Construction method and application of zoysia japonica germplasm resource fingerprint spectrum

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a construction method and application of a zoysia japonica germplasm resource fingerprint spectrum, and the construction method comprises the following steps: collecting different varieties of zoysia japonica, carrying out PCR amplification by using an SSR molecular marker primer group to obtain an electrophoresis result, numbering according to the lengths of DNA fragments obtained from different varieties of zoysia japonica in the electrophoresis result; the method comprises the following steps: taking zoysia japonica of which the fingerprint spectrum is to be constructed, performing PCR amplification by utilizing an SSR molecular marker primer group to obtain an electrophoresis result, finding out corresponding numbers according to the length of DNA fragments in the electrophoresis result, and sequencing to generate the zoysia japonica germplasm resource fingerprint spectrum. According to the construction method, the zoysia japonica germplasm resources can be accurately, efficiently and stably identified, and a theoretical basis and technical support are provided for related researches such as zoysia japonica germplasm resource identification, variety identification and molecular marker-assisted breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and specifically relates to a method for constructing a fingerprint map of Zoysia japonica germplasm resources and an application thereof. Background Art

[0002] Zoysia Willd. is a perennial herb. Zoysia has a wide distribution range, so different types of resources have been formed. Zoysia germplasm resources in my country are widely distributed and can grow in deserts and saline-alkali land environments. It is recognized as an excellent warm-season lawn grass in the world. There are five species: Japanese Zoysia (Zoysia japonica), Big Spike Zoysia (Zoysia macrostachya), Chinese Zoysia (Zoysia sinica), Grooved Leaf Zoysia (Zoysia matrella), and Slender Leaf Zoysia (Zoysia tenuifolia). Different Zoysia germplasm resources have obvious differences in environmental adaptability, stress resistance, feeding performance and other characteristics.

[0003] Zoysia is widely planted and has many varieties. With the acceleration of breeding process, the number of its varieties is also increasing rapidly. The identification of Zoysia varieties, genetic diversity analysis and the construction of DNA fingerprint database can objectively and comprehensively understand the current status of Zoysia varieties, which is of great significance for variety management, variety breeding, and germplasm collection and protection. The sources of germplasm resources among various populations of Zoysia are relatively chaotic. The reason may be that the exchange of germplasm resources between countries and regions has resulted in the source of some varieties not being their place of origin. Zoysia is monoecious and has the problem of self-pollination, which poses certain obstacles to obtaining excellent hybrids. Therefore, it is necessary to establish a method to identify Zoysia germplasm from different varieties or from different places, and to establish a unique characteristic fingerprint for each variety or species, so as to achieve the purpose of identification, classification, and even quality control.

[0004] DNA fingerprinting is drawn on the basis of molecular markers. It is characterized by rich polymorphism, rapidity, accuracy, and high individual specificity and environmental stability. It is a powerful tool for identifying varieties and strains, and is also very suitable for the identification of germplasm resources. However, so far, the use of SSR markers to construct DNA fingerprints of Zoysia japonica and provide corresponding barcode and QR code information has not been reported at home and abroad. Summary of the invention

[0005] The purpose of the present invention is to provide a method for constructing a fingerprint map of Zoysia japonica germplasm resources and its application. The construction method can accurately, efficiently and stably identify Zoysia japonica germplasm resources, and provide a theoretical basis and technical support for related research such as Zoysia japonica germplasm resource identification, variety discrimination and molecular marker-assisted breeding.

[0006] The invention provides a method for constructing a fingerprint map of Zoysia japonica germplasm resources, comprising the following steps: collecting different varieties of Zoysia japonica, performing PCR amplification using an SSR molecular marker primer set to obtain electrophoresis results, and numbering them according to the lengths of DNA fragments obtained from different varieties of Zoysia japonica in the electrophoresis results; taking Zoysia japonica for which a fingerprint map is to be constructed, performing PCR amplification using an SSR molecular marker primer set to obtain electrophoresis results, finding corresponding numbers according to the lengths of DNA fragments in the electrophoresis results and sorting them, and generating a fingerprint map of Zoysia japonica germplasm resources.

[0007] As a preferred embodiment, the SSR molecular marker primer set includes the following 5 pairs of primers: Zj003 as shown in SEQ ID NO.1 and SEQ ID NO.2, Zj082 as shown in SEQ ID NO.3 and SEQ ID NO.4, Zj083 as shown in SEQ ID NO.5 and SEQ ID NO.6, Zj177 as shown in SEQ ID NO.7 and SEQ ID NO.8, and Zj228 as shown in SEQ ID NO.9 and SEQ ID NO.10.

[0008] As a preferred embodiment, the five pairs of primers are used to distinguish the Zoysia species including: 'Suzhi No. 5', 'Suzhi No. 6', 'Suzhi No. 7', 'Suzhi No. 8', 'Suzhi No. 9', 'Suzhi No. 10' and 'Suzhi No. 12'.

