CN110551844B - Sugarcane cultivar genome SSR molecular marker development method and application - Google Patents
Sugarcane cultivar genome SSR molecular marker development method and application Download PDFInfo
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Abstract
The invention aims to develop an SSR marker primer based on R570 haploid complete genome data of sugarcane cultivars consisting of 4460 BAC library segments, which is designed, synthesized and verified by using a high-polymorphism primitive type, has the advantages of stable amplification result, clear and distinguishable electrophoretic bands and high polymorphic sites, and can be widely applied to the genetic diversity analysis, new variety identification and protection, and the construction of DNA fingerprint and genetic linkage map of sugarcane cultivars.
Description
Technical Field
The invention belongs to the technical field of sugarcane molecular breeding, and relates to SSR marker development and application based on sugarcane full genome data.
Background
Sugarcane (Sacchrum spp. Hybrid) is an important C4 plant, has strong adaptability, high biomass and high photosynthesis efficiency, can continuously plant and fix low-carbon crops with more than CO2 for many years, and is one of important sugar crops (accounting for 80 percent of the total sugar amount in the world) and biological energy crops (accounting for 40 percent of the biological ethanol in the world). In 1887, Soltwedel and J B Harrison, J R Bovell found sugarcane seeds that produced seedlings in java and west indian barbus laboratories, respectively, opening the history of sexual crosses of sugarcane (lujun 39573. The modern sugarcane cultivar is produced by interspecific hybridization of sugarcane ancestor tropical species (Saccharum officinarum L., 2n =80, x =10) and secant seed (Saccharum spontanium L., 2n = 40-128, x =8), and a sugarcane hybrid in a real sense is produced, and the bred new variety is the modern sugarcane cultivar. Due to the complex genetic background of high polyploidy and aneuploidy of sugarcane cultivars, and the complexity exceeding that of most and even all other crops, sugarcane genetics, breeding, genomic sequencing, etc. face enormous difficulties (Piperidis et al 2010; Wang et al 2017).
Since Simple Sequence Repeats (SSRs) are highly polymorphic, are widely distributed over the genome of eukaryotes (Piperidis et al 2010; Wang et al 2017), and are randomly distributed (Smith and Devey 1994), but are more biased toward low-repeat, gene-rich regions (Morgante et al 2002). Because the SSR locus has high error rate during DNA replication, a great amount of length variation (Michael Klintschar et al 2004) can be generated in or among species, and the SSR molecular marker with high polymorphism and good repeatability can be developed and screened out and can be widely applied to the fields of variety fingerprint identification, genetic diversity analysis, genetic map construction, genetic positioning or analysis of important characters (genes) and the like of various animals and plants. However, compared with other mode crops such as gramineous crops and the like, the development of the SSR molecular markers of the sugarcane and the construction of genetic linkage maps are relatively laggard, and related reports at home and abroad are relatively few. Compared with other gramineous crops, the developed SSR markers of the sugarcane have fewer markers and low polymorphism, and cannot meet the requirements of sugarcane molecular marker-assisted breeding, genetic mapping and other works. In recent years, the application of the SSR molecular markers to sugarcane cultivars is gradually developed, but the number of the SSR molecular markers of the sugarcane cultivars which can be publicly obtained at present is limited, and large-scale development of the SSR molecular markers cannot be carried out because genome sequence determination of the sugarcane cultivars is not completed at present. The traditional SSR marker development method has the defects of manpower and material resource consumption and low efficiency, and particularly for the polyploid sugarcane, the development difficulty is increased.
Based on the existence of higher gene collinearity and sequence conservation of sugarcane and sorghum, the haploid genome of sugarcane is close to the genome size of sorghum. Recently, Garsmeur et al (2008) aligned BACs of R570 sugarcane varieties with sorghum genomes using whole genome analysis (WGPTM) techniques, determined a minimal marker pathway for BACs consisting of 4660 sugarcane BACs library fragments covering sugarcane haploid genome euchromatin. The invention is based on the haploid whole genome sequence of the sugarcane cultivar, utilizes a bioinformatics method to analyze the characteristics and the distribution rule of SSR sites on a genome, designs and synthesizes SSR primers, verifies the polymorphism of SSR molecular markers, and provides molecular marker support for the research on molecular fingerprint, genetic diversity among varieties and genetic mechanism of important agronomic characters of the sugarcane cultivar and the development of molecular breeding research.
