CN110551844B - Sugarcane cultivar genome SSR molecular marker development method and application - Google Patents

Abstract

The object of the present invention lies in a kind of whole genome data of sugarcane cultivar R570 haploid composed of 4460 BAC library fragments, and the SSR marker primers designed, synthesized and verified by using the primitive type with high polymorphism, and the developed primers should be It has the advantages of stable amplification results, clearly distinguishable electrophoresis bands, and high polymorphism sites, and can be widely used in the analysis of genetic diversity of sugarcane cultivars, the identification and protection of new varieties, and the construction of DNA fingerprints and genetic linkage maps.

Description

A kind of sugarcane cultivar genome SSR molecular marker development method and application

technical field

The invention belongs to the technical field of sugarcane molecular breeding, and relates to the development and application of SSR markers based on sugarcane whole genome data.

Background technique

Sugarcane (Sacchrum spp. Hybrid) is an important C4 plant with strong adaptability, high biomass, high photosynthesis efficiency, continuous planting for many years and a low-carbon crop with more CO2 fixation. It is an important sugar crop in the world. (80% of the world's total sugar) and bioenergy crops (40% of the world's bioethanol). In 1887, Soltwedel and J B Harrison, J R Bovell found that sugarcane seeds could produce seedlings in Java and Barbados, West India respectively, which opened the history of sexual hybridization of sugarcane (Luo Junsu 1992). The modern sugarcane cultivar is an interspecific cross between the ancestors of sugarcane, tropical species (Saccharum officinarum L., 2n=80, x=10) and Saccharumspontaneum L., 2n=40~128, x=8. Genuine sugarcane hybrids are produced, and the new varieties bred are modern sugarcane cultivars. Due to the complex genetic background of high polyploidy and aneuploidy of sugarcane cultivars, and the complexity exceeds that of most or all other crops, the genetics, breeding and genome sequencing of sugarcane are facing enormous difficulties (Piperidis et al. . 2010; Wang et al. 2017).

Because Simple Sequence Repeats (SSR) are highly polymorphic, widely distributed in eukaryotic genomes (Piperidis et al. 2010; Wang et al. 2017), and randomly distributed (Smith and Devey) 1994), but prefers low-repetition, gene-rich regions (Morgante et al. 2002). Due to the high rate of errors in DNA replication at SSR sites, a large amount of variation in length can be generated within or between species (Michael Klintschar et al. 2004). Good SSR molecular markers can be widely used in various animal and plant species fingerprint identification, genetic diversity analysis, genetic map construction and genetic location or analysis of important traits (genes). However, compared with other model crops such as grasses, sugarcane SSR molecular marker development and genetic linkage map construction are relatively backward, and related domestic and foreign reports are relatively few. Compared with other grass crops, sugarcane has developed SSR markers with few markers and low polymorphisms, which cannot meet the requirements of sugarcane molecular marker-assisted breeding and genetic mapping. In recent years, the application of SSR molecular markers in sugarcane cultivars has also been gradually expanded, but the number of SSR molecular markers publicly available in sugarcane cultivars is limited. Molecular labeling is not possible. However, the traditional SSR marker development method has many shortcomings, which is labor-intensive, material-intensive, and inefficient, especially for polyploid sugarcane, which is more difficult to develop.

Based on the high gene collinearity and sequence conservation between sugarcane and sorghum, and the haploid genome of sugarcane is close to the size of sorghum genome. More recently, Garsmeur et al. (2008) used whole-genome analysis (WGPTM) to align the BACs of the R570 sugarcane cultivar with the sorghum genome and identified an overlying sugarcane haploid genome euchromatic consisting of 4660 sugarcane BACs library fragments Minimal labelling path for qualitative BACs. Based on the haploid whole genome sequence of sugarcane cultivar, the invention uses bioinformatics methods to analyze the characteristics and distribution rules of SSR sites on the genome, design and synthesize SSR primers, and verify the polymorphism of SSR molecular markers, and is a sugarcane cultivar. Molecular fingerprints, genetic diversity among varieties, genetic mechanism research of important agronomic traits, and molecular breeding research provide molecular marker support.

SUMMARY OF THE INVENTION

The object of the present invention lies in a kind of whole genome data of sugarcane cultivar R570 haploid composed of 4460 BAC library fragments, using MISA software to carry out whole genome scanning, and designing, synthesizing and The validated SSR marker primers should have the advantages of stable amplification results, clearly distinguishable electrophoresis bands, and high polymorphism sites, and can be widely used in the analysis of genetic diversity of sugarcane cultivars, the identification and protection of new varieties, Construction of DNA fingerprints and genetic linkage maps.