[0009] As a preferred embodiment, the five pairs of primers are used to distinguish the zoysia grass varieties with specific bands; the specific bands of the 'Suzhi No. 5' include: bands 134 and 144 amplified by primer pair Zj003, referred to as 134 / 144 (Zj003), bands 215 and 225 amplified by primer pair Zj083, referred to as 215 / 225 (Zj083); the specific bands of the 'Suzhi No. 6' The specific bands of 'Suzhi No. 7' include: bands 137 and 144 amplified by primer pair Zj003, referred to as 137 / 144 (Zj003), band 259 amplified by primer pair Zj082, referred to as 259 / 259 (Zj082), and bands 227 and 233 amplified by primer pair Zj228, referred to as 227 / 233 (Zj228); the specific bands of 'Suzhi No. 7' include: bands 137 and 144 amplified by primer pair Zj003, referred to as 137 / 144 (Zj003), bands 259 amplified by primer pair Zj082, referred to as 259 / 259 (Zj082), and bands 227 and 233 amplified by primer pair Zj228, referred to as 227 / 233 (Zj228). The band 214 obtained by amplification is referred to as 214 / 214 (Zj083), and the bands 224 and 230 obtained by amplification using primer pair Zj177 are referred to as 224 / 230 (Zj177); the specific bands of the 'Suzhi No. 8' include: bands 217 and 233 obtained by amplification using primer pair Zj177, referred to as 217 / 233 (Zj177); the specific bands of the 'Suzhi No. 9' include: bands 217 and 233 obtained by amplification using primer pair Zj177, referred to as 217 / 233 (Zj177); The bands 230 and 236 amplified by primer pair Zj177 are referred to as 230 / 236 (Zj177); the specific bands of the 'Suzhi No. 10' include: band 250 amplified by primer pair Zj082, referred to as 250 / 250 (Zj082); the specific bands of the 'Suzhi No. 12' include: bands 230 and 239 amplified by primer pair Zj228, referred to as 230 / 239 (Zj228).

[0010] As a preferred solution, the fingerprint spectrum includes: a barcode and / or a QR code.

[0011] As a preferred solution, the read information of the barcode and / or QR code includes: variety-specific DNA fragments, variety name, origin, germplasm type and characteristic property information.

[0012] As a preferred solution, the characteristic property information includes: one or more of paternal and maternal parent information, cultivation institution, morphological characteristics, planting time, planting method and usage information.

[0013] As a preferred solution, the PCR amplification system, in 15 μL, includes: 1 μL DNA, 1.5 μL 10× buffer, 1.5 μL MgCl2, 0.3 μL dNTP, 0.15 μL each of upstream and downstream primers, 0.3 μL Taq enzyme and the remaining ddH2O.

[0014] As a preferred embodiment, the PCR amplification program includes: PCR amplification program is: 94°C pre-denaturation for 3 minutes; 94°C denaturation for 15 seconds, 55°C annealing for 15 seconds, 72°C extension for 30 seconds, 35 cycles; and finally 94°C extension for 3 minutes.

[0015] The present invention provides application of the construction method in identification of Zoysia japonica germplasm resources.

[0016] Beneficial effects: The present invention provides a method for constructing a fingerprint map of Zoysia japonica germplasm resources, comprising the following steps: collecting different varieties of Zoysia japonica, performing PCR amplification using an SSR molecular marker primer set to obtain electrophoresis results, and numbering the DNA fragments obtained from different varieties of Zoysia japonica in the electrophoresis results according to their lengths; taking the Zoysia japonica for which a fingerprint map is to be constructed, performing PCR amplification using an SSR molecular marker primer set to obtain electrophoresis results, finding the corresponding numbers and sorting them according to the lengths of the DNA fragments in the electrophoresis results, and generating a fingerprint map of Zoysia japonica germplasm resources. The present invention uses a DNA fingerprint map code constructed using a Zoysia japonica SSR molecular marker primer set, generates corresponding barcode and QR code information, and establishes unique characteristic fingerprint map information for each variety or type, so as to achieve the purpose of identification, classification, and even quality control, and provides a feasible solution for the screening of Zoysia japonica germplasm resources, library construction, and differentiation of counterfeit products. The construction method of the present invention can accurately, efficiently and stably identify Zoysia japonica germplasm resources; the SSR molecular marker primer set also has good polymorphism and versatility, providing technical guarantee for the identification of Zoysia japonica germplasm resources and variety utilization.

[0017] The present invention provides an application of the construction method in the identification of Zoysia japonica germplasm resources, and uses a DNA fingerprint code constructed by a Zoysia japonica SSR molecular marker primer set to generate corresponding barcode and two-dimensional code information to establish unique characteristic fingerprint information for each variety or type, which can be used to distinguish different varieties to achieve the purpose of identification, classification, and even quality control, thereby reducing costs and improving detection efficiency, overcoming the defects and shortcomings of the prior art in the identification of Zoysia japonica germplasm resources, and providing a feasible solution for the screening of Zoysia japonica germplasm resources, library construction, and differentiation of counterfeit and imitation products. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The peak graph of the amplified bands of the zoysia grass variety 'Suzhi 5' by the primer Zj003 selected from the capillary electrophoresis amplification results in Table 1 is used to illustrate the capillary electrophoresis results;

[0019] Figure 2 The peak graph of the amplified bands of the zoysia grass variety 'Suzhi No. 6' by the primer Zj003 selected from the capillary electrophoresis amplification results in Table 1 is used to illustrate the capillary electrophoresis results;

[0020] Figure 3 The peak graph of the amplified bands of the zoysia grass variety 'Suzhi 7' using primer Zj003 selected from the capillary electrophoresis amplification results in Table 1 is used to illustrate the capillary electrophoresis results;

[0021] Figure 4 The peak graph of the amplified bands of the zoysia grass variety 'Suzhi 8' using primer Zj003 selected from the capillary electrophoresis amplification results in Table 1 is used to illustrate the capillary electrophoresis results;

