A SSR molecular marker primer for distinguishing the cytoplasmic types of sugarcane noble variety and cut-hand dense variety and its application

Abstract

The present invention belongs to the technical field of sugarcane molecular breeding, and specifically relates to an SSR molecular marker and application for quickly distinguishing the cytoplasmic types of noble sugarcane and cut-hand dense sugarcane, including two pairs of SSR molecular marker core primer pairs located in the sugarcane mitochondrial genome. Based on the mitochondrial genome data of noble sugarcane and cut-hand dense sugarcane, the present invention designs and synthesizes SSR marker primers, and performs polyacrylamide gel and agarose gel electrophoresis verification in 8 noble species materials, 8 cut-hand dense sugarcane materials and 10 cultivated species materials, and screens out 2 pairs of primers with stable amplification results, clear electrophoresis bands, and polymorphism between species, which can effectively distinguish the cytoplasmic types of noble sugarcane and cut-hand dense sugarcane. Agarose gel electrophoresis can also achieve the same effect, confirming that the molecular marker is easy to operate, has high repeatability and reliability, and can be effectively used for sugarcane genetic analysis and variety identification.

Description

Technical Field

[0001] The invention belongs to the technical field of sugarcane molecular breeding, and particularly relates to an SSR molecular marker for quickly distinguishing the cytoplasmic types of sugarcane noble varieties and cut-hand dense varieties and an application thereof. Background Art

[0002] The genus Saccharum includes the species Saccharum officinale ( Saccharum spontaneum )、Wild species with large stems( Saccharum robustum ) two wild species, and the noble species ( Saccharum officinarum ), Indian species ( Saccharum barberi )、Chinese species( Saccharum sinense ) and ear-eating species ( Saccharum edule ) four cultivated varieties. The nobleization theory is an important theoretical basis for sexual hybridization breeding of sugarcane and the utilization of wild germplasm resources. Most modern sugarcane cultivated varieties are the products of nobleization breeding, containing 75-85% noble species blood, 15-25% cut-hand dense species blood and 5-10% interspecific recombination, which also makes the genome of sugarcane cultivated varieties highly complex, becoming one of the most difficult genomes to decipher in the plant kingdom.

[0003] Sugarcane noble breeding is to use noble species as the female parent and cut-hand dense species as the male parent for hybridization, and use the obtained hybrid offspring as the male parent for continuous backcrossing with noble species. This process can not only pass on the high stress resistance and strong tillering genes of cut-hand dense species to the hybrid offspring, but also quickly increase the noble species blood ratio in the hybrid offspring through the unique chromosome 2n+n inheritance mode (that is, noble species are transmitted by 2n gametes, and cut-hand dense are transmitted by n gametes), effectively retaining the high-yield and high-sugar genes. Therefore, using typical noble species materials as parents for hybridization can effectively promote the process of sugarcane nobleness.

[0004] Mitochondria in eukaryotic cells are believed to originate from ancient endosymbiotic events. Their genomes are archaeal remnant genomes that are independent of the nuclear genome and are usually maternally inherited. The plant mitochondrial genome has long tended to integrate DNA from various sources through intracellular gene transfer and horizontal transfer. The genome size (from 66 kb to 11.3 Mb) and genome structure (1 to 128 circular chromosomes) show great variation among species. However, the mutation rate of the plant mitochondrial genome is much lower than that of the nuclear genome or chloroplast genome, and it is highly conserved within the species, which can be well used for the development of interspecific molecular markers. SSR markers are an ideal molecular marker with the advantages of co-dominance, high polymorphism, abundant quantity, uniform distribution, and easy detection. They are widely used in the construction of fingerprint maps, gene positioning, variety identification, and molecular marker-assisted breeding of many crops.

