A method for constructing a fingerprint of zoysia germplasm resources and application thereof

Abstract

The application belongs to the technical field of molecular biology, and particularly relates to a construction method of a Zoysia japonica germplasm resource fingerprint and application thereof. The construction method comprises the following steps: collecting different varieties of Zoysia japonica, performing PCR amplification by using a SSR molecular marker primer group, obtaining an electrophoresis result, and numbering according to the lengths of DNA fragments of different varieties of Zoysia japonica in the electrophoresis result; taking Zoysia japonica to be constructed into a fingerprint, performing PCR amplification by using the SSR molecular marker primer group, obtaining an electrophoresis result, finding corresponding numbers according to the lengths of DNA fragments in the electrophoresis result, and sorting to generate a Zoysia japonica germplasm resource fingerprint. The construction method can accurately, efficiently and stably identify Zoysia japonica germplasm resources, and provides a theoretical basis and technical support for related researches such as Zoysia japonica germplasm resource identification, variety discrimination and molecular marker assisted breeding.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology, and particularly relates to a construction method of a Zoysia Willd. germplasm resource fingerprint and application thereof. BACKGROUND

[0002] Zoysia Willd. belongs to perennial herbaceous plants. Zoysia Willd. has a wide distribution range, and thus different resource types are formed. Zoysia Willd. germplasm resources in China have a wide distribution range and can grow in desert and saline-alkali environments, and are recognized as excellent warm-season turfgrasses in the world. There are five species of Zoysia Willd. in the prior art, i.e., Zoysia japonica, Zoysia macrostachya, Zoysia sinica, Zoysia matrella and Zoysia tenuifolia. Different Zoysia Willd. germplasm resources have obvious differences in environmental adaptability, stress resistance and forage performance.

[0003] Zoysia Willd. is widely planted and has a large number of varieties, and with the acceleration of breeding process, the number of varieties is rapidly increasing. Identification of Zoysia Willd. varieties, genetic diversity analysis and construction of a DNA fingerprint database can objectively and comprehensively understand the current Zoysia Willd. variety status, and have important significance for variety management, variety breeding and germplasm resource collection and protection. The source places of Zoysia Willd. germplasm resources of different groups are relatively chaotic, which may be caused by the exchange of germplasm resources between countries and between regions, resulting in that the source places of some varieties are not their original places. Zoysia Willd. is monoecious and has the problem of selfing, which causes certain obstacles in obtaining excellent hybrid varieties. Therefore, it is necessary to establish a method for identifying Zoysia Willd. germplasm from different varieties or different places, to establish unique characteristic fingerprints for each variety or species, so as to achieve the purposes of identification, classification and even quality control.

[0004] DNA fingerprinting is drawn on the basis of molecular markers, and has the characteristics of rich polymorphism, rapidness, accuracy, high individual specificity and environmental stability. It is a powerful tool for identifying varieties and lines, and is also very suitable for germplasm resource identification. However, so far, there is no report on the construction of Zoysia Willd. DNA fingerprinting by using SSR markers and the provision of corresponding bar codes and two-dimensional code information at home and abroad. SUMMARY

[0005] The application aims to provide a method for constructing a St. Augustine grass germplasm fingerprint and application thereof, which can accurately, efficiently and stably identify St. Augustine grass germplasm, and provide a theoretical basis and technical support for St. Augustine grass germplasm identification, variety discrimination and molecular marker assisted breeding and other related researches.

[0006] The application provides a method for constructing a St. Augustine grass germplasm fingerprint, which comprises the following steps: collecting different varieties of St. Augustine grass, performing PCR amplification by using a SSR molecular marker primer group, obtaining an electrophoresis result, and numbering according to the lengths of DNA fragments obtained from different varieties of St. Augustine grass in the electrophoresis result; taking St. Augustine grass to be constructed into a fingerprint, performing PCR amplification by using the SSR molecular marker primer group, obtaining an electrophoresis result, finding corresponding numbers and performing sequencing according to the lengths of DNA fragments in the electrophoresis result, and generating a St. Augustine grass germplasm fingerprint.

[0007] As a preferred solution, the SSR molecular marker primer group comprises the following five 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 solution, the five pairs of primers are used to distinguish the following varieties of St. Augustine grass: 'SuZhi 5', 'SuZhi 6', 'SuZhi 7', 'SuZhi 8', 'SuZhi 9', 'SuZhi 10' and 'SuZhi 12'.