The technical scheme of the present invention is: based on the SSR primers developed based on the whole genome haploid sequence, a total of 27,241 SSR sites are found, and the primers are designed, characterized in that a total of 50 pairs of SSR primers of TG and CA motif types are selected, The number of repetitions was at the SSR site between TG (11-69) and CA (23-38), and the primers were designed and synthesized, and 20 pairs of primers were screened. The sequence is shown in SEQ ID No.1-40 .

The application of the above-mentioned SSR core primer set in sugarcane genetic diversity analysis, new variety identification and protection, DNA fingerprint map and genetic linkage map construction.

Beneficial effects of the present invention: the 20 pairs of SSR core primers screened out by the present invention are mainly SSR primers of TG primitive and AG primitive type, with wider coverage, rich polymorphism information and clear and easy band pattern in sugarcane cultivars Interpretation, and suitable for polyacrylamide gel electrophoresis and capillary electrophoresis detection platform detection and analysis, mainly used for sugarcane genetic diversity analysis, variety identification and protection, DNA fingerprinting and genetic linkage map construction, not only for the protection of breeders. Legitimate rights and interests, and combating the spread and sale of counterfeit sugarcane varieties are also conducive to promoting the improvement of the level of sugarcane genetics and breeding and the development and growth of the sugarcane industry.

Description of drawings

Fig. 1 is the electrophoretic pattern of PCR amplification on 4 tested sugarcane materials of some SSR primers, wherein lanes 1-4, 5-8, 9-12, 13-16 are primers FAFUR-S1, FAFUR-S2, FAFUR- S3, FAFUR-S4.

Figure 2 is the electrophoresis map of PCR amplification of SSR primer (FAFUR-S32) on 24 tested sugarcane materials, where M: represents 50 bp Marker.

Figure 3 is the UPGMA cluster analysis of 24 Saccharum materials based on 20 pairs of SSR molecular markers.

Detailed ways

1. Development of genome-wide SSR primers for sugarcane

1.1 Acquisition of sugarcane whole genome sequencing data

Whole-genome data of sugarcane cultivar R570 were obtained through the public database of the EMBL-European Institute of Bioinformatics, accession number ERZ 654945, or also at the Sugarcane Genome Center of the French Agricultural Research Institute (http://sugacane-genome.cirad. fr).

1.2 The search of SSR sequences in the whole genome sequence of sugarcane and the design of SSR primers

In this study, the MISA (Microsatellite identification tool) software was used to scan the genome sequence of sugarcane cultivars. The software was downloaded from http://pgrc.ipk-gatersleben.de/misa/. The parameters were set in the configuration file, and the nucleotide repeat motif ( The motifs are mononucleotide repeats MDRs, dinucleotide repeats DNRs, trinucleotide repeats TNRs, tetranucleotide repeats TtNRs, pentanucleotide repeats PNRs, and hexanucleotide repeats HNRs. 10, 12, 15, 16, 15, 18. The primers are designed by truncating 200 bp sequences on both sides of the SSR site. The MISA software also provides an interface tool with the batch design primer Primer3, which can convert the SSR sequences identified by MISA into the format required by Primer3, thereby facilitating batch design of primers. Primers were designed online with Primer3 (http://frodo.wi.mit.edu/primer3/). The primer design parameters are: primer length: 18-28bp; annealing temperature: 55-65℃; amplicon size: 100-500 bp; GC content: 45-65%.

1.3 Screening of genome-wide SSR primers in sugarcane cultivars

4 and 24 Saccharum materials were selected to detect the amplification efficiency of SSR markers and their polymorphisms in the population. The 50 pairs of primers synthesized were preliminarily amplified in the genomic DNA of 4 materials. According to the amplification results, primers with stable amplification results, high specificity and rich polymorphisms were screened out. The PCR reaction system was 25 μL, including 2.0 μL of 25 ng/μL DNA sample, 2.5 μL of 10× PCR buffer (Mg ²⁺ plus), 1.2 μL of 25 mmol L ^-1 dNTPs, 0.5 μL and 0.5 U of each 10 mol L ^-1 primer. μL ^-1 Taq enzyme 0.1 μL, and finally make up 25 μL with ddH ₂ O. The PCR amplification program was pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 65°C for 30 s, extension at 72°C for 30 s, for a total of 10 cycles, the annealing temperature was reduced by 0.7°C for each cycle; denaturation at 94°C for 30 s, 55 Annealing at ℃ for 30 s, extension at 72 ℃ for 30 s, a total of 25 cycles; the final extension at 72 ℃ for 7 min, and storage at 4 ℃. Reagents such as Taq enzyme and dNTP were purchased from Beijing Kangwei Century Biotechnology Co., Ltd. All PCR products were separated in a 6% polyacrylamide gel, electrophoresed on a DNA analyzer (Beijing Liuyi, DYCZ-20C) under a constant pressure of 160 V for 1.5 h, and after electrophoresis, a nucleic acid dye ( GelStain, purchased from Beijing Quanshijin Biotechnology Co., Ltd., item number: GS101-01), was stained, photographed and preserved by soaking method. If a 100-350bp amplification product is detected, it is an effective amplification primer. If there are different allele amplifications among the four sugarcane materials, it is regarded as a primer with high polymorphism.