[0005] In recent years, SSR molecular markers have been gradually applied to sugarcane, but the number of publicly available sugarcane SSR molecular markers is limited, especially the lack of markers developed based on organelle genomes. Based on the sugarcane mitochondrial genome, the present invention uses bioinformatics to analyze the distribution characteristics and regularities of SSR, designs and synthesizes interspecific SSR primers between sugarcane cut-hand dense and noble species, verifies the polymorphism of primers through experiments, and then develops related markers, which changes the shortcomings of traditional SSR molecular marker development methods that consume manpower and material resources and are inefficient. Summary of the invention

[0006] The purpose of the present invention is to provide a set of SSR marker primers associated with the sugarcane mitochondrial genome, which are particularly suitable for sugarcane cytoplasm type identification and genetic analysis, and their applications, in view of the lack of SSR marker primers developed by scanning the sugarcane mitochondrial genome in the prior art. Specifically, the mitochondrial genome types of the noble species and the cut-hand dense species where the SSR is located are distinguished, and then the maternal genetic background source of the sugarcane is distinguished, providing technical support for sugarcane variety identification and modern sugarcane molecular breeding.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] In the first aspect, an SSR molecular marker for distinguishing the cytoplasmic types of sugarcane noble species and cut-hand dense species is provided, including two pairs of polymorphic primers, the nucleotide sequences of the primers and the positions of the primers in the sugarcane mitochondrial genome are as follows:

[0009] Primer pair 1:

[0010] SsSSR5.m1.(TATTG)14-F: 5'-CCTTGGCTTGGAAAGAGTGA-3'; SEQ ID NO.15;

[0011] SsSSR5.m1.(TATTG)14-R: 5'-GGGAAAGGATATCGATGGGT-3'; SEQ ID NO.16;

[0012] Location in the mitochondrial genome: chromosome 1 of the species.

[0013] Primer pair 2:

[0014] SsSSR18.m2.(ATAAT)17-F: 5'-TTGACCAGCATCAGGTATCG-3'; SEQ ID NO.17;

[0015] SsSSR18.m2.(ATAAT)17-R: 5'-AAAAGAGGATCGCCCCTAGT-3'; SEQ ID NO.18;

[0016] Mitochondrial genome location: chromosome 2 of the species.

[0017] The present invention also provides the use of the SSR molecular marker primers for distinguishing the cytoplasmic types of sugarcane noble species and cut-hand dense species in sugarcane genetic analysis or identification.

[0018] Preferably, the method for sugarcane genetic analysis or identification comprises the following steps:

[0019] (1) Based on the mitochondrial genome data of the sugarcane noble and saccharum officinale that had been sequenced, the Perl script in the MISA software package was used to batch scan the SSR loci of the mitochondrial genomes of the sugarcane noble and saccharum officinale.

[0020] (2) Using the SSR locus information, 150 bp of flanking sequences before and after the SSR start and end sites were extracted from the mitochondrial genome, and each SSR sequence was compared with the mitochondrial genome sequence by BLASTN. The hit sequences screened out were the candidate SSR loci in the present invention.

[0021] (3) Primers were designed on both sides of the candidate SSR locus using the batch primer design software Primer3. After the primer design was completed, the primers were extracted and the duplicate primers were removed.

[0022] (4) The designed primers were used to perform e-PCR simulation amplification on the mitochondrial genomes of the sugarcane noble variety and the cut-hand dense variety to screen for SSRs that existed in both the sugarcane noble variety and the cut-hand dense variety but had different fragment sizes. These SSRs were the interspecific SSRs between the sugarcane noble variety and the cut-hand dense variety, which could distinguish their maternal genetic backgrounds.

[0023] (5) Eight representative samples of sugarcane noble varieties, eight samples of cut-hand dense varieties, and ten samples of cultivated varieties were selected, and their genomic DNA was extracted. The extracted genomic DNA was amplified by PCR using sugarcane species-specific SSR primers to obtain the amplified products.

[0024] (6) The obtained amplified product can be verified by polyacrylamide gel electrophoresis experiment or agarose gel electrophoresis experiment, and the sugarcane noble variety or the cut-hand dense variety can be determined according to the position of the band.

