Method for identifying genetic relationship of different zoysia japonica germplasm

Abstract

The invention discloses a method for identifying the genetic relationship of different zoysia japonica germplasms, and belongs to the technical field of identification of the genetic relationship of the zoysia japonica germplasms, primer sequences are shown as SEQ ID NO.11-SEQ ID NO.12. The primer is used for preparing a kit, the genetic relationship of the zoysia japonica can be specifically identified, and a foundation is laid for follow-up research on the genetic performance of the zoysia japonica.

Description

Technical Field

[0001] The invention relates to the technical field of identification of phylogenetic relationships of Zoysia japonica germplasms, and more particularly to a method for identifying phylogenetic relationships of different Zoysia japonica germplasms. Background Art

[0002] Zoysia grass (Zoysia Willd.) belongs to the perennial low-growing herbaceous plant of the Poaceae family. It is generally divided into 11 species, multiple varieties and variants. Most of them can be used to build sports lawns, recreational lawns and soil conservation lawns. There are 5 species, 2 varieties and 1 variant of Zoysia grass plants distributed in my country. The five species are Z. japonica Steud., Z. tenuiflia willd. ex Trin., Z. matrella (L.) Merr., Z. macrostachya Franch. et Sva., and Z. sinica Hance; the two variants are Z. japonica var. pollida Nakai et Honda and Z. sinica var. nipponica Ohwi.; and one variant is Z. japonica f. macrostachya HD Dung et L J Gong. Among them, Z. japonica is the species with the widest distribution and the largest application area in the genus, and is also the representative species of the genus Zoysia in China.

[0003] In recent years, molecular marker technology has been increasingly used in the study of lawn grass germplasm resources. It is also widely used in the study of Zoysia plants. EST-SSR (Expressed Sequence Tag-Simplesequencerepeat) is a molecular marker developed based on expressed sequence tags for microsatellites, which has a higher translation ability than genomic SSR markers. Since EST-SSR markers are a type of functional marker, they are obtained based on the inferred function of EST sequences. At present, EST-SSR is widely used in plant genomics research, genetic map construction, comparative mapping, and evaluation of genetic diversity. It is rarely used in the study of Zoysia germplasm resources.

[0004] Therefore, how to apply EST-SSR to molecular markers of Zoysia japonica and develop primers for identifying the genetic relationship of different Zoysia japonica germplasms is a technical problem that technicians in this field urgently need to solve. Summary of the invention

[0005] In view of this, based on the many advantages of EST-SSR molecular markers, the present invention adopts the method of EST-SSR molecular markers to study the genetic variation of Zoysia germplasm from different geographical origins, explores the genetic diversity of Zoysia germplasm resources at the molecular level, and lays a good foundation for the study of Zoysia germplasm resources.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for identifying the relationship between different Zoysia japonica germplasms, using a kit for detection, the process comprising:

[0008] 1) Use any 3 pairs of primers from the 20 pairs of EST-SSR primers screened to perform PCR amplification of EST-SSR on 43 Zoysia germplasms to obtain polymorphic bands and observe the number of alleles amplified by each pair of primers;

[0009] 2) Based on the polymorphic bands of EST-SSR primers, the genetic diversity of 43 Zoysia accessions was studied using NTSYS software to determine the genetic coefficient and analyze the distance of genetic relationship;

[0010] The kit includes any one or a combination of three primers shown in SEQ ID NO.1 to SEQ ID NO.40; specifically:

[0011]

[0012]

[0013] Preferably, the primer sequences are shown in SEQ ID NO.11 to SEQ ID NO.12;

[0014] Upstream primer F: 5'-TGCTCAAACTTGCTTTCCTG-3', as shown in SEQ ID NO.11;

[0015] Downstream primer R: 5'-CCGTGCAATCGTAGTACCAG-3', as shown in SEQ ID NO.12.

[0016] Preferably, any two sets of primers among the primers described in SEQ ID NO.1 to SEQ ID NO.10 and SEQ ID NO.13 to SEQ ID NO.40.

[0017] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses a method for identifying the kinship between different Zoysia grass germplasms. This method can identify the kinship between different Zoysia grass germplasms, laying a foundation for subsequent research on the genetic properties of Zoysia grass. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0019] Figure 1 The attached figure is the UPGMA cluster diagram of 43 Zoysia accessions analyzed by EST-SSR. DETAILED DESCRIPTION

[0020] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Example 1 Primer screening

[0022] 1) DNA extraction and verification

[0023] The DNA of 43 Zoysia germplasms was extracted by the modified CTAB method (the sources of the 43 Zoysia germplasm resources can be found in He Xiao et al., Variation of Morphological Characteristics of Zoysia Germplasm Resources, 2024, 22(13), pp. 4364-4376; see Table 1 for details). The purity and quality of the extracted DNA were tested by 1% agarose gel electrophoresis, and the DNA concentration and OD value were determined using a nucleic acid protein meter. The DNA was diluted to about 50 ng / μL and finally stored in a -20°C refrigerator for later use.

[0024] Table 143 Sources of Zoysia germplasm resources

[0025]

[0026]

[0027]

[0028]

[0029] 2) Primer screening

[0030] A total of 125 pairs of EST-SSR primers were selected. Six Zoysia japonica germplasms (No. 4, No. 9, No. 31, No. 37, No. 40, No. 42) from different sources were selected to screen the 125 pairs of primers. Finally, 20 pairs of EST-SSR markers with clear bands, good polymorphism, and obvious main bands were screened. The selected primer sequences are shown in Table 2.