[0022] Figure 5 The peak graph of the amplified bands of the zoysia grass variety 'Suzhi 9' with the primer Zj003 selected from the capillary electrophoresis amplification results in Table 1 is used to illustrate the capillary electrophoresis results;

[0023] Figure 6 The peak graph of the amplified bands of the zoysia grass variety 'Suzhi No. 10' using the primer Zj003 selected from the capillary electrophoresis amplification results in Table 1 is used to illustrate the capillary electrophoresis results;

[0024] Figure 7 The peak graph of the amplified bands of the zoysia grass variety 'Suzhi No. 12' using the primer Zj003 selected from the capillary electrophoresis amplification results in Table 1 is used to illustrate the capillary electrophoresis results;

[0025] Figure 8 The barcode DNA fingerprints constructed are: 'Suzhi No. 5', 'Suzhi No. 6', 'Suzhi No. 7' and 'Suzhi No. 8' from left to right;

[0026] Fig. 9 The barcode DNA fingerprints constructed are: 'Suzhi No. 9', 'Suzhi No. 10' and 'Suzhi No. 12' from left to right;

[0027] Fig.10 The two-dimensional code DNA fingerprint map constructed; from left to right are 'Suzhi No. 5', 'Suzhi No. 6' and 'Suzhi No. 7';

[0028] Fig.11 The two-dimensional code DNA fingerprint map constructed; from left to right are 'Suzhi No. 8', 'Suzhi No. 9', 'Suzhi No. 10' and 'Suzhi No. 12';

[0029] Fig.12 Read the actual picture of the variety in the DNA fingerprint information of 'Suzhi No. 5'; the left one is 'Suzhi No. 5' and the right one is 'Suzhi No. 6';

[0030] Fig.13 Read the actual picture of the varieties in the DNA fingerprint information; the left one is 'Suzhi No. 7' and the right one is 'Suzhi No. 8';

[0031] Fig.14 Read the actual picture of the varieties in the DNA fingerprint information; the left one is 'Suzhi No. 9' and the right one is 'Suzhi No. 10';

[0032] Fig.15 Read the actual picture of the variety in the DNA fingerprint information of 'Suzhi No. 12'. DETAILED DESCRIPTION

[0033] The invention provides a method for constructing a fingerprint map of Zoysia japonica germplasm resources, comprising the following steps: collecting different varieties of Zoysia japonica, performing PCR amplification using an SSR molecular marker primer set to obtain electrophoresis results, and numbering them according to the lengths of DNA fragments obtained from different varieties of Zoysia japonica in the electrophoresis results; taking Zoysia japonica for which a fingerprint map is to be constructed, performing PCR amplification using an SSR molecular marker primer set to obtain electrophoresis results, finding corresponding numbers according to the lengths of DNA fragments in the electrophoresis results and sorting them, and generating a fingerprint map of Zoysia japonica germplasm resources.

[0034] As a preferred embodiment, the method for constructing a fingerprint map of Zoysia japonica germplasm resources of the present invention comprises the following steps: Step 1, collecting different Zoysia japonica varieties and numbering them, respectively extracting DNA of the different Zoysia japonica varieties, performing PCR amplification on the obtained DNA using an SSR molecular marker primer set, and detecting the obtained amplification product using capillary electrophoresis; according to the electrophoresis results, taking the Zoysia japonica number as the horizontal coordinate and the DNA fragment length as the vertical coordinate, recording the detection results of the DNA amplification bands of different Zoysia japonica varieties at each intersection of the horizontal and vertical coordinates, marking those with DNA amplification bands, and not marking those without DNA amplification bands;

[0035] Take the Zoysia japonica for which the fingerprint is to be constructed, perform PCR amplification using the SSR molecular marker primer set, obtain the electrophoresis result, find the corresponding number according to the length of the DNA fragment in the electrophoresis result and sort it, and generate the Zoysia japonica germplasm resource fingerprint. The fingerprint of the present invention may include: a barcode and / or a two-dimensional code.

[0036] Step 2, using the method for constructing SSR fingerprints of different Zoysia grass varieties described in step 1 to obtain the SSR fingerprint of the Zoysia grass to be tested, comparing the SSR fingerprint of the Zoysia grass to be tested with the SSR fingerprints of the different Zoysia grass varieties, and determining the variety of the Zoysia grass to be tested based on the comparison result.

[0037] As a preferred embodiment, the SSR molecular marker primer set may include the following 5 pairs of primers: Zj003 as shown in SEQID NO.1 and SEQ ID NO.2, Zj082 as shown in SEQ ID NO.3 and SEQ ID NO.4, Zj083 as shown in SEQID NO.5 and SEQ ID NO.6, Zj177 as shown in SEQ ID NO.7 and SEQ ID NO.8, and Zj228 as shown in SEQID NO.9 and SEQ ID NO.10.

[0038] As a preferred embodiment, the five pairs of primers are used to distinguish the varieties of Zoysia japonica, including: 'Suzhi No. 5', 'Suzhi No. 6', 'Suzhi No. 7', 'Suzhi No. 8', 'Suzhi No. 9', 'Suzhi No. 10' and 'Suzhi No. 12'. In the present invention, the five pairs of primers can be used to distinguish the varieties of Zoysia japonica with specific bands; the specific bands of 'Suzhi No. 5' include 134 / 144 (Zj003), 215 / 225 (Zj083); the specific bands of 'Suzhi No. 6' include 137 / 144 (Zj003), 259 / 259 (Zj082), 227 / 233 (Zj228); the specific bands of 'Suzhi No. 7' include The specific bands of 'Suzhi No. 8' include 217 / 233 (Zj177); the specific bands of 'Suzhi No. 9' include 230 / 236 (Zj177); the specific bands of 'Suzhi No. 10' include 250 / 250 (Zj082); the specific bands of 'Suzhi No. 12' include 230 / 239 (Zj228).

