiveness of the identification process. By using the two markers in combination, the present application effectively reduces the missed identification of true hybrids that may be caused by a single marker, as each marker can capture different genetic characteristics, thereby ensuring the comprehensiveness and accuracy of the identification.

[0067] The present application directly detects the DNA sequences of the subject grass parent and its hybrids to identify them, which overcomes the uncertainty of identification based on external morphology, and has the advantages of short identification time, high accuracy, and strong reliability.

[0068] The present application uses the SSR molecular marker technology to identify true and false hybrids of the subject grass, effectively improves the identification efficiency of grass interspecific hybrids, provides strong experimental evidence for the identification of grass hybrids, and provides important technical support for grass molecular breeding.

[0069] The above merely describes the preferred embodiments of the present application, and it should be pointed out that the above preferred embodiments should not be regarded as a limitation to the present application, and the protection scope of the present application should be defined by the scope of the claims. For those skilled in the art, several improvements and refinements can be made without departing from the spirit and scope of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.

Claims

1. A method for identifying the authenticity of a Pennisetum americanum hybrid using a SSR marker primer combination, characterized in that, The method comprises the following steps: a. DNA extraction: collecting fresh and pest-free leaves from seedling plants of a hybrid combination prepared by crossing a female parent 'GM1' and a male parent 'F-B' of elephant grass, and extracting DNA; b. PCR amplification: performing PCR amplification on the extracted DNA using a SSR marker primer combination; the SSR marker primer combination is a combination of primers Pphyb-g1 and Pphyb-w1, the primer sequence of Pphyb-g1 is shown in Seq ID NO. 1-2, and the primer sequence of Pphyb-w1 is shown in Seq ID NO. 3-4; c. Electrophoresis detection: performing polyacrylamide gel electrophoresis on the PCR amplification product to detect the amplified fragments; the identified true hybrid is a parent complement type with parent-specific bands or a paternal type with only paternal-specific bands.

2. The method of claim 1, wherein, The polyacrylamide gel electrophoresis detection comprises pre-electrophoresis detection at 200V for 30 minutes, electrophoresis detection at 300V for 1.3 hours, and silver staining with 0.1% AgNO3 solution and color development with NaOH solution.

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