A SSR marker primer developed based on the Zoysia japonica genome sequence and its application

Abstract

The present invention belongs to the field of molecular biology technology, and specifically relates to an SSR marker primer developed based on the zoysia genome sequence and its application. After identifying 36 zoysia materials using the SSR marker combination provided by the present invention, the genetic relationship between them can be determined, and different materials can be distinguished, which can provide technical support for the genetic relationship analysis between unknown new varieties and known varieties and the identification of new varieties.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to an SSR marker primer developed based on a Zoysia japonica genome sequence and an application thereof. Background Art

[0002] Zoysia willd. is a perennial herbaceous plant of the Poaceae family. Different Zoysia willd. germplasm resources have obvious differences in environmental adaptability, stress resistance, feeding performance and other characteristics. Zoysia willd. has a wide distribution range in my country and strong adaptability. It can grow in deserts and saline-alkali lands and is recognized as an excellent warm-season turf grass in the world.

[0003] Understanding the genetic diversity and genetic background of species is the premise for the rational protection and utilization of this resource. Molecular markers are genetic markers based on nucleotide sequence variation within genetic material between individuals, and are powerful tools for studying genetic diversity and genetic background of materials. Although molecular marker technologies such as RAPD, ISSR, and SRAP have been applied to the genetic diversity, genetic linkage map, and genetic background of Zoysia japonica, there are few reports on Zoysia japonica-specific primers, which leads to greater difficulties in studying the genetic diversity and genetic background of Zoysia japonica, and slow breeding progress. Therefore, enriching the Zoysia japonica-specific primer library has become a top priority.

[0004] Simple sequence repeat (SSR), also known as microsatellite DNA, is a DNA fragment composed of tandem repeat sequences with 2 to 6 nucleotides as basic units. The length is mostly 100 to 200 base pairs. It has the characteristics of wide distribution, rich polymorphism, good repeatability, and co-dominant inheritance. It has been widely used in genetic diversity and genetic background research of germplasm resources, variety identification, molecular marker-assisted breeding, etc. It is one of the preferred markers for molecular marker-assisted breeding. Genome sequencing (DNA-seq) technology is a technology that uses high-throughput sequencing technology to deeply sequence DNA in tissues or cells. It is an effective means of high-throughput and low-cost analysis of gene expression differences at the genome level. It is the main means of obtaining a large amount of transcript information with high efficiency, high throughput, and low cost. At present, there is no research report on the development of Zoysia japonica SSR markers using genome sequencing. Summary of the invention

[0005] The purpose of the present invention is to provide an SSR marker primer developed based on the zoysiagrass genome sequence, a method for studying the phylogenetic relationship of zoysiagrass and a method for identifying varieties.

[0006] The present invention provides an SSR marker combination for identifying Zoysia japonica, wherein the SSR marker combination comprises SSR01, SSR02, SSR03, SSR04, SSR05, SSR06, SSR07, SSR08, SSR09, SSR10, SSR11, SSR12, SSR13, SSR14, SSR15, SSR16, SSR17, SSR18, SSR19, SSR20, SSR21, SSR22, SSR23, SSR24, SSR25, SSR26, SSR27, SSR28, SSR29, SSR30, SSR31, SSR32, SSR33, SSR34, SSR35, SSR36, SSR37, SSR38 and SSR39;

[0007] The repeating unit of SSR01 is GCC, and the number of repetitions is 5;

[0008] The repeating unit of SSR02 is GGA, and the number of repetitions is 6;

[0009] The repeating unit of SSR03 is GAG, and the number of repetitions is 6;

[0010] The repeating unit of SSR04 is CCT, and the number of repetitions is 5;

[0011] The repeating unit of SSR05 is GGT, and the number of repeats is 7;

[0012] The repeating unit of SSR06 is GCG, and the number of repeats is 10;

[0013] The repeating unit of SSR07 is GAA, and the number of repeats is 5;

[0014] The repeating unit of SSR08 is GGA, and the number of repeats is 7;

[0015] The repeating unit of SSR09 is TGT, and the number of repeats is 7;

[0016] The repeating unit of SSR10 is CTC, and the number of repeats is 8;

[0017] The repeating unit of SSR11 is GAG, and the number of repeats is 5;

[0018] The repeating unit of SSR12 is TTC, and the number of repeats is 5;

[0019] The repeating unit of SSR13 is CGC, and the number of repeats is 5;

[0020] The repeating unit of SSR14 is TCA, and the number of repeats is 8;

[0021] The repeating unit of SSR15 is GGC, and the number of repeats is 7;

[0022] The repeating unit of SSR16 is GCG, and the number of repeats is 7;

[0023] The repeating unit of SSR17 is CGC, and the number of repeats is 5;

[0024] The repeating unit of SSR18 is CTG, and the number of repeats is 7;

[0025] The repeating unit of SSR19 is GCA, and the number of repeats is 6;

[0026] The repeating unit of SSR20 is CT, and the number of repetitions is 22;

[0027] The repeating unit of SSR21 is GAG, and the number of repeats is 7;

[0028] The repeating unit of SSR22 is AGC, and the number of repetitions is 5;

[0029] The repeating unit of SSR23 is CCA, and the number of repeats is 6;

[0030] The repeating unit of SSR24 is GGC, and the number of repetitions is 6;

[0031] The repeating unit of SSR25 is GCG, and the number of repeats is 8;

[0032] The repeating unit of SSR26 is TGC, and the number of repeats is 5;

[0033] The repeating unit of SSR27 is CCT, and the number of repeats is 5;

[0034] The repeating unit of SSR28 is TC, and the number of repetitions is 8;

[0035] The repeating unit of SSR29 is GTG, and the number of repeats is 6;

[0036] The repeating unit of SSR30 is GCC, and the number of repetitions is 5;

[0037] The repeating unit of SSR31 is GAC, and the number of repeats is 7;

[0038] The repeating unit of SSR32 is CCG, and the number of repeats is 5;

[0039] The repeating unit of SSR33 is TAC, and the number of repeats is 8;

[0040] The repeating unit of SSR34 is CGG, and the number of repeats is 6;

[0041] The repeating unit of SSR35 is TCAGC, and the number of repeats is 5;

[0042] The repeating unit of SSR36 is CCTCAG, and the number of repeats is 5;

[0043] The repeating unit of SSR37 is TGA, and the number of repeats is 5;

[0044] The repeating unit of SSR38 is CGG, and the number of repeats is 5;

[0045] The repeating unit of SSR39 is AGG, and the number of repetitions is 5.