Disclosure of Invention
The invention aims to provide sugarcane cultivar R570 haploid whole genome data consisting of 4460 BAC library segments, which is characterized in that MISA software is utilized to carry out whole genome scanning, SSR marker primers which are designed, synthesized and verified according to the reported primitive type with high polymorphism are developed, the developed primers have the advantages of stable amplification result, clear and distinguishable electrophoretic bands and high polymorphic sites, and can be widely applied to the analysis of sugarcane cultivar genetic diversity, the identification and protection of new varieties and the construction of DNA fingerprint and genetic linkage maps.
The technical scheme of the invention is as follows: the method comprises the steps of finding 27241 SSR sites based on SSR primers developed by a whole genome haploid sequence, designing the primers, and is characterized in that 50 pairs of SSR primers with TG and CA primitive types are selected, the SSR sites with the repetition times between TG (11-69) and CA (23-38) are respectively selected, the primers are designed and synthesized, and 20 pairs of primers are screened, wherein the sequences are shown as SEQ ID No. 1-40.
The SSR core primer group is applied to the aspects of sugarcane genetic diversity analysis, new variety identification and protection, and DNA fingerprint and genetic linkage map construction.
The invention has the beneficial effects that: the 20 pairs of SSR core primers screened by the invention mainly comprise SSR primers of TG primitive and AG primitive types, have wider coverage, have rich polymorphism information in sugarcane cultivars, clear band types and are easy to interpret, are suitable for polyacrylamide gel electrophoresis and capillary electrophoresis detection platform detection analysis, are mainly used for sugarcane genetic diversity analysis, variety identification and protection, DNA fingerprint and genetic linkage map construction, are beneficial to protecting legal rights and interests of breeders, fighting against the propagation and sale of counterfeit sugarcane varieties, and are also beneficial to promoting the improvement of sugarcane genetic breeding level and the development and the growth of sugarcane industry.
Drawings
FIG. 1 is a PCR amplification electropherogram of a portion of SSR primers on 4 sugarcane materials tested, in which lanes 1-4,5-8, 9-12, 13-16 are primers FAFUR-S1, FAFUR-S2, FAFUR-S3, and FAFUR-S4, respectively.
FIG. 2 is a PCR amplification electropherogram of SSR primers (FAFUR-S32) on 24 sugarcane materials tested, wherein M represents 50bp Marker.
Fig. 3 is a UPGMA cluster analysis of 24 saccharum materials based on 20 pairs of SSR molecular markers.
Detailed Description
1. Development of sugarcane whole genome SSR primers
1.1 acquisition of sugarcane Whole genome sequencing data
Sugarcane cultivar R570 complete genome data was obtained from EMBL-European bioinformatics institute public database, accession number ERZ 654945, or alternatively at the French institute of agriculture, sugarcane genome center (http:// sugar-genome.
1.2 search of SSR sequence in sugarcane whole genome sequence and design of SSR primer
The present study applied MISA (Microsallite identification tool) software to scan genomic sequences of sugarcane cultivars, which was downloaded from http:// pgrc. ipk-gateslide. de/misa/, with parameters set in the profile, nucleotide repeat motifs (motifs) being mono (monomeric repeat MDRs), di (dinucleotide repeats DNRs), tri (trinucleatide repeats TNRs), tetra (tetranucleotide repeats TtNRs), penta (tanducteotide repeats PNRs), and hexa (heterocyclic repeat HNRs), with the most sequence lengths being 10, 12, 15, 16, 15, 18, respectively. 200 bp sequences on two sides of an SSR locus are intercepted to design primers, MISA software also provides an interface tool with a batch design Primer3, the SSR sequences identified by the MISA can be converted into a format required by a Primer3, and therefore the primers can be conveniently designed in batches. Primers were designed on-line using Primer3 (http:// frodo. wi. mit. edu/Primer3/), with Primer design parameters: primer length 18-28 bp, annealing temperature 55-65 ℃; amplicon size: 100-500 bp, GC content: 45 to 65 percent.