[0009] As a preferred solution, the 5 pairs of primers are used to distinguish the specific bands of the Zoysia varieties; the specific bands of the 'SuZhi 5' include: bands 134 and 144 amplified by the primer pair Zj003, referred to as 134 / 144 (Zj003), bands 215 and 225 amplified by the primer pair Zj083, referred to as 215 / 225 (Zj083); the specific bands of the 'SuZhi 6' include: bands 137 and 144 amplified by the primer pair Zj003, referred to as 137 / 144 (Zj003), band 259 amplified by the primer pair Zj082, referred to as 259 / 259 (Zj082), bands 227 and 233 amplified by the primer pair Zj228, referred to as 227 / 233 (Zj228); the specific bands of the 'SuZhi 7' include: band 214 amplified by the primer pair Zj083, referred to as 214 / 214 (Zj083), bands 224 and 230 amplified by the primer pair Zj177, referred to as 224 / 230 (Zj177); the specific bands of the 'SuZhi 8' include: bands 217 and 233 amplified by the primer pair Zj177, referred to as 217 / 233 (Zj177); the specific bands of the 'SuZhi 9' include: bands 230 and 236 amplified by the primer pair Zj177, referred to as 230 / 236 (Zj177); the specific bands of the 'SuZhi 10' include: band 250 amplified by the primer pair Zj082, referred to as 250 / 250 (Zj082); the specific bands of the 'SuZhi 12' include: bands 230 and 239 amplified by the primer pair Zj228, referred to as 230 / 239 (Zj228).

[0010] As a preferred solution, the fingerprint includes: a bar code and / or a two-dimensional code.

[0011] As a preferred solution, the reading information of the bar code and / or two-dimensional code includes: variety-specific DNA fragments, variety names, places of origin, germplasm types, and characteristic information.

[0012] As a preferred solution, the characteristic information includes: one or more of paternal and maternal information, breeding institutions, morphological characteristics, planting times, planting methods, and use information.

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

[0014] As a preferred solution, the PCR amplification procedure comprises: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; and finally 94℃ extension for 3 min.

[0015] The application provides application of the construction method in identification of zoysia grass germplasm resources.

[0016] Beneficial effects: the application provides a construction method of a zoysia grass germplasm resource fingerprint, comprising the following steps: collecting different varieties of zoysia grass, performing PCR amplification by using a SSR molecular marker primer group to obtain an electrophoresis result, and numbering according to the lengths of DNA fragments of different varieties of zoysia grass in the electrophoresis result; taking zoysia grass to be constructed into a fingerprint, performing PCR amplification by using the SSR molecular marker primer group to obtain an electrophoresis result, finding corresponding numbers according to the lengths of DNA fragments in the electrophoresis result and sorting, and generating a zoysia grass germplasm resource fingerprint. The DNA fingerprint code constructed by using the zoysia grass SSR molecular marker primer group, the corresponding bar code and two-dimensional code information generated, and the unique characteristic fingerprint information established for each variety or species can achieve the purposes of identification, classification and even quality control, and provide a feasible scheme for screening, library construction and distinguishing of fake products of zoysia grass germplasm resources. The construction method can accurately, efficiently and stably identify zoysia grass germplasm resources; the SSR molecular marker primer group has good polymorphism and universality, and provides technical support for identification of zoysia grass germplasm resources and utilization of varieties.

[0017] The application provides application of the construction method in identification of zoysia grass germplasm resources, the DNA fingerprint code constructed by using the zoysia grass SSR molecular marker primer group, the corresponding bar code and two-dimensional code information generated, and the unique characteristic fingerprint information established for each variety or species can be used to distinguish different varieties, achieve the purposes of identification, classification and even quality control, reduce costs, improve detection efficiency, overcome the defects and deficiencies in identification of zoysia grass germplasm resources in the prior art, and provide a feasible scheme for screening, library construction and distinguishing of fake products of zoysia grass germplasm resources. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The amplification band peak graph of the primer Zj003 selected from the capillary electrophoresis amplification result in Table 1 on the zoysia grass variety 'Su Zhi 5' is used as an example to illustrate the capillary electrophoresis result.

[0019] Figure 2 The amplification band peak graph of the primer Zj003 selected from the capillary electrophoresis amplification result in Table 1 on the zoysia grass variety 'Su Zhi 6' is used as an example to illustrate the capillary electrophoresis result.