[0039] As a preferred embodiment, the fingerprint includes: a barcode and / or a QR code. The read information of the barcode and / or QR code of the present invention may include: variety-specific DNA fragments, variety name, origin, germplasm type and characteristic information. The characteristic information of the present invention may include: one or more of paternal and maternal information, cultivation institution, morphological characteristics, planting time, planting method and use information.

[0040] As a preferred embodiment, the PCR amplification system can include 1 μL of DNA, 1.5 μL of 10×buffer, 1.5 μL of MgCl2, 0.3 μL of dNTP, 0.15 μL of upstream and downstream primers, 0.3 μL of Taq enzyme and the remaining ddH2O. The PCR amplification program of the present invention can include: PCR amplification program: 94°C pre-denaturation for 3 min; 94°C denaturation for 15 s, 55°C annealing for 15 s, 72°C extension for 30 s, 35 cycles; and finally 94°C extension for 3 min.

[0041] The present invention provides an application of the above-mentioned construction method in the identification of Zoysia japonica germplasm resources, using the DNA fingerprint code constructed by the Zoysia japonica SSR molecular marker primer set, and the corresponding barcode and two-dimensional code information to establish unique characteristic fingerprint information for each variety or type.

[0042] In order to further illustrate the present invention, the construction method of a Zoysia japonica germplasm resource fingerprint map provided by the present invention and its application are described in detail below in combination with the embodiments, but they should not be understood as limiting the protection scope of the present invention.

[0043] Example 1

[0044] 1. Build fingerprint code

[0045] The SSR marker primer sets Zj003, Zj082, Zj083, Zj177, and Zj228 for identifying Zoysia japonica varieties, and the specific nucleotide sequences corresponding to the primer pairs are shown in Table 1 .

[0046] Table 1 SSR markers and their primer sequences

[0047]

[0048] 24 samples of Zoysia japonica genomic DNA were extracted, and the material information is shown in Table 2. The Zoysia japonica genomic DNA was amplified by PCR using the above-mentioned SSR marker primer set to obtain PCR products; the PCR products were subjected to capillary electrophoresis; and the electrophoresis bands were analyzed.

[0049] In capillary electrophoresis, the concentration of Zoysia japonica genomic DNA was 20 ng / μL; the PCR reaction system was 15 μL, including: 1 μL DNA, 1.5 μL 10× buffer, 1.5 μL MgCl2 (25 mmol), 0.3 μL dNTP (10 mmol), 0.15 μL upstream and downstream primers (10 μmol each), 0.3 μL Taq enzyme (5 μ / μL), and ddH2O to 15 μL; the PCR amplification program was: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 30 s, 35 cycles; at the end of the cycle, finally extension at 94°C for 3 min.

[0050] Table 224 Zoysia grass material information

[0051]

[0052]

[0053] 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.

[0054] The results of amplification of 24 Zoysia varieties using SSR marker primer sets Zj003, Zj082, Zj083, Zj177, and Zj228, and the total number of polymorphic bands, polymorphic fragment size, and coding obtained by SSR fluorescent labeling capillary electrophoresis were summarized.

[0055] Table 324 SSR fingerprint codes of Zoysia japonica

[0056]

[0057] The DNA fingerprint was constructed using the polymorphic fragments and coding information of capillary electrophoresis. The capillary electrophoresis results were coded with numbers and letters according to Table 3, and the permutations and combinations of the five primer pairs Zj003, Zj083, Zj082, Zj177 and Zj228 were used for the DNA fingerprint.

[0058] 2. Construct and apply the core germplasm DNA fingerprint

[0059] The above five pairs of SSR primers and electrophoresis conditions were used to analyze seven Zoysia japonica varieties. The information of the seven Zoysia japonica varieties is shown in Table 4, and the amplification results are shown in Tables 5 and Figure 1 to Figure 7, 7 Zoysia varieties had specific bands: the specific bands of 'Suzhi 5' were 134 / 144 (Zj003), 215 / 225 (Zj083); the specific bands of 'Suzhi 6' were 137 / 144 (Zj003), 259 / 259 (Zj082), 227 / 233 (Zj228); the specific bands of 'Suzhi 7' were 214 / 21 4(Zj083), 224 / 230(Zj177); the specific band of 'Suzhi No. 8' is 217 / 233(Zj177); the specific band of 'Suzhi No. 9' is 230 / 236(Zj177); the specific band of 'Suzhi No. 10' is 250 / 250(Zj082); the specific band of 'Suzhi No. 12' is 230 / 239(Zj228).

[0060] Table 47 Zoysia grass material information

[0061]

[0062] 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.

[0063] Table 5 Amplification results of 7 Zoysia species

[0064] Material Name Zj003 Zj082 Zj083 Zj177 Zj228 Suzhi No.5 134 / 144 248 / 248 215 / 225 217 / 230 230 / 236 Suzhi No.6 137 / 144 259 / 259 225 / 225 218 / 224 227 / 233 Suzhi No.7 131 / 143 248 / 248 214 / 214 224 / 230 227 / 241 Suzhi No.8 134 / 134 240 / 240 213 / 213 217 / 233 227 / 241 Suzhi No.9 131 / 144 248 / 248 215 / 215 230 / 236 227 / 231 Suzhi No.10 135 / 143 250 / 250 213 / 215 218 / 224 227 / 241 Suzhi No.12 144 / 147 240 / 259 213 / 215 218 / 224 230 / 239

[0065] Figure 1 to Figure 7 It can be seen that the SSR primer set has the characteristics of good repeatability, clear bands and high polymorphism.