[0031] Table 2 Sequences of 20 pairs of primers screened

[0032]

[0033]

[0034]

[0035] 3) PCR amplification and electrophoresis detection

[0036] The PCR reaction system is shown in Table 3. The amplification process was carried out on a PCR instrument, and the amplification program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 45 s, extension at 72°C for 1 min, and 35 cycles; finally extension at 72°C for 5 min, and storage at 4°C. The PCR amplification product was detected by 8% polyacrylamide gel electrophoresis. After the electrophoresis was completed, it was stained with AgNO3, developed with NaOH and formaldehyde, and finally photographed and recorded.

[0037] Table 3 PCR amplification reaction system

[0038]

[0039] The results were as follows: 43 Zoysia germplasm DNAs were amplified using the 20 pairs of EST-SSR primers screened, and a total of 91 bands were obtained, of which 78 were polymorphic bands, with a polymorphic band ratio of 86.65% (Table 4). The bands amplified by a single primer ranged from 1 to 8, with an average of 4.55 bands amplified by each primer, and an average of 3.90 polymorphic bands amplified by each primer. Among them, the primer numbered ES-091 amplified the most bands, which were 8. There were 10 primers with the highest polymorphic band ratio: ES-001, ES-007, ES-009, ES-013, ES-056, ES-066, ES-089, ES-092, ES-097, and ES-100, all of which were 100%. There was one primer with the lowest polymorphic band ratio: ES-021, with a polymorphism of 50%.

[0040] Table 4 EST-SSR primer amplification results

[0041]

[0042] Example 2 Analysis of genetic diversity of EST-SSR populations of Zoysia germplasm

[0043] EST-SSR PCR amplification (Table 5) was performed on 43 Zoysia germplasms using 20 pairs of EST-SSR primers screened in Example 1. Clear and stable electrophoresis bands were statistically analyzed, with a band assigned a value of 1, no band assigned a value of 0, and missing a value of 9. A database was established for band polymorphism comparison and cluster analysis. POPGEN32 software was used to perform statistical analysis on the genetic diversity of Zoysia germplasm, and the number of alleles (Na), allele frequency, Shannon information index (I), observed heterozygosity Ho, expected heterozygosity He, and Nei gene diversity index (H) were calculated. NTSYS software was used to perform UPGMA system clustering on 43 materials to analyze the genetic differentiation relationship between materials and between geographical provenances.

[0044] Results A total of 75 alleles were amplified, and each primer pair detected 1-6 alleles, with an average of 3.75 alleles detected by each EST-SSR primer pair. The observed heterozygosity of the 20 EST-SSR pairs ranged from 0 to 1, with an average of 0.193, and the expected heterozygosity ranged from 0 to 0.786, with an average of 0.559. The maximum observed heterozygosity of ES-013 was 1, and the maximum expected heterozygosity of ES-070 was 0.786. The Shaanno information index of the 20 EST-SSR primer pairs ranged from 0 to 1.561, with an average of 0.986. The largest Shaanno information index was ES-012, which indicated that ES-012 had a high efficiency in polymorphism detection.

[0045] Table 5 EST-SSR genetic diversity analysis of 43 Zoysia accessions

[0046]

[0047]

[0048]

[0049] Example 3 Genetic Similarity Coefficient Analysis

[0050] Based on the polymorphic bands of 20 pairs of EST-SSR primers, the genetic diversity of 43 Zoysia materials was studied using NTSYS software. The results showed that the genetic similarity coefficients between the materials were between 0.530 and 0.864, with an average of 0.711. The two materials with the largest genetic coefficient (0.864) were No. 20 and No. 19, and the two materials with the smallest genetic coefficient (0.530) were No. 4 and No. 36.

[0051] Example 4 Cluster analysis to verify the function of primers

[0052] The 43 Zoysia accessions were clustered using the UPGMA method. Figure 1 As shown in the figure, 43 Zoysia germplasms were divided into 4 groups at 0.80: Class I includes 28 materials numbered 1 and 13, of which 12 are from Hainan, China, 9 are from Shandong, China, 3 are from Jiangsu, China, 1 is from Anhui, China, 1 is from Fujian, China, 1 is from Guangdong, China, and 1 is from Sri Lanka; Class II includes 13 materials numbered 30 and 31, of which 5 are from Shandong, China, 3 are from Hainan, China, 2 are from Jiangsu, China, 2 are from Guangdong, China, and 1 is from Zhejiang, China; Class III includes 1 material numbered 24 from Zhejiang, China, and Class IV has only 1 material numbered 42 from Shanghai, China.

[0053] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0054] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for identifying the relationship between different Zoysia japonica germplasms, characterized in that: The test is performed using a kit, and the process includes: 1) Use any 3 pairs of primers from the 20 pairs of EST-SSR primers screened to perform PCR amplification of EST-SSR on 43 Zoysia germplasms to obtain polymorphic bands and observe the number of alleles amplified by each pair of primers; 2) Based on the polymorphic bands of EST-SSR primers, the genetic diversity of 43 Zoysia accessions was studied using NTSYS software to determine the genetic coefficient and analyze the distance of genetic relationship; The kit includes any one or a combination of three primers shown in SEQ ID NO.1 to SEQ ID NO.40; specifically:

2. The method for identifying the relationship between different Zoysia japonica germplasms according to claim 1, characterized in that: The primer sequences are shown in SEQ ID NO.11 to SEQ ID NO.12; Upstream primer F: 5'-TGCTCAAACTTGCTTTCCTG-3', as shown in SEQ ID NO.11; Downstream primer R: 5'-CCGTGCAATCGTAGTACCAG-3', as shown in SEQ ID NO.

12.

3. The method for identifying the relationship between different Zoysia japonica germplasms according to claim 1, characterized in that: Any two sets of primers among the primers described in SEQ ID NO.1 to SEQ ID NO.10 and SEQ ID NO.13 to SEQ ID NO.40.

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