[0046] The present invention also provides a set of primer combinations for amplifying the SSR marker combination described in the above technical solution, including Zj-SSR01, Zj-SSR02, Zj-SSR03, Zj-SSR04, Zj-SSR05, Zj-SSR06, Zj-SSR07, Zj-SSR08, Zj-SSR09, Zj-SSR10, Zj-SSR11, Zj-SSR12, Zj-SSR13, Zj-SSR14, Zj-SSR15, Zj-SSR16, Zj-SSR17, Zj-SSR18, SR18, Zj-SSR19, Zj-SSR20, Zj-SSR21, Zj-SSR22, Zj-SSR23, Zj-SSR24, Zj-SSR25, Zj-SSR26, Zj-SSR27, Zj-SSR28, Zj -SSR29, Zj-SSR30, Zj-SSR31, Zj-SSR32, Zj-SSR33, Zj-SSR34, Zj-SSR35, Zj-SSR36, Zj-SSR37, Zj-SSR38 and Zj-SSR39;

[0047] The Zj-SSR01 includes Zj-SSR01-F and Zj-SSR01-R, the nucleotide sequence of Zj-SSR01-F is shown in SEQ ID No.1, and the nucleotide sequence of Zj-SSR01-R is shown in SEQ ID No.2;

[0048] The Zj-SSR02 includes Zj-SSR02-F and Zj-SSR02-R, the nucleotide sequence of Zj-SSR02-F is shown in SEQ ID No.3, and the nucleotide sequence of Zj-SSR02-R is shown in SEQ ID No.4;

[0049] The Zj-SSR03 includes Zj-SSR03-F and Zj-SSR03-R, the nucleotide sequence of the Zj-SSR03-F is shown in SEQ ID No.5, and the nucleotide sequence of the Zj-SSR03-R is shown in SEQ ID No.6;

[0050] The Zj-SSR04 includes Zj-SSR04-F and Zj-SSR04-R, the nucleotide sequence of the Zj-SSR04-F is shown in SEQ ID No.7, and the nucleotide sequence of the Zj-SSR04-R is shown in SEQ ID No.8;

[0051] The Zj-SSR05 includes Zj-SSR05-F and Zj-SSR05-R, the nucleotide sequence of Zj-SSR05-F is shown in SEQ ID No.9, and the nucleotide sequence of Zj-SSR05-R is shown in SEQ ID No.10;

[0052] The Zj-SSR06 includes Zj-SSR06-F and Zj-SSR06-R, the nucleotide sequence of Zj-SSR06-F is shown in SEQ ID No.11, and the nucleotide sequence of Zj-SSR06-R is shown in SEQ ID No.12;

[0053] The Zj-SSR07 includes Zj-SSR07-F and Zj-SSR07-R, the nucleotide sequence of Zj-SSR07-F is shown in SEQ ID No.13, and the nucleotide sequence of Zj-SSR07-R is shown in SEQ ID No.14;

[0054] The Zj-SSR08 includes Zj-SSR08-F and Zj-SSR08-R, the nucleotide sequence of the Zj-SSR08-F is shown in SEQ ID No.15, and the nucleotide sequence of the Zj-SSR08-R is shown in SEQ ID No.16;

[0055] The Zj-SSR09 includes Zj-SSR09-F and Zj-SSR09-R, the nucleotide sequence of Zj-SSR09-F is shown in SEQ ID No.17, and the nucleotide sequence of Zj-SSR09-R is shown in SEQ ID No.18;

[0056] The Zj-SSR10 includes Zj-SSR10-F and Zj-SSR10-R, the nucleotide sequence of the Zj-SSR10-F is shown in SEQ ID No.19, and the nucleotide sequence of the Zj-SSR10-R is shown in SEQ ID No.20;

[0057] The Zj-SSR11 includes Zj-SSR11-F and Zj-SSR11-R, the nucleotide sequence of Zj-SSR11-F is shown in SEQ ID No.21, and the nucleotide sequence of Zj-SSR11-R is shown in SEQ ID No.22;

[0058] The Zj-SSR12 includes Zj-SSR12-F and Zj-SSR12-R, the nucleotide sequence of Zj-SSR12-F is shown in SEQ ID No.23, and the nucleotide sequence of Zj-SSR12-R is shown in SEQ ID No.24;

[0059] The Zj-SSR13 includes Zj-SSR13-F and Zj-SSR13-R, the nucleotide sequence of Zj-SSR13-F is shown in SEQ ID No.25, and the nucleotide sequence of Zj-SSR13-R is shown in SEQ ID No.26;

[0060] The Zj-SSR14 includes Zj-SSR14-F and Zj-SSR14-R, the nucleotide sequence of Zj-SSR14-F is shown in SEQ ID No.27, and the nucleotide sequence of Zj-SSR14-R is shown in SEQ ID No.28;

[0061] The Zj-SSR15 includes Zj-SSR15-F and Zj-SSR15-R, the nucleotide sequence of the Zj-SSR15-F is shown in SEQ ID No.29, and the nucleotide sequence of the Zj-SSR15-R is shown in SEQ ID No.30;

[0062] The Zj-SSR16 includes Zj-SSR16-F and Zj-SSR16-R, the nucleotide sequence of Zj-SSR16-F is shown in SEQ ID No.31, and the nucleotide sequence of Zj-SSR16-R is shown in SEQ ID No.32;

[0063] The Zj-SSR17 includes Zj-SSR17-F and Zj-SSR17-R, the nucleotide sequence of the Zj-SSR17-F is shown in SEQ ID No.33, and the nucleotide sequence of the Zj-SSR17-R is shown in SEQ ID No.34;

[0064] The Zj-SSR18 includes Zj-SSR18-F and Zj-SSR18-R, the nucleotide sequence of Zj-SSR18-F is shown in SEQ ID No.35, and the nucleotide sequence of Zj-SSR18-R is shown in SEQ ID No.36;

[0065] The Zj-SSR19 includes Zj-SSR19-F and Zj-SSR19-R, the nucleotide sequence of the Zj-SSR19-F is shown in SEQ ID No.37, and the nucleotide sequence of the Zj-SSR19-R is shown in SEQ ID No.38;

[0066] The Zj-SSR20 includes Zj-SSR20-F and Zj-SSR20-R, the nucleotide sequence of the Zj-SSR20-F is shown in SEQ ID No.39, and the nucleotide sequence of the Zj-SSR20-R is shown in SEQ ID No.40;

[0067] The Zj-SSR21 includes Zj-SSR21-F and Zj-SSR21-R, the nucleotide sequence of the Zj-SSR21-F is shown in SEQ ID No.41, and the nucleotide sequence of the Zj-SSR21-R is shown in SEQ ID No.42;

[0068] The Zj-SSR22 includes Zj-SSR22-F and Zj-SSR22-R, the nucleotide sequence of the Zj-SSR22-F is shown in SEQ ID No.43, and the nucleotide sequence of the Zj-SSR22-R is shown in SEQ ID No.44;

[0069] The Zj-SSR23 includes Zj-SSR23-F and Zj-SSR23-R, the nucleotide sequence of the Zj-SSR23-F is shown in SEQ ID No.45, and the nucleotide sequence of the Zj-SSR23-R is shown in SEQ ID No.46;

[0070] The Zj-SSR24 includes Zj-SSR24-F and Zj-SSR24-R, the nucleotide sequence of the Zj-SSR24-F is shown in SEQ ID No.47, and the nucleotide sequence of the Zj-SSR24-R is shown in SEQ ID No.48;

[0071] The Zj-SSR25 includes Zj-SSR25-F and Zj-SSR25-R, the nucleotide sequence of the Zj-SSR25-F is shown in SEQ ID No.49, and the nucleotide sequence of the Zj-SSR25-R is shown in SEQ ID No.50;

[0072] The Zj-SSR26 includes Zj-SSR26-F and Zj-SSR26-R, the nucleotide sequence of the Zj-SSR26-F is shown in SEQ ID No.51, and the nucleotide sequence of the Zj-SSR26-R is shown in SEQ ID No.52;

[0073] The Zj-SSR27 includes Zj-SSR27-F and Zj-SSR27-R, the nucleotide sequence of the Zj-SSR27-F is shown in SEQ ID No.53, and the nucleotide sequence of the Zj-SSR27-R is shown in SEQ ID No.54;

[0074] The Zj-SSR28 includes Zj-SSR28-F and Zj-SSR28-R, the nucleotide sequence of the Zj-SSR28-F is shown in SEQ ID No.55, and the nucleotide sequence of the Zj-SSR28-R is shown in SEQ ID No.56;

[0075] The Zj-SSR29 includes Zj-SSR29-F and Zj-SSR29-R, the nucleotide sequence of the Zj-SSR29-F is shown in SEQ ID No.57, and the nucleotide sequence of the Zj-SSR29-R is shown in SEQ ID No.58;