1.4 Reading of polymorphic site data

SSR site: add an underscore to the primer name followed by the size of the amplified fragment, for example: EST40_152 counts the target fragment. Indicates that the primer EST40 has a polymorphic fragment at the position of 150 bp, and the effective fragment range is from 100 bp to 500 bp. "1" indicates the presence, "0" indicates the absence, and "-" indicates that the band is missing.

Example 1:

A total of 4,460 sequences were downloaded from the Sugarcane Genome Center of the French Agricultural Research Institute (http://sugacane-genome.cirad.fr) using the above method, 27,241 SSR sites were found, and 22,932 pairs of SSR primers were successfully designed. The specific operations are as follows :

From 22932 pairs of designed primers, SSR primers with TG and CA motif types were searched, 50 pairs were randomly selected, and 4 sugarcane materials (cultivar R570, cultivar ROC1, tropical species LA purple and cut hand dense) were used. species SES208) to verify the amplification efficiency (see Table 1). The amplification results showed that: a total of 45 pairs of primers could amplify clear amplification bands, the remaining 5 pairs of primers had no amplification bands or the amount of amplification products was weak, and another 35 pairs of primers showed more than 4 materials. The polymorphism rate was 70% (35/50). Among them, there were 28 pairs of TG repeat primers and 7 pairs of AG repeat primers. Fig. 1 is the electrophoresis pattern of PCR amplification on 4 tested sugarcane materials of some SSR primers, lanes 1-4, 5-8, 9-12, 13-16 are primers FAFUR-S1, FAFUR-S2, FAFUR-S3 respectively , FAFUR-S4 amplification results.

Table 1 Information on the four tested sugarcane germplasms

Example 2:

In order to further verify the polymorphism of the SSR primer pairs identified in this study, 20 pairs of SSR primers with higher polymorphism were selected from the 35 pairs of primers screened above, and 18 backbone parents in my country (their bloodlines came from tropical species, 2-4 species of D. scuti, 2-4 species of wild species and Indian species) (see Table 2), 2 ancestors of sugarcane (S. S. 208 S. S. 208 and D. LA purple) and 4 world species Genetic diversity analysis and polymorphism evaluation of SSR primers were performed on important sugarcane cultivars (LCP85-384, R570, ROC 16 and ROC 22). The results showed that 20 pairs of primers showed obvious polymorphisms in 24 sugarcane experimental materials. A total of 95 alleles were amplified, and each pair amplified 1-7 alleles, and each pair of primers amplified on average. 4.75 alleles were obtained. Figure 2 shows the electrophoretic patterns of PCR amplification of 2 SSR primers on 24 tested sugarcane materials, and the data of the electrophoretic patterns is read to establish a 0-1 data matrix. At the same time, UPGMA software was used to analyze the data matrix, and the phylogenetic tree was generated after cluster analysis. The results are shown in Figure 3.

It can be seen from Figure 3 that the genetic similarity coefficient between the tested materials is between 0.40 and 0.82, and when the genetic similarity coefficient is 0.525, the 24 sugarcane materials can be divided into 5 types, the first type contains 2 sugarcane cultivars Co1001 and Co419; the second type has 19 sugarcane materials; the third type has only 1 tropical species type LA purple; the fourth type has 1 sugarcane cultivar material CP28-11; The SES 208 was separated from other sugarcane cultivars and the tropical species (LA purple) earlier when the similarity coefficient was 0.4, indicating that the SES 208 had a distant genetic relationship with the sugarcane cultivars. According to the analysis results of Zhang Qiong et al. (2009) and others, CP28-11 has the blood relationship of tropical species (0.5), Cuoshoumi (0.125) and Indian species (0.375), and the genetic relationship is between Cuoshoumi and tropical species , the results of this study are basically consistent with the above studies. The tropical species (LA purple) was separated from other sugarcane cultivars at a similarity coefficient of 0.525, followed by the Indian species Co1001 and Co419 at a similarity coefficient of 0.551. There is also rich genetic diversity among sugarcane cultivars.

Table 2 Germplasm information of 24 sugarcane cultivated for testing

Table 3 Information table of 20 pairs of SSR primers with amplification polymorphisms

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

SEQUENCE LISTING

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Claims (2)

1. The SSR primer composition for the genome of the sugarcane cultivar is characterized by comprising 20 pairs of SSR primers with amplification polymorphism, wherein the sequences of the SSR primers are shown as SEQ ID No. 1-40.

2. Use of an SSR primer composition of claim 1 in the analysis of sugarcane cultivar genetic diversity, identification and protection of new varieties, construction of DNA fingerprinting and genetic linkage maps.

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