1.3 screening of Whole genome SSR primers for sugarcane cultivars
4 and 24 parts of Saccharum material were selected, respectively, for detecting the amplification efficiency of SSR markers and their polymorphisms in the population. The 50 pairs of synthesized primers are subjected to preliminary amplification on the genomic DNA of 4 parts of materials, and primers with stable amplification result, high specificity and rich polymorphism are screened according to the amplification result. PCR reaction 25. mu.L, wherein 2.0. mu.L of 25 ng/. mu.L DNA sample contains 10 XPCR buffer (Mg)2+ plus) 2.5 μL、25 mmol L -1 dNTPs 1.2μL、10 mol L-1Primers were 0.5. mu.L and 0.5U. mu.L, respectively-1Taq enzyme 0.1. mu.L, finally ddH2O make up to 25. mu.L. The PCR amplification program comprises pre-denaturation at 94 ℃ for 5 min, denaturation at 94 ℃ for 30S, annealing at 65 ℃ for 30S and extension at 72 ℃ for 30S, 10 cycles, annealing temperature reduction of 0.7 ℃ per cycle, denaturation at 94 ℃ for 30S, annealing at 55 ℃ for 30S and extension at 72 ℃ for 30S, 25 cycles, extension at 72 ℃ for 7 min and storage at 4 ℃. Taq enzyme, dNTP and other reagents are purchased from Beijing kang, a century Biotechnology Co., Ltd. All PCR products were separated in 6% polyacrylamide gel, electrophoresed for 1.5 hours under 160V constant pressure on a DNA analyzer (DYCZ-20C) and stained, photographed and stored by a dye-bath method using nucleic acid dye (GelStain, available from Beijing Quanyujin Biotechnology Co., Ltd., product number: GS 101-01) after electrophoresis. If the amplification products of 100-350bp are detected, the primers are effective amplification primers, and if different allelic genes are amplified among 4 saccharum materials, the primers with high polymorphism are considered.
1.4 reading of polymorphic site data
SSR sites: the size of the amplified fragment is followed by the primer name followed by an underline, for example: EST40_152 was counted for the target fragment. The EST40 primer has a polymorphism fragment at the position of 150bp, the effective fragment range is 100 bp to 500 bp, 1 represents the existence, 0 represents the nonexistence, and the '-' represents the deletion of band.
Example 1:
the method is applied to download a sequence 4460 from a genome center (http:// sugar-genome. cirad. fr) of sugarcane of the French agriculture institute in total, 27241 SSR sites are found, and SSR primer 22932 pairs are successfully designed, and the specific operation is as follows:
from 22932 pairs of primers designed, SSR primers of TG and CA motif types were searched, 50 pairs of them were randomly selected, and amplification efficiency verification was performed using 4 Saccharum materials (cultivar R570, cultivar ROC1, tropical species LA purple and Customidium secret SES 208) (see Table 1). The amplification result shows that: a total of 45 primers can amplify clear amplification bands, the other 5 primers have no amplification bands or have weak amplification product amount, and the other 35 primers show polymorphism on 4 materials, and the polymorphism rate is 70 percent (35/50), wherein 28 pairs of primers of TG repeat type and 7 pairs of primers of AG repeat type. FIG. 1 shows the PCR amplification electropherograms of the SSR primers on 4 sugarcane materials tested, and lanes 1-4,5-8, 9-12, and 13-16 show the results of the amplification with primers FAFUR-S1, FAFUR-S2, FAFUR-S3, and FAFUR-S4, respectively.