[0076] The Zj-SSR30 includes Zj-SSR30-F and Zj-SSR30-R, the nucleotide sequence of the Zj-SSR30-F is shown in SEQ ID No.59, and the nucleotide sequence of the Zj-SSR30-R is shown in SEQ ID No.60;

[0077] The Zj-SSR31 includes Zj-SSR31-F and Zj-SSR31-R, the nucleotide sequence of the Zj-SSR31-F is shown in SEQ ID No.61, and the nucleotide sequence of the Zj-SSR31-R is shown in SEQ ID No.62;

[0078] The Zj-SSR32 includes Zj-SSR32-F and Zj-SSR32-R, the nucleotide sequence of Zj-SSR32-F is shown in SEQ ID No.63, and the nucleotide sequence of Zj-SSR32-R is shown in SEQ ID No.64;

[0079] The Zj-SSR33 includes Zj-SSR33-F and Zj-SSR33-R, the nucleotide sequence of the Zj-SSR33-F is shown in SEQ ID No.65, and the nucleotide sequence of the Zj-SSR33-R is shown in SEQ ID No.66;

[0080] The Zj-SSR34 includes Zj-SSR34-F and Zj-SSR34-R, the nucleotide sequence of the Zj-SSR34-F is shown in SEQ ID No.67, and the nucleotide sequence of the Zj-SSR34-R is shown in SEQ ID No.68;

[0081] The Zj-SSR35 includes Zj-SSR35-F and Zj-SSR35-R, the nucleotide sequence of the Zj-SSR35-F is shown in SEQ ID No.69, and the nucleotide sequence of the Zj-SSR35-R is shown in SEQ ID No.70;

[0082] The Zj-SSR36 includes Zj-SSR36-F and Zj-SSR36-R, the nucleotide sequence of the Zj-SSR36-F is shown in SEQ ID No.71, and the nucleotide sequence of the Zj-SSR36-R is shown in SEQ ID No.72;

[0083] The Zj-SSR37 includes Zj-SSR37-F and Zj-SSR37-R, the nucleotide sequence of the Zj-SSR37-F is shown in SEQ ID No.73, and the nucleotide sequence of the Zj-SSR37-R is shown in SEQ ID No.74;

[0084] The Zj-SSR38 includes Zj-SSR38-F and Zj-SSR38-R, the nucleotide sequence of the Zj-SSR38-F is shown in SEQ ID No.75, and the nucleotide sequence of the Zj-SSR38-R is shown in SEQ ID No.76;

[0085] The Zj-SSR39 includes Zj-SSR39-F and Zj-SSR39-R, the nucleotide sequence of the Zj-SSR39-F is shown in SEQ ID No.77, and the nucleotide sequence of the Zj-SSR39-R is shown in SEQ ID No.78.

[0086] The present invention also provides the use of the SSR marker combination described in the above technical solution or the primer combination described in the above technical solution in identifying and / or distinguishing Zoysia japonica varieties.

[0087] The present invention also provides a method for identifying Zoysia japonica varieties, comprising the following steps:

[0088] Extracting Zoysia japonica genomic DNA; using the primer combination described in the above technical solution to perform PCR amplification on the Zoysia japonica genomic DNA to obtain PCR products; performing acrylamide gel electrophoresis and capillary electrophoresis on the PCR products; and analyzing the electrophoresis bands.

[0089] The present invention also provides a kit for identifying Zoysia japonica varieties, wherein the kit comprises the primer combination described in the above technical solution.

[0090] The present invention also provides the use of the SSR marker combination described in the above technical solution, the primer combination described in the above technical solution, or the kit described in the above technical solution in the phylogenetic analysis of Zoysia japonica.

[0091] The present invention also provides the use of the SSR marker combination described in the above technical solution, the primer combination described in the above technical solution, or the kit described in the above technical solution in the maternal tracing of Zoysia japonica.

[0092] The present invention also provides the use of the SSR marker combination described in the above technical solution, the primer combination described in the above technical solution, or the kit described in the above technical solution in the genetic pedigree analysis and / or genetic map construction of Zoysia japonica.

[0093] The present invention also provides the use of the SSR marker combination described in the above technical solution, the primer combination described in the above technical solution, or the kit described in the above technical solution in the protection of Zoysia japonica germplasm resources and / or assisted breeding.

[0094] Beneficial effects:

[0095] The present invention provides an SSR marker combination for identifying Zoysia japonica. After identifying 36 Zoysia japonica materials using the SSR marker combination provided by the present invention, the genetic relationship between them can be determined, and different materials can be distinguished, which can provide technical support for the genetic relationship analysis between unknown new varieties and known varieties and the identification of new varieties.

[0096] The SSR marker primers provided by the present invention are derived from the genome sequence of Zoysia japonica. The primers are rich in polymorphism, stable in amplification, and easy to identify amplified bands. They are new markers that exist stably and fill the gap in the development of SSR primers based on the genome of Zoysia japonica. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0098] Figure 1The peak graph of the amplified bands of the primer Zj-SSR4 selected from the capillary electrophoresis amplification results in Table 4 for the zoysia grass variety Z0413-1 is used to illustrate the capillary electrophoresis results;

[0099] Figure 2 The peak graph of the amplified bands of the primer Zj-SSR4 selected from the capillary electrophoresis amplification results in Table 4 for the zoysia grass variety Z0417-10 is used to illustrate the capillary electrophoresis results;

[0100] Figure 3 The peak graph of the amplified bands of the primer Zj-SSR4 selected from the capillary electrophoresis amplification results in Table 4 for the zoysia grass variety Z0232-5 is used to illustrate the capillary electrophoresis results;

[0101] Figure 4 The peak graph of the amplified bands of the primer Zj-SSR4 selected from the capillary electrophoresis amplification results in Table 4 for the zoysia grass variety Z123 is used to illustrate the capillary electrophoresis results;

[0102] Figure 5 This is the cluster analysis diagram of 36 Zoysia japonica variety resources using 39 pairs of SSR primers;

[0103] Figure 6 This is the PCoA plot of 39 pairs of SSR primers for 36 Zoysia materials. DETAILED DESCRIPTION

[0104] The present invention provides an SSR marker combination for identifying Zoysia japonica, wherein the SSR marker combination comprises SSR01, SSR02, SSR03, SSR04, SSR05, SSR06, SSR07, SSR08, SSR09, SSR10, SSR11, SSR12, SSR13, SSR14, SSR15, SSR16, SSR17, SSR18, SSR19, SSR20, SSR21, SSR22, SSR23, SSR24, SSR25, SSR26, SSR27, SSR28, SSR29, SSR30, SSR31, SSR32, SSR33, SSR34, SSR35, SSR36, SSR37, SSR38 and SSR39;