[0020] Figure 3 The amplification band peak chart of Zoysia japonica variety 'SuZhi No.7' by primer Zj003 selected from the capillary electrophoresis amplification results in Table 1 is shown to illustrate the capillary electrophoresis results;

[0021] Figure 4 The amplification band peak chart of Zoysia japonica variety 'SuZhi No.8' by primer Zj003 selected from the capillary electrophoresis amplification results in Table 1 is shown to illustrate the capillary electrophoresis results;

[0022] Figure 5 The amplification band peak chart of Zoysia japonica variety 'SuZhi No.9' by primer Zj003 selected from the capillary electrophoresis amplification results in Table 1 is shown to illustrate the capillary electrophoresis results;

[0023] Figure 6 The amplification band peak chart of Zoysia japonica variety 'SuZhi No.10' by primer Zj003 selected from the capillary electrophoresis amplification results in Table 1 is shown to illustrate the capillary electrophoresis results;

[0024] Figure 7 The amplification band peak chart of Zoysia japonica variety 'SuZhi No.12' by primer Zj003 selected from the capillary electrophoresis amplification results in Table 1 is shown to illustrate the capillary electrophoresis results;

[0025] Figure 8 The bar code DNA fingerprint map constructed; wherein from left to right in turn are 'SuZhi No.5', 'SuZhi No.6', 'SuZhi No.7' and 'SuZhi No.8';

[0026] Figure 9 The bar code DNA fingerprint map constructed; wherein from left to right in turn are 'SuZhi No.9', 'SuZhi No.10' and 'SuZhi No.12';

[0027] Figure 10 The two-dimensional code DNA fingerprint map constructed; wherein from left to right in turn are 'SuZhi No.5', 'SuZhi No.6' and 'SuZhi No.7';

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

[0029] Figure 12 The variety real object chart in the 'SuZhi No.5' DNA fingerprint map information is read; wherein the left is 'SuZhi No.5' and the right is 'SuZhi No.6';

[0030] Figure 13 The variety real object chart in the DNA fingerprint map information is read; wherein the left is 'SuZhi No.7' and the right is 'SuZhi No.8';

[0031] Figure 14 Read the variety of physical map in the DNA fingerprint information; Wherein the left is 'Su Zhi 9', right is 'Su Zhi 10';

[0032] Figure 15 Read the variety of physical map in the DNA fingerprint information of 'Su Zhi 12'. DETAILED DESCRIPTION

[0033] The application provides a kind of zoysia germplasm fingerprint construction method, comprising the following steps: collecting different varieties of zoysia, using SSR molecular marker primer group to carry out PCR amplification, obtain electrophoresis result, according to the length of DNA fragment obtained in electrophoresis result of different varieties of zoysia Numbering;Take the zoysia to be constructed fingerprint, using SSR molecular marker primer group to carry out PCR amplification, obtain electrophoresis result, according to the length of DNA fragment in electrophoresis result Find the corresponding number and sort, generate zoysia germplasm fingerprint.

[0034] As a preferred embodiment, the zoysia germplasm fingerprint construction method of the application comprises the following steps: step 1, collect different zoysia varieties and number, extract DNA of the different zoysia varieties respectively, use SSR molecular marker primer group to carry out PCR amplification on the obtained DNA, and detect the obtained amplification product by capillary electrophoresis;According to the electrophoresis result, take the zoysia number as the abscissa, the DNA fragment length as the ordinate, record the detection results of the DNA amplification bands of different zoysia varieties at each abscissa and ordinate intersection, mark the DNA amplification bands, and do not mark the DNA amplification bands.

[0035] Take the zoysia to be constructed fingerprint, using SSR molecular marker primer group to carry out PCR amplification, obtain electrophoresis result, according to the length of DNA fragment in electrophoresis result Find the corresponding number and sort, generate zoysia germplasm fingerprint. The fingerprint of the application can include: barcode and / or two-dimensional code.

[0036] Step 2, obtain the SSR fingerprint of the test zoysia by using the zoysia SSR fingerprint construction method of step 1, compare the SSR fingerprint of the test zoysia with the SSR fingerprint of the different zoysia varieties, and determine the variety of the test zoysia according to the comparison result.

[0037] As a preferred embodiment, the SSR molecular marker primer set can include 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.

[0038] As a preferred embodiment, the 5 pairs of primers used to distinguish the St. Augustine varieties include: ‘SuZhi 5’, ‘SuZhi 6’, ‘SuZhi 7’, ‘SuZhi 8’, ‘SuZhi 9’, ‘SuZhi 10’ and ‘SuZhi 12’. In the present application, the 5 pairs of primers can be used to distinguish the St. Augustine varieties with specific bands; the specific bands of ‘SuZhi 5’ include 134 / 144 (Zj003), 215 / 225 (Zj083); the specific bands of ‘SuZhi 6’ include 137 / 144 (Zj003), 259 / 259 (Zj082), 227 / 233 (Zj228); the specific bands of ‘SuZhi 7’ include 214 / 214 (Zj083), 224 / 230 (Zj177); the specific bands of ‘SuZhi 8’ include 217 / 233 (Zj177); the specific bands of ‘SuZhi 9’ include 230 / 236 (Zj177); the specific bands of ‘SuZhi 10’ include 250 / 250 (Zj082); the specific bands of ‘SuZhi 12’ include 230 / 239 (Zj228).