[0066] The codes corresponding to the five pairs of SSR primers are combined in the above order to form the application core germplasm DNA fingerprint code, that is, the string DNA molecular ID number. For example, the amplification results of "Suzhi No. 5" are compared with the amplification results in Table 5 to find the number corresponding to the amplification results in Table 3. From Table 5, it is found that the amplified fragment of "Suzhi No. 5" using primer pair Zj003 is 134 / 144, and the ranking number in the polymorphic fragment gradient of primer Zj003 in Table 3 is 6, that is, 6 is the first code of the DNA fingerprint; from Table 5, it is found that the amplified fragment using primer Zj082 is 248 / 248, and the ranking number in the polymorphic fragment gradient of primer Zj082 in Table 3 is 8, that is, 8 is the second code of the DNA fingerprint, and the rest of the codes are deduced in the same way, generating the DNA molecular ID number 68B15 of "Suzhi No. 5". Table 6 shows the DNA fingerprint codes generated for 7 Zoysia varieties.

[0067] Table 67 DNA fingerprint codes of Zoysia japonica

[0068] Material Name SSR fingerprinting Suzhi No.5 68B15 Suzhi No.6 8DE42 Suzhi No.7 18893 Suzhi No.8 42523 Suzhi No.9 28AA1 Suzhi No.10 79643 Suzhi No.12 C5646

[0069] The DNA fingerprint code (DNA molecular ID code) was imported into the online barcode generator (https: / / www.ecjson.com / barcode / ). Based on the three forms of string, barcode and QR code, 7 barcode DNA molecular ID cards of the applied core germplasm were successfully constructed. The online software forage QR code generator (http: / / cli.im / ) was used to encode each variety, and the variety name, origin, germplasm type, characteristics and other information of each variety were entered together to generate the QR barcode of the tested Zoysia japonica germplasm. The barcode DNA fingerprint is shown in Figure 8 and Fig. 9 , QR code DNA fingerprint map see Fig.10 and Fig.11 Scan the corresponding QR code DNA fingerprint map to read the following information about Zoysia japonica.

[0070] (1) DNA fingerprint information of 'Suzhi No. 5'

[0071] Place of Origin: Nanjing, Jiangsu

[0072] Germplasm type: National approved variety

[0073] Features:

[0074] This variety is a new variety bred by the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province, using the hybrid zoysia grass 'Suzhi No. 1' and Zoysia ditchii, and passed the national approval in 2018. This variety has significant hybrid vigor, higher density, finer texture, longer green period than Zoysia ditchii, softer texture and better uniformity than Zoysia ditchii, and can be used for the construction of ornamental lawns, public green spaces, sports field lawns and soil conservation lawns in the middle and lower reaches of the Yangtze River and the south, especially for recreational lawns, ornamental lawns and public green spaces in the middle and lower reaches of the Yangtze River and the south.

[0075] The morphological characteristics of this variety are as follows: natural grass layer height 8.77cm; dark green leaves, soft texture, average leaf length 5.33cm, width 0.14cm; well-developed stolons, stolons internode length 1.85cm, diameter 1.0mm, high lawn density, every 100cm 2 The number of upright branches is 953.6. The height of reproductive branches is 4.75cm, the length of inflorescence is 1.62cm, the number of inflorescence spikelets is 16.60, the length of spikelet is 2.80mm, and the width is 0.8mm.

[0076] The planting time is from May to September, which is suitable for vegetative propagation. Pay attention to leveling the land when planting, removing stones and other debris. Keep the soil moist in the first two weeks after planting to promote rooting.

[0077] See the actual picture of 'Suzhi No. 5' Fig.12 Center left.

[0078] (2) DNA fingerprint information of 'Suzhi No. 6'

[0079] Place of Origin: Nanjing, Jiangsu

[0080] Germplasm type: National approved variety

[0081] Features:

[0082] This variety is a new variety bred by the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province, using the hybridization of Zoysia japonica and Zoysia ditchii. It passed the national approval in 2023. The most prominent features of this variety are its high density, fine texture, strong salt tolerance, and fast turf growth speed. 'Suzhi No. 6' hybrid Zoysia japonica can be planted on saline-alkali land with a total salt content of 8-10‰. It is an excellent grass variety for ecological restoration of saline-alkali land.

[0083] The morphological characteristics of this variety are as follows: the natural average grass layer height is 11.04cm; the average leaf length is 6.30cm and the width is 1.70mm; the stolons are well developed, and the internode length of the stolons is 1.17cm and the diameter is 1.10mm; the lawn density is high, with 100cm per 100cm 2 The number of upright branches is 553.6. The reproductive branch height is 4.94cm, the inflorescence length is 1.71cm, and the width is 1.20mm; the stolons are light purple, and the anthers and glume are green.

[0084] The planting time is from May to September, which is suitable for vegetative propagation. Pay attention to leveling the land when planting, removing stones and other debris. Keep the soil moist in the first two weeks after planting to promote rooting.

[0085] The actual picture of 'Suzhi No. 6' is shown in Fig.12 Center right.