[0105] The repeating unit of SSR01 is GCC, which is repeated 5 times; the repeating unit of SSR02 is GGA, which is repeated 6 times; the repeating unit of SSR03 is GAG, which is repeated 6 times; the repeating unit of SSR04 is CCT, which is repeated 5 times; the repeating unit of SSR05 is GGT, which is repeated 7 times; the repeating unit of SSR06 is GCG, which is repeated 10 times; the repeating unit of SSR07 is GAA, which is repeated 5 times; the repeating unit of SSR08 is GGA, which is repeated 7 times; the repeating unit of SSR09 is TGT, which is repeated 7 times; the repeating unit of SSR10 is CTC, which is repeated 10 times; The repeating unit of SSR11 is GAG, and the repeating number is 5; the repeating unit of SSR12 is TTC, and the repeating number is 5; the repeating unit of SSR13 is CGC, and the repeating number is 5; the repeating unit of SSR14 is TCA, and the repeating number is 8; the repeating unit of SSR15 is GGC, and the repeating number is 7; the repeating unit of SSR16 is GCG, and the repeating number is 7; the repeating unit of SSR17 is CGC, and the repeating number is 5; the repeating unit of SSR18 is CTG, and the repeating number is 7; the repeating unit of SSR19 is GCA, and the repeating number is 6; the repeating unit of SSR20 is The repeating unit of SSR21 is GAG, which is repeated 7 times; the repeating unit of SSR22 is AGC, which is repeated 5 times; the repeating unit of SSR23 is CCA, which is repeated 6 times; the repeating unit of SSR24 is GGC, which is repeated 6 times; the repeating unit of SSR25 is GCG, which is repeated 8 times; the repeating unit of SSR26 is TGC, which is repeated 5 times; the repeating unit of SSR27 is CCT, which is repeated 5 times; the repeating unit of SSR28 is TC, which is repeated 8 times; the repeating unit of SSR29 is GTG, which is repeated 6 times; the repeating unit of SSR30 is The repeating unit of SSR31 is GAC, and the number of repetitions is 7; the repeating unit of SSR32 is CCG, and the number of repetitions is 5; the repeating unit of SSR33 is TAC, and the number of repetitions is 8; the repeating unit of SSR34 is CGG, and the number of repetitions is 6; the repeating unit of SSR35 is TCAGC, and the number of repetitions is 5; the repeating unit of SSR36 is CCTCAG, and the number of repetitions is 5; the repeating unit of SSR37 is TGA, and the number of repetitions is 5; the repeating unit of SSR38 is CGG, and the number of repetitions is 5; the repeating unit of SSR39 is AGG, and the number of repetitions is 5.

[0106] The present invention also provides a set of primer combinations for amplifying the SSR marker combination described in the above technical solution, including Zj-SSR01, Zj-SSR02, Zj-SSR03, Zj-SSR04, Zj-SSR05, Zj-SSR06, Zj-SSR07, Zj-SSR08, Zj-SSR09, Zj-SSR10, Zj-SSR11, Zj-SSR12, Zj-SSR13, Zj-SSR14, Zj-SSR15, Zj-SSR16, Zj-SSR17, Zj-SSR18, Zj-SSR19, Zj-SSR20 Zj-SSR35, Zj-SSR36, Zj-SSR37, Zj-SSR38 and Zj-SSR39; the specific nucleotide sequence of the primer combination is shown in Table 1.

[0107] Table 1 SSR markers and their primer sequences

[0108]

[0109]

[0110]

[0111]

[0112] The SSR primers provided by the present invention can be applied to the fields of Zoysia japonica variety identification, genetic pedigree analysis, genetic map construction, protection of germplasm resources and assisted breeding: 39 pairs of SSR primers were used to perform genetic diversity analysis on 36 Zoysia japonica varieties, which have the characteristics of good repeatability, clear spectrum bands, high polymorphism, etc., and can be widely used in Zoysia japonica variety identification, high-density genetic linkage map construction, diversity analysis and molecular-assisted breeding.

[0113] The present invention also provides the use of the SSR marker combination described in the above technical solution or the primer combination described in the above technical solution in identifying and / or distinguishing Zoysia japonica varieties.

[0114] The preferred varieties are shown in Table 3.

[0115] The present invention also provides a method for identifying Zoysia japonica varieties, comprising the following steps:

[0116] Extracting genomic DNA from Zoysia japonica; using the primer combination described in the above technical solution to perform PCR amplification on the genomic DNA from Zoysia japonica to obtain PCR products; performing acrylamide gel electrophoresis and capillary electrophoresis on the PCR products; and analyzing the electrophoresis bands. In the present invention, the analysis of the electrophoresis bands preferably includes: distinguishing the variety from other varieties according to the size of the specific amplified fragments of different Zoysia japonica varieties shown in Table 6, thereby achieving the purpose of variety identification.

[0117] In the present invention, the concentration of the genomic DNA of Zoysia japonica in the acrylamide gel electrophoresis is preferably 20 ng / μL; the PCR amplification system, based on 10 μL, preferably includes the following components: 1 μL of genomic DNA of Zoysia japonica, 0.4 μL of upstream and downstream primers, 5 μL of 2×T5 SuperMix, 3.2 μL of ddH 2 O; the PCR amplification program includes: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 28 cycles; and final extension at 72°C for 7 min; the concentration of the acrylamide gel is preferably 8%.

[0118] In the present invention, in the capillary electrophoresis, the concentration of Zoysia japonica genomic DNA is preferably 20 ng / μL; the PCR reaction system is 15 μL, including: DNA 1 μL, 10*buffer 1.5 μL, MgCl 2 (25mmol) 1.5μL, dNTP (10mmol) 0.3μL, upstream and downstream primers (10umol) 0.15μL, Taq enzyme (5u / μL) 0.3μL, ddH 2 O to 15 μL; PCR amplification program: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; cycle end, final extension at 94℃ for 3 min 。

[0119] In the present invention, the method for analyzing the kinship of Zoysia japonica comprises the following steps: after amplifying the DNA of different varieties of Zoysia japonica using the primer combination described in the above technical solution, the PCR product is displayed as a band value by capillary electrophoresis, and the band value is input into the software PolyGeneV1.7 for cluster analysis. Those with closer kinship will appear on the same evolutionary branch and can be divided into the same group.

[0120] The present invention also provides a zoysia genome chip, which contains the SSR marker combination described in the above technical solution.

[0121] The present invention also provides the use of the SSR marker combination described in the above technical solution, the primer combination described in the above technical solution, or the kit described in the above technical solution in the phylogenetic analysis of Zoysia japonica.

[0122] The present invention also provides the use of the SSR marker combination described in the above technical solution, the primer combination described in the above technical solution, or the kit described in the above technical solution in the maternal tracing of Zoysia japonica.

[0123] The present invention also provides the use of the SSR marker combination described in the above technical solution, the primer combination described in the above technical solution, or the kit described in the above technical solution in the genetic pedigree analysis and / or genetic map construction of Zoysia japonica.

[0124] The present invention also provides the use of the SSR marker combination described in the above technical solution, the primer combination described in the above technical solution, or the kit described in the above technical solution in the protection of Zoysia japonica germplasm resources and / or assisted breeding.

[0125] To further illustrate the present invention, an SSR marker primer developed based on the Zoysia genome sequence and its application provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0126] Example 1

[0127] Development of SSR Primers for Highly Polymorphic Zoysia japonica

[0128] Screening of Unigene sequences rich in SSR sites: Based on the reported sequencing results and annotations of the Zoysia genome, Unigene was assembled after strict filtering and used as background data for subsequent SSR primer development. MISA software was used to search for SSR sites in Unigene, and the search criteria were: mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide, with a minimum number of repetitions of 10, 6, 5, 5, 5, and 5 times, respectively. 18,981 SSR sites were found in the Unigene sequence of the Zoysia genome. The types of SSR sites detected in the genome included mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeat unit types and mixed SSRs. Among them, there were more sites with mono- and tri-nucleotide repeat units, with 7,806 and 6,805 sites, respectively, and the frequencies of occurrence were 41.14% and 35.85%, respectively.

[0129] PrimerPremier 5.0 was used to design SSR primers. The repeating units of the primer design selection sites were dinucleotides, trinucleotides, tetranucleotides, pentanucleotides and hexanucleotides, with a minimum number of repetitions of 6, 5, 5, 5 and 5 times, respectively. The primer length was controlled at 17-25 bp, and the expected product length was 100-280 bp. 400 pairs of primers were randomly selected for amplification.