[0039] As a preferred embodiment, the fingerprint includes a bar code and / or a two-dimensional code. The reading information of the bar code and / or two-dimensional code of the present application can include: variety-specific DNA fragments, variety names, places of origin, germplasm types and characteristic information. The characteristic information of the present application can include one or more of the following: paternal and maternal information, breeding institutions, morphological characteristics, planting time, planting methods and use information.

[0040] As a preferred embodiment, the system of the PCR amplification can include, in 15 μL, 1 μL of DNA, 1.5 μL of 10x buffer, 1.5 μL of MgCl2, 0.3 μL of dNTP, 0.15 μL of each of the upstream and downstream primers, 0.3 μL of Taq enzyme, and the rest of ddH2O. The PCR amplification procedure of the application can include: the PCR amplification procedure is: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 55℃ recombination for 15 s, 72℃ extension for 30 s, 35 cycles; finally, 94℃ extension for 3 min.

[0041] The application provides application of the construction method in identification of zoysia grass germplasm resources, and establishes unique characteristic fingerprint information for each variety or species by using a DNA fingerprint code constructed by a zoysia grass SSR molecular marker primer group and corresponding bar code and two-dimensional code information.

[0042] In order to further illustrate the application, a zoysia grass germplasm fingerprint construction method and application thereof provided by the application are described in detail below in combination with examples, but they should not be understood as limiting the protection scope of the application.

[0043] Example 1

[0044] 1. Constructing a fingerprint code

[0045] The SSR marker primer group Zj003, Zj082, Zj083, Zj177 and Zj228 for identifying zoysia grass varieties and the specific nucleotide sequences of primer pairs corresponding thereto are shown in Table 1.

[0046] Table 1: SSR markers and primer sequences

[0047]

[0048] Twenty-four zoysia grass genomic DNAs were extracted, and the material information is shown in Table 2. The zoysia grass genomic DNAs were subjected to PCR amplification by using the above-mentioned SSR marker primer group 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 the Zoysia japonica genomic DNA is 20 ng / μL; the PCR reaction system is 15 μL, including: DNA 1 μL, 10×buffer 1.5 μL, MgCl2(25 mmol) 1.5 μL, dNTP(10 mmol) 0.3 μL, the upstream and downstream primers each (10 μmol) 0.15 μL, Taq enzyme (5 μ / μL) 0.3 μL, ddH2O to 15 μL; the PCR amplification procedure is: 94℃ pre-denaturation 3 min; 94℃ denaturation 15 s, 55℃ recombination 15 s, 72℃ extension 30 s, 35 cycles; after the cycle, finally 94℃ extension 3 min.

[0050] Table 2 Information of 24 Zoysia japonica materials

[0051]

[0052]

[0053] Note: The above germplasm resources are unique to the applicant, and are guaranteed to be released to the public for verification experiments within twenty years from the application date. The public can purchase and obtain them from Jiangsu Institute of Botany, Chinese Academy of Sciences.

[0054] The results of amplification of 24 Zoysia japonica varieties by the SSR marker primer group Zj003, Zj082, Zj083, Zj177 and Zj228, the total number of polymorphic bands, the size of the polymorphic fragments and the coding summary obtained by SSR fluorescence labeling capillary electrophoresis.

[0055] Table 3 24 Zoysia japonica SSR fingerprint codes

[0056]

[0057] The capillary electrophoresis polymorphic fragments and coding information are used to construct the DNA fingerprint. The capillary electrophoresis results are coded by numbers and English letters according to Table 3, and the arrangement combination of the 5 pairs of primers Zj003, Zj083, Zj082, Zj177 and Zj228 is used for the DNA fingerprint.

[0058] 2, Application core germplasm DNA fingerprint

[0059] The 5 pairs of SSR primers and the electrophoresis conditions are used to analyze 7 Zoysia japonica varieties, and the information of the 7 Zoysia japonica varieties is shown in Table 4, the amplification results are shown in Table 5 and Figures 1-7, 7 parts of the St. Augustine grass varieties have specific bands: the specific bands of 'SuZhi 5' are 134 / 144 (Zj003), 215 / 225 (Zj083); the specific bands of 'SuZhi 6' are 137 / 144 (Zj003), 259 / 259 (Zj082), 227 / 233 (Zj228); the specific bands of 'SuZhi 7' are 214 / 214 (Zj083), 224 / 230 (Zj177); the specific bands of 'SuZhi 8' are 217 / 233 (Zj177); the specific bands of 'SuZhi 9' are 230 / 236 (Zj177); the specific bands of 'SuZhi 10' are 250 / 250 (Zj082); the specific bands of 'SuZhi 12' are 230 / 239 (Zj228).