[0025] Preferably, the PCR amplification reaction system is: a total system of 20 μL, including 1 μL of 50 ng / μL DNA sample, 0.5 μL of 10 μmol upstream and downstream primers, 10 μL of 2×Rapid Taq Master Mix, and 8 μL of double distilled water.

[0026] Preferably, the PCR amplification reaction procedure is:

[0027] Pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 10 s, for a total of 34 cycles; final extension at 72°C for 5 min, and storage at 4°C.

[0028] Preferably, the steps of the polyacrylamide gel electrophoresis experiment are:

[0029] Prepare 9% polyacrylamide gel; perform electrophoresis at 160V constant voltage for 150 minutes to separate PCR products; use bubble staining method for staining, and take photos and observe on a gel imaging system.

[0030] Preferably, the steps of the agarose gel electrophoresis experiment are:

[0031] Prepare 3% agarose gel; perform electrophoresis at 130V constant voltage for 30 minutes to separate PCR products; finally, take photos and observe on a gel imaging system.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention provides two pairs of SSR molecular markers for distinguishing the cytoplasmic types of the noble sugarcane variety and the cut-hand dense variety, which can clearly distinguish the position of the SSR in the mitochondrial genome, and then analyze the maternal genetic background of the noble sugarcane variety and the cut-hand dense variety, and analyze the origin and evolution process of modern sugarcane from the perspective of molecular markers, providing new ideas and basis for the evolutionary analysis and variety identification of modern sugarcane. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] 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.

[0035] Figure 1 This is the polymorphism verification of 12 pairs of candidate SSR primers in representative materials of the species Cut Hand Mi and the species Gao Gui. The odd-numbered lanes are the species Cut Hand Mi NP-X, the even-numbered lanes are the species Gao Gui Fuguo No. 1, and M is a 50 bp DNA Ladder.

[0036] Figure 2 This is the polyacrylamide gel electrophoresis result of primer SsSSR5.m1.(TATTG)14 on 26 sugarcane materials, among which 1-8 are noble varieties, 9-16 are cut-hand dense varieties, 17-26 are cultivated varieties, and M is 50 bp DNA Ladder.

[0037] Figure 3This is the polyacrylamide gel electrophoresis result of primer SsSSR18.m2.(ATAAT)17 on 26 sugarcane materials, among which 1-8 are noble varieties, 9-16 are cut-hand dense varieties, 17-26 are cultivated varieties, and M is 50 bp DNA Ladder.

[0038] Figure 4 This is the agarose gel electrophoresis result of primer SsSSR5.m1.(TATTG)14 on 24 sugarcane materials, among which 1-8 are noble species, 9-16 are cut-hand dense species, 17-24 are cultivated species, and M is 50 bp DNA Ladder.

[0039] Figure 5 This is the agarose gel electrophoresis result of primer SsSSR18.m2.(ATAAT)17 on 24 sugarcane materials, among which 1-8 are noble varieties, 9-16 are cut-hand dense varieties, 17-24 are cultivated varieties, and M is 50 bp DNA Ladder. DETAILED DESCRIPTION

[0040] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention. Example 1

[0041] 1. Searching for SSR sequences in the mitochondrial genome sequences of sugarcane noble and cut-hand dense varieties and designing and validating SSR primers

[0042] 1.1 SSR locus scanning

[0043] The Perl script in the Micro Satellite identification tool-MISA software package was used to batch scan the SSR loci of the sugarcane mitochondrial genome (https: / / sugarcane.gxu.edu.cn / sgdv3 / download). The standard for whether it is SSR is set as follows:

[0044] 1) Unit 1 should be repeated at least ten times, Unit 2 should be repeated at least seven times, Unit 3 should be repeated at least six times, Unit 4 should be repeated at least five times, Unit 5 should be repeated at least four times, and Unit 6 should be repeated at least four times;

[0045] 2) When there are two or more SSRs (including two), and the interval between adjacent SSRs does not exceed 100bp, they are combined into a composite SSR.