[0130] Young leaves of the six Zoysia materials in Table 2 were collected, and the DNA of the samples to be tested was extracted by the CTAB method. The operation was carried out according to the CTAB method for extracting plant DNA. The obtained DNA was diluted to 20 ng / μl, and the DNA concentration and quality of the samples were detected by ELISA and agarose gel electrophoresis. The mother solution was stored in a -20°C low-temperature refrigerator.

[0131] Table 26 Zoysia grass material information

[0132]

[0133] Note: The above germplasm resources are unique to the applicant and are guaranteed to be distributed to the public for verification experiments within 20 years from the date of application. The public can purchase them from the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province.

[0134] The DNA of the six Zoysia materials extracted was used as a template and PCR amplification was performed using the above 400 pairs of SSR primers. The PCR reaction system was 10 μL, including:

[0135] 1 μL 20 ng / μL template DNA, 0.4 μL upstream and downstream primers, 5 μL 2×T5 SuperMix (Beijing Qingke Biotechnology Co., Ltd. (Nanjing)), 3.2 μL ddH 2 O.

[0136] The reaction procedure was 94℃ pre-denaturation for 5min; 94℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 30s, 28 cycles, and finally 72℃ extension for 7min. The PCR products were subjected to 8% acrylamide vertical plate electrophoresis to obtain 44 pairs of SSR marker primers with clear bands, high polymorphism and good repeatability.

[0137] The 44 pairs of primers obtained were used to perform PCR amplification and acrylamide gel electrophoresis on the 36 materials in Table 2 to further verify the polymorphism and stability of the 44 pairs of primers in the identification of Zoysia japonica varieties. The PCR reaction system and PCR amplification conditions were the same as above. According to the identification results, 39 pairs of primers with high polymorphism and good stability were finally screened out as candidate primers for Example 2 (as shown in SEQ ID NO.1 to SEQ ID NO.78 in Table 1).

[0138] Example 2

[0139] Application of 39 pairs of SSR primers in identification and genetic diversity analysis of 36 Zoysia japonica varieties

[0140] 1. Extract genomic DNA from the 36 Zoysia materials in Table 3 and adjust the concentration to 20 ng / μl.

[0141] 2. PCR amplification was performed on the genomic DNA of 36 Zoysia materials extracted with 39 pairs of primers obtained in Example 1. The PCR reaction system was 15 μL, including: DNA 1 μL, 10* buffer 1.5 μL, MgCl 2 (25mmol) 1.5μL, dNTP (10mmol) 0.3μL, upstream and downstream primers (10μmol each) 0.15μL, Taq enzyme (5u / μL) 0.3μL, ddH 2 Add 3% HO to 15 μL.

[0142] The reaction procedure was as follows: 94°C pre-denaturation for 3 min; 94°C denaturation for 15 s, 55°C renaturation for 15 s, 72°C extension for 30 s, 35 cycles; after the cycle, the final extension was at 94°C for 3 min. 。

[0143] Table 336 Zoysia grass material information

[0144]

[0145]

[0146] Note: The above germplasm resources are unique to the applicant and are guaranteed to be distributed to the public for verification experiments within 20 years from the date of application. The public can purchase them from the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province.

[0147] 3. The PCR products were analyzed by capillary electrophoresis to display the band values. The band values ​​were counted by EXCEL 2016, and the genetic distance, principal coordinates and other related analyses were performed using the software PolyGene V1.7. The unrooted UPGMA evolutionary tree was constructed using PolyGene V1.7 software, and the cluster analysis diagram of 36 Zoysia species was obtained. The cluster analysis results are shown in Figure 1 shown.

[0148] Depend on Figure 1It can be seen that the 36 varieties can be divided into 3 groups using the primer pairs provided by the present invention. The first group (Cluster I) includes Z0404-3, 20238-1, Z143, Z0417-10, Z0417-4, Z0417-1, Z0417-7, Z077, Z128, Z125, Z0417-11, and Z152; the second group (Cluster II) includes Z0403-9, Z0409-1, Z176, Z0415-3, The third group (Cluster III) includes Z0232-3, Z0402-8, Z0232-5, Z0217-1, Z136, Z137, Z229, Z228, Z135, Z145, and Z22-2.

[0149] 4. PCR amplification was performed on 36 Zoysia materials using the 39 pairs of SSR primers in Example 2, and genetic diversity analysis was performed on the SSR population of the Zoysia materials, as shown in Table 4.

[0150] Table 4 Genetic diversity analysis of 36 Zoysia japonica materials by SSR markers

[0151]

[0152]

[0153] Ho represents the observed heterozygosity; He represents the expected heterozygosity; PIC represents the polymorphic information content; Ae represents the effective number of alleles; Ar represents the allelic richness; I represents the Shannon diversity index. These data can be used to evaluate the polymorphism of primers and their resolution of materials.

[0154] It can be seen from Table 4 that the 39 pairs of marker primers developed based on the present invention have a PIC range of (0.099-0.866), an I range of (0.215-2.287), a Ho range of (0.111-1.000), a He range of (0.105-0.878), a Ae range of (1.117-8.203), an Ar range of (2-14), and a Fst range of (0.011-0.741). In addition, 211 reliable polymorphic bands were identified, and the average number of polymorphic bands per pair of primers was 5. It can be seen that the SSR primers developed by the present invention have rich polymorphism.

[0155] 5. The genetic similarity (GS) between germplasms was calculated using the PolyGene V1.7 program, and then principal coordinate analysis (PCoA) was performed. The results are as follows: Figure 2 shown.

[0156] Depend on Figure 2 It can be seen that the 36 Zoysia japonica germplasm resources in Table 3 can be divided into three groups according to genetic differences: the first group (Cluster I), the second group (Cluster II), and the third group (Cluster III); among them, the first group (Cluster I) is mainly distributed in the first and second quadrants and the distribution is relatively concentrated, the second group (Cluster II) is mostly distributed in the fourth quadrant, and the third group (Cluster III) is relatively concentrated in the third quadrant.

[0157] The genetic diversity index of each population was calculated using GenAlEx6.502, and the results are shown in Table 5 below.

[0158] Table 5 Genetic diversity index of each population

[0159]

[0160] N represents the number of individuals in the population; Na represents the number of different alleles; Ne represents the number of effective alleles. Mean represents the mean, and SE represents the standard error.

[0161] As shown in Table 11, the genetic diversity in Cluster II was the highest (He = 0.705), followed by Cluster I (He = 0.675) and Cluster III (He = 0.646).

[0162] 6. The PCR products were displayed by capillary electrophoresis. Different varieties can be distinguished well according to the value of the amplified band. The capillary electrophoresis results showed that the 36 materials identified all had their specific primer bands. Any specific primer band of each variety can distinguish the variety from other varieties, thus achieving the purpose of variety identification. The specific amplified fragments of SSR primers for identifying different varieties are shown in Table 6.

[0163] Table 6 SSR primers and specific amplified fragment sizes for identifying different Zoysia species (unit: bp)

[0164]

[0165]

[0166] Note: Some materials will show two different values ​​(i.e., two bands) after capillary electrophoresis, indicating that the material is heterozygous. Some materials will show one value, indicating that the material is homozygous. In the table, " / " is used to separate the two bands amplified by the primer.

[0167] From the above examples, it can be seen that after identifying 36 Zoysia japonica materials using the SSR marker combination provided by the present invention, the genetic relationship between them can be determined and different materials can be distinguished, providing technical support for the genetic relationship analysis between unknown new varieties and known varieties and the identification of new varieties.