[0060] Table 4 Information of 7 St. Augustine grass materials

[0061]

[0062] Note: The above germplasm resources are unique to the applicant, and are guaranteed to be released to the public for verification experiments within 20 years from the application date. The public can purchase and obtain them from Jiangsu Institute of Botany, Chinese Academy of Sciences.

[0063] Table 5 Amplification results of 7 St. Augustine grass varieties

[0064] Material name Zj003 Zj082 Zj083 Zj177 Zj228 SuZhi 5 134 / 144 248 / 248 215 / 225 217 / 230 230 / 236 SuZhi 6 137 / 144 259 / 259 225 / 225 218 / 224 227 / 233 SuZhi 7 131 / 143 248 / 248 214 / 214 224 / 230 227 / 241 SuZhi 8 134 / 134 240 / 240 213 / 213 217 / 233 227 / 241 SuZhi 9 131 / 144 248 / 248 215 / 215 230 / 236 227 / 231 SuZhi 10 135 / 143 250 / 250 213 / 215 218 / 224 227 / 241 SuZhi 12 144 / 147 240 / 259 213 / 215 218 / 224 230 / 239

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

[0066] According to the above order, the coding combination corresponding to the 5 pairs of SSR primer sets is combined to form the application core germplasm DNA fingerprint code, i.e. the string DNA molecular identity card number. For example, for 'SuZhi 5', find the number corresponding to the amplification result in Table 3 from the amplification result in Table 5, find that the amplification fragment of 'SuZhi 5' with primer pair Zj003 is 134 / 144 from Table 5, and the ordering number in the polymorphic fragment gradient of primer Zj003 in Table 3 is 6, i.e. 6 is the first code of the DNA fingerprint map; find that the amplification fragment with primer Zj082 is 248 / 248 from Table 5, and the ordering number in the polymorphic fragment gradient of primer Zj082 in Table 3 is 8, i.e. 8 is the second code of the DNA fingerprint map. The rest of the codes are sequentially generated, and the DNA molecular identity card number of 'SuZhi 5' is 68B15. Table 6 is the DNA fingerprint code generated by 7 St. Augustine grass varieties.

[0067] Table 6 DNA fingerprint code of 7 St. Augustine grasses

[0068] Material name SSR fingerprinting (SSR fingerprinting) SuZhi 5 68B15 SuZhi 6 8DE42 SuZhi 7 18893 SuZhi 8 42523 SuZhi 9 28AA1 SuZhi 10 79643 SuZhi 12 C5646

[0069] The DNA fingerprint code (DNA molecular identity code) was introduced into online barcode generation (https: / / www.ecjson.com / barcode / ), based on string, barcode, and two-dimensional code three forms, and seven barcodes of DNA molecular identity certificates of application core germplasm were successfully constructed. Each variety was coded using online software grass two-dimensional code generator (http: / / cli.im / ), and the variety name, origin, germplasm type, characteristic property, and other information of each variety were recorded to generate two-dimensional barcodes of the test Zoysia japonica germplasm, in which the barcode DNA fingerprint is shown in Figure 8 and Figure 9 , and the two-dimensional code DNA fingerprint is shown in Figure 10 and Figure 11 . Scanning the corresponding two-dimensional code DNA fingerprint can read the following information of Zoysia japonica.

[0070] (1) DNA fingerprint information of 'Su Zhi 5'

[0071] Origin: Nanjing, Jiangsu

[0072] Germplasm type: National approved variety

[0073] Characteristic property:

[0074] This variety is a new variety of Zoysia japonica hybridized by 'Su Zhi 1' and Zoysia matrella developed by Jiangsu Province and the Institute of Botany of the Chinese Academy of Sciences, which was approved by the state in 2018. This variety has significant heterosis, higher density, finer texture, and longer green period than Zoysia japonica, and better uniformity and softer texture than Zoysia matrella. It can be used for ornamental turf, public green space, sports field turf, and soil conservation turf in the middle and lower reaches of the Yangtze River and the south of China, especially for leisure turf, ornamental turf, and public green space in the middle and lower reaches of the Yangtze River and the south of China.

[0075] The morphological characteristics of this variety are as follows: natural grass layer height 8.77 cm; leaf color dark green, texture flexible, average leaf length 5.33 cm, width 0.14 cm; stolon developed, stolon internode length 1.85 cm, diameter 1.0 mm, turf density high, 953.6 erect branches per 100 cm 2 Reproductive branch height 4.75 cm, spike length 1.62 cm, spikelet number 16.60, spikelet length 2.80 mm, width 0.8 mm.

[0076] Planting time: May to September, suitable for vegetative propagation, pay attention to leveling the land and removing stones and other sundries, and keep the soil moist for two weeks after planting to promote rooting.