[0046] 1.2 Sequence extraction

[0047] The physical position of each SSR site on the mitochondrial genome sequence was calculated, and the flanking sequences of 150 bp before and after the SSR start and end sites were cut from the sugarcane genome using the SSR position information. Each SSR sequence was compared with the mitochondrial genome sequence by BLASTN (E value 1e-5), with a sequence similarity of 100% and a 100% alignment of the flanking sequence length. The hit sequences screened out were the candidate SSR sites in the present invention.

[0048] 1.3 Primer design

[0049] Primer3 software was used to design primers on both sides of the SSR locus. The parameters were set as follows: primer length 18-23 bp, optimal 20 bp, GC content between 40% and 60%, product length 100-300 bp, and the SSR locus must be included in the product sequence. After the design was completed, the primer sequence was extracted and the duplicate primers were removed.

[0050] 1.4 Primer verification

[0051] Using Electronic PCR (https: / / www.animalgenome.org / bioinfo / resources /

[0052] manuals / ePCR.html) The designed primers were simulated by electronic PCR amplification on the mitochondrial genome, and the primers that could amplify specific fragments on the mitochondrial genome were extracted as candidate SSR primers. Finally, 35 primer pairs were obtained for the noble species and 30 primer pairs were obtained for the cut-hand dense species. After that, the candidate SSR primers were compared on the mitochondrial genomes of the two sugarcane species to screen for SSRs that existed in both the noble and cut-hand dense species but had different product sizes, that is, interspecific mitochondrial SSRs between the noble and cut-hand dense species of sugarcane. A total of 12 pairs of SSR primers were obtained for subsequent verification (Table 1).

[0053]

[0054]

[0055] As shown in SEQ ID NOs. 1~24.

[0056] 2. Screening of interspecific mitochondrial SSR primers between sugarcane noble and cut-handed varieties

[0057] 2.1 Extraction of genomic DNA

[0058] Eight representative sugarcane noble varieties, eight cut-hand dense varieties, and ten cultivated varieties were selected (Table 2; variety source Lei LIU, Wang H, Li Y, et al. Genome-wide development of interspecific microsatellite markers for Saccharum officinarum and Saccharum spontaneum [J]. Journal of Integrative Agriculture, 2022, 21(11): 3230-3244.) to detect the amplification efficiency and interspecific specificity of sugarcane mitochondrial SSR markers. The CTAB method was used to extract the genomic DNA of the above sugarcane materials.

[0059]

[0060]

[0061] 2.2 PCR amplification

[0062] The synthesized primers were used to amplify the extracted sugarcane genomic DNA as a template. According to the amplification results, the primers with stable amplification results, high interspecies specificity and rich polymorphism were screened. The PCR reaction system was 20 μL, including 1 μL of 50 ng / μL DNA sample, 0.5 μL of 10 μmol upstream and downstream primers, 10 μL of 2×Rapid Taq Master Mix, and 8 μL of double distilled water (ddH2O). The PCR amplification program was 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 56℃ annealing for 15 s, and 72℃ extension for 10 s, for a total of 34 cycles; 72℃ final extension for 5 min, and storage at 4℃. 2×Rapid Taq Master Mix 10 μL was purchased from Nanjing Novozyme Biotechnology Co., Ltd., with the catalog number P222.

[0063] 2.3 Polyacrylamide gel electrophoresis

[0064] All PCR products were separated in 9% polyacrylamide gel and electrophoresed for 2h30min at a constant voltage of 160V. After electrophoresis, they were stained with 3× nucleic acid dye by bubble dyeing, and then photographed and stored on a gel imaging system. 10000*SolarRed nucleic acid dye was purchased from Beijing Solar Red Technology Co., Ltd., with the catalog number G5560.