[0168] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A SSR marker combination for identifying Zoysia japonica, characterized in that: The SSR marker combination includes SSR01, SSR02, SSR03, SSR04, SSR05, SSR06, SSR07, SSR08, SSR09, SSR10, SSR11, SSR12, SSR13, SSR14, SSR15, SSR16, SSR17, SSR18, SSR19, SSR20, SSR21, SSR22, SSR23, SSR24, SSR25, SSR26, SSR27, SSR28, SSR29, SSR30, SSR31, SSR32, SSR33, SSR34, SSR35, SSR36, SSR37, SSR38 and SSR39; The repeating unit of the SSR01 is GCC, and the amplification primers of the SSR01 include Zj-SSR01-F having a nucleotide sequence as shown in SEQ ID No.1 and Zj-SSR01-R having a nucleotide sequence as shown in SEQ ID No.2; The repeating unit of the SSR02 is GGA, and the amplification primers of the SSR02 include Zj-SSR02-F with a nucleotide sequence as shown in SEQ ID No.3 and Zj-SSR02-R with a nucleotide sequence as shown in SEQ ID No.4; The repeating unit of the SSR03 is GAG, and the amplification primers of the SSR03 include Zj-SSR03-F having a nucleotide sequence as shown in SEQ ID No.5 and Zj-SSR03-R having a nucleotide sequence as shown in SEQ ID No.6; The repeating unit of the SSR04 is CCT, and the amplification primers of the SSR04 include Zj-SSR04-F having a nucleotide sequence as shown in SEQ ID No.7 and Zj-SSR04-R having a nucleotide sequence as shown in SEQ ID No.8; The repeating unit of SSR05 is GGT, and the amplification primers of SSR05 include Zj-SSR05-F with a nucleotide sequence as shown in SEQ ID No.9 and Zj-SSR05-R with a nucleotide sequence as shown in SEQ ID No.10; The repeating unit of SSR06 is GCG, and the amplification primers of SSR06 include Zj-SSR06-F with a nucleotide sequence as shown in SEQ ID No.11 and Zj-SSR06-R with a nucleotide sequence as shown in SEQ ID No.12; The repeating unit of the SSR07 is GAA, and the amplification primers of the SSR07 include Zj-SSR07-F having a nucleotide sequence as shown in SEQ ID No.13 and Zj-SSR07-R having a nucleotide sequence as shown in SEQ ID No.14; The repeating unit of the SSR08 is GGA, and the amplification primers of the SSR08 include Zj-SSR08-F having a nucleotide sequence as shown in SEQ ID No.15 and Zj-SSR08-R having a nucleotide sequence as shown in SEQ ID No.16; The repeating unit of the SSR09 is TGT, and the amplification primers of the SSR09 include Zj-SSR09-F having a nucleotide sequence as shown in SEQ ID No. 17 and Zj-SSR09-R having a nucleotide sequence as shown in SEQ ID No. 18; The repeating unit of the SSR10 is CTC, and the amplification primers of the SSR10 include Zj-SSR10-F having a nucleotide sequence as shown in SEQ ID No.19 and Zj-SSR10-R having a nucleotide sequence as shown in SEQ ID No.20; The repeating unit of the SSR11 is GAG, and the amplification primers of the SSR11 include Zj-SSR11-F having a nucleotide sequence as shown in SEQ ID No.21 and Zj-SSR11-R having a nucleotide sequence as shown in SEQ ID No.22; The repeating unit of the SSR12 is TTC, and the amplification primers of the SSR12 include Zj-SSR12-F having a nucleotide sequence as shown in SEQ ID No.23 and Zj-SSR12-R having a nucleotide sequence as shown in SEQ ID No.24; The repeating unit of the SSR13 is CGC, and the amplification primers of the SSR13 include Zj-SSR13-F having a nucleotide sequence as shown in SEQ ID No.25 and Zj-SSR13-R having a nucleotide sequence as shown in SEQ ID No.26; The repeating unit of the SSR14 is TCA, and the amplification primers of the SSR14 include Zj-SSR14-F having a nucleotide sequence as shown in SEQ ID No.27 and Zj-SSR14-R having a nucleotide sequence as shown in SEQ ID No.28; The repeating unit of the SSR15 is GGC, and the amplification primers of the SSR15 include Zj-SSR15-F having a nucleotide sequence as shown in SEQ ID No.29 and Zj-SSR15-R having a nucleotide sequence as shown in SEQ ID No.30; The repeating unit of the SSR16 is GCG, and the amplification primers of the SSR16 include Zj-SSR16-F having a nucleotide sequence as shown in SEQ ID No.31 and Zj-SSR16-R having a nucleotide sequence as shown in SEQ ID No.32; The repeating unit of the SSR17 is CGC, and the amplification primers of the SSR17 include Zj-SSR17-F having a nucleotide sequence as shown in SEQ ID No.33 and Zj-SSR17-R having a nucleotide sequence as shown in SEQ ID No.34; The repeating unit of the SSR18 is CTG, and the amplification primers of the SSR18 include Zj-SSR18-F having a nucleotide sequence as shown in SEQ ID No.35 and Zj-SSR18-R having a nucleotide sequence as shown in SEQ ID No.36; The repeating unit of the SSR19 is GCA, and the amplification primers of the SSR19 include Zj-SSR19-F having a nucleotide sequence as shown in SEQ ID No.37 and Zj-SSR19-R having a nucleotide sequence as shown in SEQ ID No.38; The repeating unit of the SSR20 is CT, and the amplification primers of the SSR20 include Zj-SSR20-F having a nucleotide sequence as shown in SEQ ID No.39 and Zj-SSR20-R having a nucleotide sequence as shown in SEQ ID No.40; The repeating unit of the SSR21 is GAG, and the amplification primers of the SSR21 include Zj-SSR21-F having a nucleotide sequence as shown in SEQ ID No.41 and Zj-SSR21-R having a nucleotide sequence as shown in SEQ ID No.42; The repeating unit of the SSR22 is AGC, and the amplification primers of the SSR22 include Zj-SSR22-F having a nucleotide sequence as shown in SEQ ID No.43 and Zj-SSR22-R having a nucleotide sequence as shown in SEQ ID No.44; The repeating unit of the SSR23 is CCA, and the amplification primers of the SSR23 include Zj-SSR23-F having a nucleotide sequence as shown in SEQ ID No.45 and Zj-SSR23-R having a nucleotide sequence as shown in SEQ ID No.46; The repeating unit of the SSR24 is GGC, and the amplification primers of the SSR24 include Zj-SSR24-F having a nucleotide sequence as shown in SEQ ID No.47 and Zj-SSR24-R having a nucleotide sequence as shown in SEQ ID No.48; The repeating unit of the SSR25 is GCG, and the amplification primers of the SSR25 include Zj-SSR25-F having a nucleotide sequence as shown in SEQ ID No.49 and Zj-SSR25-R having a nucleotide sequence as shown in SEQ ID No.50; The repeating unit of the SSR26 is TGC, and the amplification primers of the SSR26 include Zj-SSR26-F having a nucleotide sequence as shown in SEQ ID No.51 and Zj-SSR26-R having a nucleotide sequence as shown in SEQ ID No.52; The repeating unit of the SSR27 is CCT, and the amplification primers of the SSR27 include Zj-SSR27-F having a nucleotide sequence as shown in SEQ ID No.53 and Zj-SSR27-R having a nucleotide sequence as shown in SEQ ID No.54; The repeating unit of the SSR28 is TC, and the amplification primers of the SSR28 include Zj-SSR28-F having a nucleotide sequence as shown in SEQ ID No.55 and Zj-SSR28-R having a nucleotide sequence as shown in SEQ ID No.56; The repeating unit of the SSR29 is GTG, and the amplification primers of the SSR29 include Zj-SSR29-F having a nucleotide sequence as shown in SEQ ID No.57 and Zj-SSR29-R having a nucleotide sequence as shown in SEQ ID No.58; The repeating unit of the SSR30 is GCC, and the amplification primers of the SSR30 include Zj-SSR30-F having a nucleotide sequence as shown in SEQ ID No.59 and Zj-SSR30-R having a nucleotide sequence as shown in SEQ ID No.60; The repeating unit of the SSR31 is GAC, and the amplification primers of the SSR31 include Zj-SSR31-F having a nucleotide sequence as shown in SEQ ID No.61 and Zj-SSR31-R having a nucleotide sequence as shown in SEQ ID No.62; The repeating unit of the SSR32 is CCG, and the amplification primers of the SSR32 include Zj-SSR32-F having a nucleotide sequence as shown in SEQ ID No.63 and Zj-SSR32-R having a nucleotide sequence as shown in SEQ ID No.64; The repeating unit of the SSR33 is TAC, and the amplification primers of the SSR33 include Zj-SSR33-F having a nucleotide sequence as shown in SEQ ID No.65 and Zj-SSR33-R having a nucleotide sequence as shown in SEQ ID No.66; The repeating unit of the SSR34 is CGG, and the amplification primers of the SSR34 include Zj-SSR34-F having a nucleotide sequence as shown in SEQ ID No.67 and Zj-SSR34-R having a nucleotide sequence as shown in SEQ ID No.68; The repeating unit of the SSR35 is TCAGC, and the amplification primers of the SSR35 include Zj-SSR35-F having a nucleotide sequence as shown in SEQ ID No.69 and Zj-SSR35-R having a nucleotide sequence as shown in SEQ ID No.70; The repeating unit of the SSR36 is CCTCAG, and the amplification primers of the SSR36 include Zj-SSR36-F having a nucleotide sequence as shown in SEQ ID No.71 and Zj-SSR36-R having a nucleotide sequence as shown in SEQ ID No.72; The repeating unit of the SSR37 is TGA, and the amplification primers of the SSR37 include Zj-SSR37-F having a nucleotide sequence as shown in SEQ ID No.73 and Zj-SSR37-R having a nucleotide sequence as shown in SEQ ID No.74; The repeating unit of the SSR38 is CGG, and the amplification primers of the SSR38 include Zj-SSR38-F having a nucleotide sequence as shown in SEQ ID No.75 and Zj-SSR38-R having a nucleotide sequence as shown in SEQ ID No.76; The repeating unit of the SSR39 is AGG, and the amplification primers of the SSR39 include Zj-SSR39-F having a nucleotide sequence as shown in SEQ ID No.77 and Zj-SSR39-R having a nucleotide sequence as shown in SEQ ID No.