[0077] The physical map of 'SuZhi 5' is shown in Figure 12 left.

[0078] (2) DNA fingerprint information of 'SuZhi 6'

[0079] Origin: Nanjing, Jiangsu

[0080] Germplasm type: National approved variety

[0081] Characteristics:

[0082] This variety is a new variety bred by the Chinese Academy of Sciences, Institute of Botany, Jiangsu Province, by hybridizing Zoysia japonica and Zoysia matrima, and was approved by the state in 2023. The most outstanding feature of this variety is its high density, fine texture, strong salt tolerance, and fast establishment speed. 'SuZhi 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: natural average grass layer height 11.04 cm; average leaf length 6.30 cm, width 1.70 mm; well-developed stolon, stolon internode length 1.17 cm, diameter 1.10 mm; high turf density, 553.6 erect branches per 100 cm 2 . Reproductive branch height 4.94 cm, inflorescence length 1.71 cm, width 1.20 mm; stolon color light purple, anther and glume green.

[0084] Planting time: May to September, suitable for vegetative propagation, pay attention to leveling the land, removing stones and other debris, and keeping the soil moist for the first two weeks after planting to promote root growth.

[0085] The physical map of 'SuZhi 6' is shown in Figure 12 right.

[0086] (3) DNA fingerprint information of 'SuZhi 7'

[0087] Origin: Nanjing, Jiangsu

[0088] Germplasm type: Plant new variety protection variety

[0089] Characteristics:

[0090] This variety is a new variety bred by the Chinese Academy of Sciences, Institute of Botany, Jiangsu Province, by hybridizing Zoysia japonica and Zoysia matrima, and was approved by the state in 2023. The most outstanding feature of this variety is its high density, fine texture, strong salt tolerance, and fast establishment speed. 'SuZhi 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.

[0091] The morphological characteristics of this variety are as follows: the natural grass layer height is 13.2 cm on average; the average length of leaf blade is 4.41 cm, the width is 3.40 mm, the pubescence on upper and lower leaf blade surfaces is sparse and nearly non-existent; the stolon is developed, the stolon internode length is 2.20 cm, and the diameter is 1.20 mm; the turf density is high, and the number of erect branches per 100 cm 2 of Z0415-3 is 396.65. The height of reproductive branch is 4.99 cm, the length of spike is 1.80 cm, the width is 1.30 mm, the average number of spikelets per spike is 23.3; the stolon color and anther are purple, and the glume is dark purple.

[0092] The planting time can be from May to September, and the vegetative propagation is mainly used, and the seed propagation can also be used. When planting, the land should be leveled, and the stones and other sundries should be removed, and the soil should be kept wet for two weeks after planting to promote rooting.

[0093] The physical map of 'SuZhi 7' is shown in Figure 13 the left of the middle.

[0094] (4) DNA fingerprint information of 'SuZhi 8'

[0095] Origin: Nanjing, Jiangsu

[0096] Germplasm type: nationally approved variety

[0097] Characteristics:

[0098] This variety is a new variety bred by hybridizing 'SuZhi 1' with Zoysia japonica, which was approved by the state in 2015. The variety has the excellent characteristics of developed stolons and underground stems, fast growth, high turf quality, strong salt tolerance, high spike density and seed yield, disease and insect resistance, drought resistance, and low maintenance cost. It can be used for ornamental turf, public green space, sports field turf, soil conservation turf, and saline-alkali land turf planting, especially for the selection of grass species for saline-alkali land improvement.

[0099] The morphological characteristics of this variety are as follows: the natural grass layer height is 12.5 cm; the leaf color is dark green, the texture is flexible, the average length of leaf blade is 8.16 cm, and the width is 3.50 mm; the stolon is developed, the stolon internode length is 2.92 cm, and the diameter is 1.30 mm, the turf density is high, and the number of erect branches per 100 cm 2 of Z0415-3 is 451.2. Compared with Gaohe, the outstanding characteristics of Z0415-3 are also in the high spike density, the spike density is 3760 / m 2 , the average height of reproductive branch is 26.5 cm, the average length of spike is 3.44 cm, the average width of spike is 2.0 mm, the length and width of spikelet 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] Planting time is from May to September. This is an excellent variety of zoysia grass that can be propagated both asexually and sexually. When planting, be sure to level the soil and remove debris such as rocks. Keep the soil moist for the first two weeks after planting to promote rooting.

[0101] The actual picture of 'Suzhi No. 8' is shown in Figure 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 superior new strain developed by the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province, by hybridizing Zoysia japonica and Zoysia dulcifolia. It features dark green leaves, high turf value, well-developed runners and rhizomes, rapid growth, and strong disease and salt tolerance. Compared to existing varieties, its grass layer height, density, and texture are intermediate, placing it in the middle. It is suitable for various lawn applications, including landscaping, sports field construction, water conservancy slope protection, and saline-alkali land restoration in Beijing and areas south of the capital.