[0086] (3) DNA fingerprint information of 'Suzhi No. 7'

[0087] Place of Origin: Nanjing, Jiangsu

[0088] Germplasm type: Plant variety protection variety

[0089] Features:

[0090] This variety is a new variety bred by the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province, using the hybridization of Zoysia japonica and Zoysia sinensis. It obtained the new plant variety right in 2023. It has excellent characteristics such as low grass layer, high density, good uniformity, and fast growth rate. It can be used for ornamental lawns, public green spaces, sports lawns, saline-alkali land greening and soil conservation lawns, especially the preferred grass variety for sports lawns such as football fields.

[0091] The morphological characteristics of this variety are as follows: the average height of the natural grass layer is 13.2cm; the average length of the leaves is 4.41cm, the width is 3.40mm, and the hairs on the upper and lower surfaces of the leaves are sparse and almost absent; the stolons are well developed, and the internode length of the stolons is 2.20cm and the diameter is 1.20mm; the lawn density is high, with 100cm per 100cm 2 The number of upright branches is 396.65. The reproductive branch height is 4.99cm, the inflorescence length is 1.80cm, the width is 1.30mm, and the average number of spikelets per spike is 23.3; the color of the stolons and anthers are purple, and the husk is dark purple.

[0092] The planting time is from May to September. Vegetative propagation is the main method, and seed propagation is also possible. When planting, pay attention to leveling the land, removing stones and other debris, and keep the soil moist in the first two weeks after planting to promote rooting.

[0093] See the actual picture of 'Suzhi No. 7' Fig.13 Center left.

[0094] (4) DNA fingerprint information of 'Suzhi No. 8'

[0095] Place of Origin: Nanjing, Jiangsu

[0096] Germplasm type: National approved variety

[0097] Features:

[0098] This variety is a new variety bred by the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province, using the hybrid zoysia of 'Suzhi No. 1' and the long-flowered zoysia. It passed the national approval in 2015. This variety has excellent characteristics such as well-developed stolons and underground stems, fast growth, high lawn quality, strong salt tolerance, high inflorescence density and seed yield, disease and insect resistance, drought resistance, and low maintenance costs. It can be used for ornamental lawns, public green spaces, sports field lawns, soil conservation lawns, and saline-alkali land lawns, especially as a preferred grass species for saline-alkali land improvement.

[0099] The morphological characteristics of this variety are as follows: the natural grass layer height is 12.5cm; the leaf color is dark green, the texture is flexible, the average leaf length is 8.16cm, and the width is 3.50mm; the runners are well developed, and the length and diameter of the runner internodes are 2.92cm and 1.30mm respectively. The lawn density is relatively high, with 100cm per 100cm 2 The number of upright branches is 451.2. Compared with other varieties, the outstanding feature of Z0415-3 is that its inflorescence density is higher, with an inflorescence density of 3760 / m 2 The average height of the reproductive branches is 26.5 cm, the average length of the inflorescence is 3.44 cm, the average width of the inflorescence is 2.0 mm, the length and width of the spikelets are 4.13 mm and 1.22 mm respectively, the number of grains per spike is 25.6, and the thousand-grain weight is 0.85 g.

[0100] The planting time is from May to September. It is an excellent variety of zoysia grass that can be propagated asexually or sexually. When planting, pay attention to leveling the land, removing stones and other debris, and keep the soil moist in the first two weeks after planting to promote rooting.

[0101] See the actual picture of 'Suzhi No. 8' Fig.13 Center right.

[0102] (5) DNA fingerprint information of 'Suzhi No. 9'

[0103] Place of Origin: Nanjing, Jiangsu

[0104] Germplasm type: excellent new strain

[0105] Features:

[0106] This variety is a new excellent strain bred by the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province, using the hybridization of Zoysia japonica and Zoysia ditchii. It has the excellent characteristics of dark green leaves, high lawn value, well-developed stolons and underground stems, fast growth, strong disease resistance and salt tolerance. Compared with existing varieties, the grass layer height, density and texture are between existing varieties, and it belongs to the intermediate type material of existing varieties. It is suitable for the construction of various types of lawns such as garden greening, sports field construction, water conservancy slope protection, saline-alkali land ecological restoration, etc. in Beijing and the south.

[0107] The morphological characteristics of this variety are as follows: the average height of the natural grass layer is 17.2cm; the average length of the leaves is 6.85cm, the width is 3.40mm, the hairs on the upper surface of the leaves are sparse, and the hairs on the lower surface are almost absent; the density of the ligule is sparse or almost absent; the stolons and underground stems are well developed, the internode length of the stolons is 2.71cm, and the diameter is 1.37mm; the lawn density is 100cm 2 The number of upright branches is 261.08. The reproductive branch height is 9.89cm, the inflorescence length is 2.49cm, the width is 1.24mm, and the average number of spikelets per spike is 34; the color of the stolons is light purple, the anthers are purple, and the husks are purple.

[0108] The planting time is from May to September, and vegetative propagation is the main method. Pay attention to leveling the land and removing stones and other debris when planting. Keep the soil moist in the first two weeks after planting to promote rooting.

[0109] The actual picture of 'Suzhi No. 9' can be found at Fig.14 Center left.