78.

2. A set of primer combinations for amplifying the SSR marker combination according to claim 1, characterized in that: Including Zj-SSR01, Zj-SSR02, Zj-SSR03, Zj-SSR04, Zj-SSR05, Zj-SSR06, Zj-SSR07, Zj-SSR08, Zj-SSR09, Zj-SS R10, Zj-SSR11, Zj-SSR12, Zj-SSR13, Zj-SSR14, Zj-SSR15, Zj-SSR16, Zj-SSR17, Zj-SSR18, Zj-SSR19, Zj- SSR20, Zj-SSR21, Zj-SSR22, Zj-SSR23, Zj-SSR24, Zj-SSR25, Zj-SSR26, Zj-SSR27, Zj-SSR28, Zj-SSR29, Z j-SSR30, Zj-SSR31, Zj-SSR32, Zj-SSR33, Zj-SSR34, Zj-SSR35, Zj-SSR36, Zj-SSR37, Zj-SSR38 and Zj-SSR39; The Zj-SSR01 includes Zj-SSR01-F and Zj-SSR01-R, the nucleotide sequence of Zj-SSR01-F is shown in SEQ ID No.1, and the nucleotide sequence of Zj-SSR01-R is shown in SEQ ID No.2; The Zj-SSR02 includes Zj-SSR02-F and Zj-SSR02-R, the nucleotide sequence of Zj-SSR02-F is shown in SEQ ID No.3, and the nucleotide sequence of Zj-SSR02-R is shown in SEQ ID No.4; The Zj-SSR03 includes Zj-SSR03-F and Zj-SSR03-R, the nucleotide sequence of Zj-SSR03-F is shown in SEQ ID No.5, and the nucleotide sequence of Zj-SSR03-R is shown in SEQ ID No.6; The Zj-SSR04 includes Zj-SSR04-F and Zj-SSR04-R, the nucleotide sequence of Zj-SSR04-F is shown in SEQ ID No.7, and the nucleotide sequence of Zj-SSR04-R is shown in SEQ ID No.8; The Zj-SSR05 includes Zj-SSR05-F and Zj-SSR05-R, the nucleotide sequence of the Zj-SSR05-F is shown in SEQ ID No.9, and the nucleotide sequence of the Zj-SSR05-R is shown in SEQ ID No.10; The Zj-SSR06 includes Zj-SSR06-F and Zj-SSR06-R, the nucleotide sequence of Zj-SSR06-F is shown in SEQ ID No.11, and the nucleotide sequence of Zj-SSR06-R is shown in SEQ ID No.12; The Zj-SSR07 includes Zj-SSR07-F and Zj-SSR07-R, the nucleotide sequence of Zj-SSR07-F is shown in SEQ ID No.13, and the nucleotide sequence of Zj-SSR07-R is shown in SEQ ID No.14; The Zj-SSR08 includes Zj-SSR08-F and Zj-SSR08-R, the nucleotide sequence of Zj-SSR08-F is shown in SEQ ID No.15, and the nucleotide sequence of Zj-SSR08-R is shown in SEQ ID No.16; The Zj-SSR09 includes Zj-SSR09-F and Zj-SSR09-R, the nucleotide sequence of Zj-SSR09-F is shown in SEQ ID No.17, and the nucleotide sequence of Zj-SSR09-R is shown in SEQ ID No.18; The Zj-SSR10 includes Zj-SSR10-F and Zj-SSR10-R, the nucleotide sequence of the Zj-SSR10-F is shown in SEQ ID No.19, and the nucleotide sequence of the Zj-SSR10-R is shown in SEQ ID No.20; The Zj-SSR11 includes Zj-SSR11-F and Zj-SSR11-R, the nucleotide sequence of Zj-SSR11-F is shown in SEQ ID No.21, and the nucleotide sequence of Zj-SSR11-R is shown in SEQ ID No.22; The Zj-SSR12 includes Zj-SSR12-F and Zj-SSR12-R, the nucleotide sequence of Zj-SSR12-F is shown in SEQ ID No. 23, and the nucleotide sequence of Zj-SSR12-R is shown in SEQ ID No. 24; The Zj-SSR13 includes Zj-SSR13-F and Zj-SSR13-R, the nucleotide sequence of Zj-SSR13-F is shown in SEQ ID No.25, and the nucleotide sequence of Zj-SSR13-R is shown in SEQ ID No.26; The Zj-SSR14 includes Zj-SSR14-F and Zj-SSR14-R, the nucleotide sequence of Zj-SSR14-F is shown in SEQ ID No. 27, and the nucleotide sequence of Zj-SSR14-R is shown in SEQ ID No. 28; The Zj-SSR15 includes Zj-SSR15-F and Zj-SSR15-R, the nucleotide sequence of the Zj-SSR15-F is shown in SEQ ID No.29, and the nucleotide sequence of the Zj-SSR15-R is shown in SEQ ID No.30; The Zj-SSR16 includes Zj-SSR16-F and Zj-SSR16-R, the nucleotide sequence of Zj-SSR16-F is shown in SEQ ID No.31, and the nucleotide sequence of Zj-SSR16-R is shown in SEQ ID No.32; The Zj-SSR17 includes Zj-SSR17-F and Zj-SSR17-R, the nucleotide sequence of the Zj-SSR17-F is shown in SEQ ID No.33, and the nucleotide sequence of the Zj-SSR17-R is shown in SEQ ID No.34; The Zj-SSR18 includes Zj-SSR18-F and Zj-SSR18-R, the nucleotide sequence of Zj-SSR18-F is shown in SEQ ID No.35, and the nucleotide sequence of Zj-SSR18-R is shown in SEQ ID No.36; The Zj-SSR19 includes Zj-SSR19-F and Zj-SSR19-R, the nucleotide sequence of Zj-SSR19-F is shown in SEQ ID No.37, and the nucleotide sequence of Zj-SSR19-R is shown in SEQ ID No.38; The Zj-SSR20 includes Zj-SSR20-F and Zj-SSR20-R, the nucleotide sequence of the Zj-SSR20-F is shown in SEQ ID No.39, and the nucleotide sequence of the Zj-SSR20-R is shown in SEQ ID No.40; The Zj-SSR21 includes Zj-SSR21-F and Zj-SSR21-R, the nucleotide sequence of the Zj-SSR21-F is shown in SEQ ID No.41, and the nucleotide sequence of the Zj-SSR21-R is shown in SEQ ID No.42; The Zj-SSR22 includes Zj-SSR22-F and Zj-SSR22-R, the nucleotide sequence of Zj-SSR22-F is shown in SEQ ID No.43, and the nucleotide sequence of Zj-SSR22-R is shown in SEQ ID No.44; The Zj-SSR23 includes Zj-SSR23-F and