[0107] The morphological characteristics of this variety are as follows: the average height of the natural grass layer is 17.2 cm; the average length of the leaves is 6.85 cm, the width is 3.40 mm, 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 runners and underground stems are well developed, with the internode length of the runner being 2.71 cm and the diameter being 1.37 mm; the lawn density is 100 cm per 100 cm. 2 The number of upright branches is 261.08. The reproductive branches are 9.89 cm tall, the inflorescence is 2.49 cm long and 1.24 mm wide, and the average number of spikelets per spike is 34. The stolons are light purple, the anthers are purple, and the glumes are purple.

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

[0109] The actual picture of 'Suzhi No. 9' is shown in Figure 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 line bred by hybridization of Zoysia japonica and Z. matrima in Jiangsu Province, China. It has dark green leaves, long green period, high value of turf, developed stolons and rhizomes, fast growth, strong cold resistance, etc. Compared with the existing varieties, the height, density and texture of the grass layer are between those of the existing varieties, and it belongs to the intermediate type material of the existing varieties. It is suitable for the construction of various lawns in Beijing and the south of China, such as landscaping, sports field construction, water conservancy slope protection, and ecological restoration of saline-alkali land.

[0115] The morphological characteristics of this variety are as follows: the average height of natural grass layer is 18.5 cm; the average length of leaf blade is 8.83 cm, and the width is 2.70 mm; the pubescence on the upper surface of the leaf blade is dense, and the pubescence on the lower surface is nearly absent; the stolon and rhizome are developed, the internode length of stolon is 2.63 cm, and the diameter is 1.39 mm; the number of erect branches per 100 cm 2 of turf density is 297.42; the height of reproductive branch is 9.03 cm, the length of spike is 2.26 cm, and the diameter is 1.37 mm; the average number of spikelets per spike is 25; the stolon color is dark purple, the anther is purple, and the glume is dark purple.

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

[0117] The physical map of 'Su Zhi 10' is shown in Figure 14 the middle right.

[0118] (7) DNA fingerprint information of 'Su Zhi 12'

[0119] Origin: Nanjing, Jiangsu

[0120] Germplasm type: new excellent line

[0121] Characteristics:

[0122] This variety is a new excellent line bred by hybridization of Zoysia japonica and Z. matrima in Jiangsu Province, China. It has dark green leaves, long green period, high value of turf, developed stolons and rhizomes, fast growth, strong cold resistance, etc. Compared with the existing varieties, the height, density and texture of the grass layer are between those of the existing varieties, and it belongs to the intermediate type material of the existing varieties. It is suitable for the construction of various lawns in Beijing and the south of China, such as landscaping, sports field construction, water conservancy slope protection, and ecological restoration of saline-alkali land.

[0123] The morphological characteristics of the present variety are as follows: natural grass layer height is 18.33 cm; average length of leaf blade is 4.96 cm, width is 2.37 mm, and the density of pubescence on upper and lower leaf blades is sparse and nearly none; stolon is developed, the average length of stolon internode is 1.93 cm, and the diameter is 1.21 mm; the turf density is high, and the number of erect branches per 100 cm 2 of the turf is 346.7. The inflorescence density is low, and the average number of inflorescences per 100 cm 2 of the turf is 2-3, the height of reproductive branch is 6.33 cm, the length of inflorescence is 1.79 cm, and the diameter of inflorescence is 1.62 mm; the color of stolon is light purple, and the color of anther and glume is purple.

[0124] The planting time can be from May to September, and the vegetative propagation is mainly used. When planting, the land should be leveled, and the stones and other sundries should be removed. In the first two weeks after planting, the soil should be kept wet to promote rooting.

[0125] The physical map of ‘SuZhi 12’ is shown in Figure 15 .

[0126] Example 2

[0127] On the basis of Example 1, in order to further verify whether the above 5 pairs of SSR primers can distinguish the 7 new varieties of St. Augustine grass from other St. Augustine grass varieties, the main cultivated variety ‘LanYin 3’ St. Augustine grass, Manila, the introduced variety ‘Diamond’, the national approved variety ‘SuZhi 1’ hybrid St. Augustine grass, ‘SuZhi 3’ hybrid St. Augustine grass, ‘SuZhi 4’ hybrid St. Augustine grass, and 11 excellent new strains selected by the unit are selected. The specific information is shown in Table 7. The method described in Example 1 is used to perform PCR amplification and capillary electrophoresis by using the above 5 pairs of SSR primers, and the SSR fingerprint map is constructed. The SSR fingerprint code of the 17 samples is compared with the SSR fingerprint code of the above 7 St. Augustine grass varieties. The above 5 pairs of SSR primers can effectively distinguish the 17 samples to be tested from the above 7 St. Augustine grass materials. The SSR amplification results of the 17 samples are shown in Table 8, and the generated fingerprint code is shown in Table 9.