[0110] (6) DNA fingerprint information of 'Suzhi No. 10'

[0111] Place of Origin: Nanjing, Jiangsu

[0112] Germplasm type: excellent new strain

[0113] Features:

[0114] This variety is a new excellent strain bred by the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province, using Zoysia japonica and Zoysia japonica to cross. It has the excellent characteristics of dark green leaves, long green period, high lawn value, well-developed stolons and underground stems, fast growth rate, strong cold resistance, etc. Compared with existing varieties, the grass layer height, density and texture are between existing varieties, and it belongs to the intermediate type material of existing varieties. It is suitable for the construction of various lawns such as garden greening, sports field construction, water conservancy slope protection, saline-alkali land ecological restoration, etc. in Beijing and the south.

[0115] The morphological characteristics of this variety are as follows: the average height of the natural grass layer is 18.5cm; the average length of the leaves is 8.83cm, the width is 2.70mm, the upper surface of the leaves is densely covered with hair, and the lower surface is almost covered with hair; the density of the ligule is sparse and almost absent; the stolons and underground stems are well developed, the internode length of the stolons is 2.63cm, and the diameter is 1.39mm; the lawn density is 100cm 2 The number of upright branches is 297.42. The reproductive branch height is 9.03cm, the inflorescence length is 2.26cm, the diameter is 1.37mm, and the average number of spikelets per spike is 25; the color of the stolons is dark purple, the anthers are purple, and the husks are dark purple.

[0116] The planting time is from May to September, and vegetative propagation is the main method. Pay attention to leveling the land and removing stones and other debris when planting. Keep the soil moist in the first two weeks after planting to promote rooting.

[0117] The actual picture of 'Suzhi No. 10' can be found at Fig.14 Center right.

[0118] (7) DNA fingerprint information of 'Suzhi No. 12'

[0119] Place of Origin: Nanjing, Jiangsu

[0120] Germplasm type: excellent new strain

[0121] Features:

[0122] This variety is a new excellent strain bred by the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province, using the hybridization of Zoysia japonica and Zoysia dulse. It has the excellent characteristics of high grass density, fine texture, good uniformity, well-developed stolons and underground stems, fast growth, strong disease resistance and salt tolerance. It is suitable for the construction of various lawns such as garden greening, sports field construction, water conservancy slope protection, saline-alkali land ecological restoration, etc. in Beijing and the south.

[0123] The morphological characteristics of this variety are as follows: the natural grass layer height is 18.33cm; the average length of the leaves is 4.96cm, the width is 2.37mm, and the density of hairs on the upper and lower surfaces of the leaves is sparse or almost non-existent; the stolons are well developed, and the average length of the stolons is 1.93cm and the diameter is 1.21mm; the lawn density is high, with 100cm per 100cm 2 The number of upright branches is 346.7. The inflorescence density is low, with 100 cm 2 There are 2 to 3 inflorescences on average, with reproductive branch height of 6.33 cm, inflorescence length of 1.79 cm and inflorescence diameter of 1.62 mm; the color of the stolons is light purple, and the anthers and glumes are purple.

[0124] The planting time is from May to September, and vegetative propagation is the main method. Pay attention to leveling the land and removing stones and other debris when planting. Keep the soil moist in the first two weeks after planting to promote rooting.

[0125] The actual picture of 'Suzhi No. 12' can be found at Fig.15 .

[0126] Example 2

[0127] On the basis of Example 1, in order to further verify whether the above 5 pairs of SSR primer sets can distinguish the 7 new varieties of Zoysia from other Zoysia varieties, the main cultivated varieties on the market, 'Lanyin No. 3' Zoysia, Manila, the introduced variety 'Diamond', the national approved varieties 'Suzhi No. 1' hybrid Zoysia, 'Suzhi No. 3' hybrid Zoysia, 'Suzhi No. 4' hybrid Zoysia, and 11 excellent new lines bred by this unit were selected. The specific information is shown in Table 7. According to the method described in Example 1, the above 5 pairs of SSR primers were used for PCR amplification and capillary electrophoresis, and SSR fingerprint maps were constructed. The SSR fingerprint map codes of the 17 samples were compared with the SSR fingerprint map codes of the above 7 Zoysia varieties. The above 5 pairs of SSR primer sets can effectively distinguish the 17 samples to be tested from the above 7 Zoysia materials. The SSR amplification results constructed from the 17 samples are shown in Table 8, and the generated fingerprint map codes are shown in Table 9.

[0128] Table 7 Information of 17 samples of Zoysia japonica to be tested

[0129]

[0130]

[0131] Table 8 SSR amplification results of 17 Zoysia species

[0132] Material No. / Name Zj003 Zj082 Zj083 Zj177 Zj228 Lanyin No.3 134 / 143 253 / 253 219 / 219 217 / 230 230 / 243 Manila 134 / 134 250 / 253 213 / 215 218 / 230 231 / 241 Diamond 144 / 150 250 / 259 217 / 226 218 / 224 233 / 233 Suzhi No.1 134 / 143 248 / 248 213 / 215 218 / 230 236 / 245 Suzhi No.3 137 / 150 240 / 250 213 / 213 218 / 233 227 / 241 Suzhi No.4 134 / 143 253 / 253 212 / 215 217 / 230 233 / 236 Z0404-3 135 / 143 241 / 241 212 / 212 218 / 230 236 / 244 Z0403-9 131 / 143 240 / 250 213 / 225 218 / 236 233 / 236 Z0201-6 131 / 150 241 / 251 215 / 215 218 / 230 227 / 231 Z0417-10 143 / 147 238 / 250 212 / 212 217 / 230 230 / 230 Z0417-4 147 / 147 240 / 253 212 / 212 217 / 230 230 / 245 Z0232-3 135 / 143 248 / 248 214 / 217 218 / 224 227 / 241 Z0417-1 134 / 143 240 / 240 212 / 219 218 / 227 230 / 230 Z0413-6 134 / 134 248 / 248 217 / 226 218 / 218 233 / 241 Z0417-11 134 / 143 240 / 240 207 / 213 218 / 230 233 / 243 Z0409-1 131 / 143 240 / 259 225 / 225 218 / 236 230 / 236 Z0238-1 141 / 150 240 / 240 212 / 212 218 / 224 233 / 244