Zj-SSR23-R, the nucleotide sequence of the Zj-SSR23-F is shown in SEQ ID No.45, and the nucleotide sequence of the Zj-SSR23-R is shown in SEQ ID No.46; The Zj-SSR24 includes Zj-SSR24-F and Zj-SSR24-R, the nucleotide sequence of Zj-SSR24-F is shown in SEQ ID No.47, and the nucleotide sequence of Zj-SSR24-R is shown in SEQ ID No.48; The Zj-SSR25 includes Zj-SSR25-F and Zj-SSR25-R, the nucleotide sequence of the Zj-SSR25-F is shown in SEQ ID No.49, and the nucleotide sequence of the Zj-SSR25-R is shown in SEQ ID No.50; The Zj-SSR26 includes Zj-SSR26-F and Zj-SSR26-R, the nucleotide sequence of Zj-SSR26-F is shown in SEQ ID No.51, and the nucleotide sequence of Zj-SSR26-R is shown in SEQ ID No.52; The Zj-SSR27 includes Zj-SSR27-F and Zj-SSR27-R, the nucleotide sequence of the Zj-SSR27-F is shown in SEQ ID No.53, and the nucleotide sequence of the Zj-SSR27-R is shown in SEQ ID No.54; The Zj-SSR28 includes Zj-SSR28-F and Zj-SSR28-R, the nucleotide sequence of the Zj-SSR28-F is shown in SEQ ID No.55, and the nucleotide sequence of the Zj-SSR28-R is shown in SEQ ID No.56; The Zj-SSR29 includes Zj-SSR29-F and Zj-SSR29-R, the nucleotide sequence of the Zj-SSR29-F is shown in SEQ ID No.57, and the nucleotide sequence of the Zj-SSR29-R is shown in SEQ ID No.58; The Zj-SSR30 includes Zj-SSR30-F and Zj-SSR30-R, the nucleotide sequence of the Zj-SSR30-F is shown in SEQ ID No.59, and the nucleotide sequence of the Zj-SSR30-R is shown in SEQ ID No.60; The Zj-SSR31 includes Zj-SSR31-F and Zj-SSR31-R, the nucleotide sequence of the Zj-SSR31-F is shown in SEQ ID No.61, and the nucleotide sequence of the Zj-SSR31-R is shown in SEQ ID No.62; The Zj-SSR32 includes Zj-SSR32-F and Zj-SSR32-R, the nucleotide sequence of Zj-SSR32-F is shown in SEQ ID No.63, and the nucleotide sequence of Zj-SSR32-R is shown in SEQ ID No.64; The Zj-SSR33 includes Zj-SSR33-F and Zj-SSR33-R, the nucleotide sequence of Zj-SSR33-F is shown in SEQ ID No.65, and the nucleotide sequence of Zj-SSR33-R is shown in SEQ ID No.66; The Zj-SSR34 includes Zj-SSR34-F and Zj-SSR34-R, the nucleotide sequence of the Zj-SSR34-F is shown in SEQ ID No.67, and the nucleotide sequence of the Zj-SSR34-R is shown in SEQ ID No.68; The Zj-SSR35 includes Zj-SSR35-F and Zj-SSR35-R, the nucleotide sequence of the Zj-SSR35-F is shown in SEQ ID No.69, and the nucleotide sequence of the Zj-SSR35-R is shown in SEQ ID No.70; The Zj-SSR36 includes Zj-SSR36-F and Zj-SSR36-R, the nucleotide sequence of the Zj-SSR36-F is shown in SEQ ID No.71, and the nucleotide sequence of the Zj-SSR36-R is shown in SEQ ID No.72; The Zj-SSR37 includes Zj-SSR37-F and Zj-SSR37-R, the nucleotide sequence of Zj-SSR37-F is shown in SEQ ID No.73, and the nucleotide sequence of Zj-SSR37-R is shown in SEQ ID No.74; The Zj-SSR38 includes Zj-SSR38-F and Zj-SSR38-R, the nucleotide sequence of the Zj-SSR38-F is shown in SEQ ID No.75, and the nucleotide sequence of the Zj-SSR38-R is shown in SEQ ID No.76; The Zj-SSR39 includes Zj-SSR39-F and Zj-SSR39-R, the nucleotide sequence of the Zj-SSR39-F is shown in SEQ ID No.77, and the nucleotide sequence of the Zj-SSR39-R is shown in SEQ ID No.

78.

3. Use of the SSR marker combination of claim 1 or the primer combination of claim 2 in identifying and / or distinguishing Zoysia japonica varieties.

4. A method for identifying Zoysia japonica varieties, characterized in that: The steps include: Extracting genomic DNA from Zoysia japonica; using the primer combination described in claim 2 to perform PCR amplification on the genomic DNA from Zoysia japonica to obtain PCR products; performing acrylamide gel electrophoresis and capillary electrophoresis on the PCR products; and analyzing the electrophoresis bands.

5. A kit for identifying Zoysia japonica varieties, characterized in that: The kit comprises the primer combination according to claim 2.

6. Use of the SSR marker combination of claim 1, the primer combination of claim 2, or the kit of claim 5 in phylogenetic analysis of Zoysia japonica.

7. Use of the SSR marker combination of claim 1, the primer combination of claim 2, or the kit of claim 5 in tracing the maternal origin of Zoysia japonica.

8. Use of the SSR marker combination of claim 1, the primer combination of claim 2, or the kit of claim 5 in genetic pedigree analysis and / or genetic map construction of Zoysia japonica.

9. Use of the SSR marker combination of claim 1, the primer combination of claim 2, or the kit of claim 5 in the protection of Zoysia japonica germplasm resources and / or assisted breeding.

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