Table 14 test sugarcane germplasm information
Example 2:
in order to further verify the polymorphism of the SSR primer pair identified by the research, 20 pairs of SSR primers with higher polymorphism are selected from the 35 pairs of primers selected from the above, and genetic diversity analysis and polymorphism evaluation of SSR primers are carried out on 18 backbone parents (the bloods margin of which are from tropical species, cleft fingerling, large-stem wild species and 2-4 species of Indian species) (see table 2), 2 sugarcane ancestors (cleft fingerling SES 208 and tropical species LA purple) and 4 worldwide important sugarcane cultivars (LCP 85-384, R570, ROC 16 and ROC 22). The results show that: the 20 pairs of primers show obvious polymorphism on 24 sugarcane experimental materials, 95 alleles are obtained through co-amplification, 1-7 alleles are amplified in each pair, and 4.75 alleles are amplified in each pair of primers on average. FIG. 2 shows the PCR amplification electropherograms of 2 SSR primers on 24 sugarcane materials to be tested, and the electropherograms are subjected to data reading to establish a 0-1 data matrix. Meanwhile, UPGMA software is used for analyzing the data matrix, and after clustering analysis, the affinity relationship evolutionary tree is generated, and the result is shown in figure 3.
As can be seen from FIG. 3, the genetic similarity coefficients between the test materials are distributed between 0.40 and 0.82, and when the genetic similarity coefficient is 0.525, 24 sugarcane materials can be divided into 5 types, wherein the first type comprises 2 sugarcane cultivars Co1001 and Co 419; the second type has 19 sugarcane materials; the third type has only 1 tropical type LA purple; the fourth has 1 sugarcane cultivar material CP 28-11; the fifth species has 1. sup. secant density species SES 208, where SES 208 separates earlier from other sugarcane cultivars and tropical species (LA purple) at a similarity coefficient of 0.4, indicating that the secant density species has a far-off relationship with sugarcane cultivars. The CP28-11 has a bloody border relationship of tropical species (0.5), cleft hand density (0.125) and indian species (0.375) according to the analysis results of the zhangong et al (2009) and the like, and the genetic relationship is between the cleft hand density and the tropical species, and the results of the present study are substantially consistent with the above-mentioned study. While the tropical species (LA purple) was separated from other sugar cane cultivars with a similarity coefficient of 0.525, followed by indian species Co1001 and Co419 with a similarity coefficient of 0.551, the results showed that indian species genetic relationships were intermediate between the tropical species and sugar cane cultivars, and also had abundant genetic diversity.
Table 224 germplasm information of test sugarcane cultivars
TABLE 320 SSR primer information Table for polymorphisms with amplification
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Fujian agriculture and forestry university
<120> development method and application of SSR molecular marker of sugarcane cultivar genome
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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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| CN108504771A (en) * | 2018-06-22 | 2018-09-07 | 福建农林大学 | A method of exploitation sugarcane SSR marker and identification Sugarcane Breeding affiliation |
| CN108841983A (en) * | 2018-06-22 | 2018-11-20 | 福建农林大学 | A kind of SSR primer of sugarcane overall length transcript profile data large-scale development |
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| CN108504771A (en) * | 2018-06-22 | 2018-09-07 | 福建农林大学 | A method of exploitation sugarcane SSR marker and identification Sugarcane Breeding affiliation |
| CN108841983A (en) * | 2018-06-22 | 2018-11-20 | 福建农林大学 | A kind of SSR primer of sugarcane overall length transcript profile data large-scale development |
Non-Patent Citations (3)
| Title |
|---|
| A mosaic monoploid reference sequence for the highly complex genome of sugarcane;Olivier Garsmeur等;《NATURE COMMUNICATIONS》;20180706;第9卷;文章编号2638 * |
| Highly Polymorphic Microsatellite DNA Markers for Sugarcane Germplasm Evaluation and Variety Identity Testing;Pingwu Liu等;《Sugar Tech》;20110622;第13卷(第2期);第129–136页 * |
| 甘蔗栽培种单倍体基因组SSR位点的发掘与应用;王恒波等;《作物学报》;20200115;第46卷(第4期);第631-642页 * |
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