[0128] Table 7 Information of 17 St. Augustine grasses to be tested

[0129]

[0130]

[0131] Table 8 SSR amplification results of 17 St. Augustine grass varieties

[0132] Material number / name Zj003 Zj082 Zj083 Zj177 Zj228 LanYin 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 1 134 / 143 248 / 248 213 / 215 218 / 230 236 / 245 SuZhi 3 137 / 150 240 / 250 213 / 213 218 / 233 227 / 241 SuZhi 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 code of 17 St. Augustine grass varieties

[0134]

[0135]

[0136] The fingerprint code of the 17 St. Augustine grass varieties in Table 9 is not found to match the fingerprint code in Table 6, proving that it does not belong to the 7 St. Augustine grass varieties in Table 6. It can be seen that the SSR molecular marker primer set and the fingerprint code provided by the application can effectively identify the 7 St. Augustine grass varieties, and provide a theoretical basis and technical support for the identification and new variety protection of the 7 St. Augustine grass varieties.

[0137] It can be seen that the DNA fingerprint code constructed by using the St. Augustine grass SSR molecular marker primer set and the corresponding bar code and two-dimensional code information generated can establish unique characteristic fingerprint information for each variety or species, and can be used to distinguish different varieties, which reduces the cost and improves the detection efficiency.

[0138] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which belongs to the protection scope of the present application.

Claims

1. A method of distinguishing between varieties of Zoysia japonica, characterised by, The method comprises the following steps: Collecting different varieties of Zoysia japonica, using a SSR molecular marker primer set to perform PCR amplification to obtain an electrophoresis result, and numbering the lengths of the DNA fragments of the different varieties of Zoysia japonica in the electrophoresis result; taking Zoysia japonica to be constructed into a fingerprint, using a SSR molecular marker primer set to perform PCR amplification to obtain an electrophoresis result, finding the corresponding number and sorting according to the lengths of the DNA fragments in the electrophoresis result, and generating a Zoysia japonica germplasm resource fingerprint; The SSR molecular marker primer set comprises the following five 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; The Zoysia japonica varieties are: 'Su Zhi 5', 'Su Zhi 6', 'Su Zhi 7', 'Su Zhi 8', 'Su Zhi 9', 'Su Zhi 10', and 'Su Zhi 12'; When the five pairs of primers are used to distinguish the Zoysia japonica varieties, specific bands are obtained; the specific bands of 'Su Zhi 5' include bands 134 and 144 obtained by amplification with primer pair Zj003 and bands 215 and 225 obtained by amplification with primer pair Zj083; the specific bands of 'Su Zhi 6' include bands 137 and 144 obtained by amplification with primer pair Zj003, band 259 obtained by amplification with primer pair Zj082, and bands 227 and 233 obtained by amplification with primer pair Zj228; the specific bands of 'Su Zhi 7' include band 214 obtained by amplification with primer pair Zj083 and bands 224 and 230 obtained by amplification with primer pair Zj177; the specific bands of 'Su Zhi 8' include bands 217 and 233 obtained by amplification with primer pair Zj177; the specific bands of 'Su Zhi 9' include bands 230 and 236 obtained by amplification with primer pair Zj177; the specific bands of 'Su Zhi 10' include band 250 obtained by amplification with primer pair Zj082; and the specific bands of 'Su Zhi 12' include bands 230 and 239 obtained by amplification with primer pair Zj228.

2. The method of claim 1, wherein, The fingerprint comprises a bar code and / or a two-dimensional code.

3. The method of claim 2, wherein, The reading information of the bar code and / or the two-dimensional code comprises variety-specific DNA fragments, variety names, places of origin, germplasm types, and characteristic information.

4. The method of claim 3, wherein, The characteristic information comprises one or more of paternal and maternal information, breeding institutions, morphological characteristics, planting times, planting methods, and use information.

5. The method of claim 1, wherein, The PCR amplification system is 15 μL, including: DNA 1 μL, 10×buffer 1.5 μL, MgCl2 1.5 μL, dNTP 0.3 μL, each of the upstream and downstream primers 0.15 μL, Taq enzyme 0.3 μL and the rest of ddH2O.

6. The method of claim 5, wherein, The PCR amplification procedure includes: the PCR amplification procedure is: 94℃ pre-denaturation 3 min; 94℃ denaturation 15 s, 55℃ recombination 15 s, 72℃ extension 30 s, 35 cycles; finally 94℃ extension 3 min.

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