[0133] Table 9 Fingerprint codes of 17 Zoysia species

[0134]

[0135]

[0136] The fingerprint codes of the 17 Zoysia species in Table 9 did not match the fingerprint codes in Table 6, proving that they did not belong to the 7 Zoysia species in Table 6. It can be seen that the SSR molecular marker primer set and fingerprint code provided by the present invention can effectively identify the 7 Zoysia species, providing a theoretical basis and technical support for the identification of the 7 Zoysia species and the protection of new varieties.

[0137] It can be seen that the DNA fingerprint code constructed using the Zoysia japonica SSR molecular marker primer set and the corresponding barcode and QR code information generated can establish unique characteristic fingerprint information for each variety or type, which can be used to distinguish different varieties, thereby reducing costs and improving detection efficiency.

[0138] 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 method for constructing a fingerprint of Zoysia japonica germplasm resources, characterized in that: The following steps are involved: Different varieties of Zoysia japonica were collected, and PCR amplification was performed using SSR molecular marker primer sets to obtain electrophoresis results, which were numbered according to the length of DNA fragments obtained from the electrophoresis results of different varieties of Zoysia japonica. Zoysia japonica for fingerprint construction was taken, and PCR amplification was performed using SSR molecular marker primer sets to obtain electrophoresis results. The corresponding numbers were found and sorted according to the length of DNA fragments in the electrophoresis results to generate a fingerprint map of Zoysia japonica germplasm resources.

2. The construction method according to claim 1, characterized in that: The SSR molecular marker primer set includes the following 5 pairs of primers: Zj003 as shown in SEQ ID NO.1 and SEQ ID NO.2, Zj082 as shown in SEQ ID NO.3 and SEQ ID NO.4, Zj083 as shown in SEQ ID NO.5 and SEQ ID NO.6, Zj177 as shown in SEQ ID NO.7 and SEQ ID NO.8, and Zj228 as shown in SEQ ID NO.9 and SEQ ID NO.

10.

3. The construction method according to claim 2, characterized in that: The five pairs of primers are used to distinguish the varieties of Zoysia japonica including: 'Suzhi No. 5', 'Suzhi No. 6', 'Suzhi No. 7', 'Suzhi No. 8', 'Suzhi No. 9', 'Suzhi No. 10' and 'Suzhi No. 12'.

4. The construction method according to claim 3, characterized in that: The five pairs of primers are used to distinguish the varieties of Zoysia japonica, and the specific bands are obtained; the specific bands of the 'Suzhi No. 5' include: bands 134 and 144 amplified by primer pair Zj003, and bands 215 and 225 amplified by primer pair Zj083; the specific bands of the 'Suzhi No. 6' include: bands 137 and 144 amplified by primer pair Zj003, band 259 amplified by primer pair Zj082, and bands 227 and 233 amplified by primer pair Zj228; the specific bands of the 'Suzhi No. 7' include: bands 137 and 144 amplified by primer pair Zj003, band 259 amplified by primer pair Zj082, and bands 227 and 233 amplified by primer pair Zj228; the specific bands of the 'Suzhi No. 7' include: bands 137 and 144 amplified by primer pair Zj003, bands ... j083 amplified band 214, and bands 224 and 230 amplified by primer pair Zj177; the specific bands of the 'Suzhi No. 8' include: bands 217 and 233 amplified by primer pair Zj177; the specific bands of the 'Suzhi No. 9' include: bands 230 and 236 amplified by primer pair Zj177; the specific bands of the 'Suzhi No. 10' include: band 250 amplified by primer pair Zj082; the specific bands of the 'Suzhi No. 12' include: bands 230 and 239 amplified by primer pair Zj228.

5. The construction method according to claim 1, characterized in that: The fingerprint spectrum includes: a bar code and / or a QR code.

6. The construction method according to claim 5, characterized in that: The read information of the barcode and / or QR code includes: variety-specific DNA fragment, variety name, origin, germplasm type and characteristic property information.

7. The construction method according to claim 6, characterized in that: The characteristic property information includes: one or more of paternal and maternal information, cultivation institution, morphological characteristics, planting time, planting method and usage information.

8. The construction method according to claim 1, characterized in that: The PCR amplification system is calculated in 15 μL and includes: 1 μL DNA, 1.5 μL 10× buffer, 1.5 μL MgCl2, 0.3 μL dNTP, 0.15 μL each of upstream and downstream primers, 0.3 μL Taq enzyme and the remaining ddH2O.

9. The construction method according to claim 8, characterized in that: The PCR amplification program includes: PCR amplification program is: 94°C pre-denaturation for 3 minutes; 94°C denaturation for 15 seconds, 55°C annealing for 15 seconds, 72°C extension for 30 seconds, 35 cycles; and finally 94°C extension for 3 minutes.

10. Application of the construction method according to any one of claims 1 to 9 in the identification of Zoysia japonica germplasm resources.

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