[0065] 2.4 Agarose gel electrophoresis

[0066] All PCR products were separated in 3% agarose gel (containing 1× nucleic acid dye) and electrophoresed at a constant voltage of 130V for 30min. After the electrophoresis, they were photographed and stored on a gel imaging system.

[0067] First, NP-X and Fuguo No. 1 were selected as representative varieties to verify the polymorphism of the above 12 pairs of SSR primers ( Figure 1 ), and then selected highly polymorphic co-dominant markers for verification in 26 Saccharum materials. According to the banding of 26 Saccharum materials, two pairs of target primers (SsSSR5.m1.(TATTG)14 and SsSSR18.m2.(ATAAT)17) were finally screened out, which had clear banding patterns and obvious interspecies specificity in the mitochondrial genome of S. serrata.

[0068] The results of polyacrylamide gel electrophoresis of two pairs of primers were as follows: Figure 2 and Figure 3 From the electrophoresis diagram, we can see that there are co-dominant markers in the noble sugarcane variety and the cut-hand dense variety, that is, there are bands in both varieties, and the corresponding fragment sizes of the bands are different; only the noble type bands exist in the cultivated variety. Therefore, the two pairs of primers are SSR molecular markers that can distinguish the cytoplasmic types of the noble sugarcane variety and the cut-hand dense variety, and can distinguish the maternal lineage background of modern sugarcane cultivated varieties. Agarose gel electrophoresis can also show the same effect as polyacrylamide gel electrophoresis ( Figure 4 , Figure 5 ), which also shows the accuracy of the developed markers and the convenience of operation.

[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A SSR molecular marker primer for distinguishing the mitochondrial genome types of sugarcane noble species and cut-hand dense species, characterized in that: The SSR molecular marker primers are primer pair 1 or primer pair 2; The nucleotide sequence of the primer pair 1 is: 5'-CCTTGGCTTGGAAAGAGTGA-3', as shown in SEQ ID NO: 15; 5′-GGGAAAGGATATCGATGGGT-3′, as shown in SEQ ID NO: 16; The nucleotide sequence of the primer pair 2 is: 5'-TTGACCAGCATCAGGTATCG-3', as shown in SEQ ID NO: 17; 5′-AAAAGAGGATCGCCCCTAGT-3′, as shown in SEQ ID NO:

18.

2. The SSR molecular marker primer according to claim 1, characterized in that The primer pair 1 is located at chromosome 1 of the species of Pseudomonas aeruginosa in the mitochondrial genome; the primer pair 2 is located at chromosome 2 of the species of Pseudomonas aeruginosa in the mitochondrial genome.

3. Use of the SSR molecular marker primers described in claim 1 or 2 in genetic analysis or identification of sugarcane noble and cut-hand dense varieties.

4. The use according to claim 3, characterized in that: The application comprises the following steps: (1) extracting genomic DNA of sugarcane varieties, and performing PCR amplification on the extracted genomic DNA using the SSR molecular marker primers to obtain an amplified product; (2) Performing polyacrylamide gel electrophoresis or agarose gel electrophoresis on the amplified product obtained in step (1) to determine whether it is a noble variety of sugarcane or a cut-hand dense variety based on the position of the bands.

5. The use according to claim 4, characterized in that: The PCR amplification reaction system is as follows: a total system of 20 μL, including 1 μL of 50 ng / μL DNA sample, 0.5 μL of 10 μmol upstream and downstream primers, 10 μL of 2×Rapid Taq Master Mix, and 8 μL of double distilled water.

6. The use according to claim 4, characterized in that: The procedure of the PCR amplification reaction is: Pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 10 s, for a total of 34 cycles; final extension at 72°C for 5 min, and storage at 4°C.

7. The use according to claim 4, characterized in that: The steps of the polyacrylamide gel electrophoresis experiment are: Prepare 9% polyacrylamide gel; perform electrophoresis at 160V constant voltage for 150 minutes to separate PCR products; use bubble staining method for staining, and take photos and observe on a gel imaging system.

8. The use according to claim 4, characterized in that: The steps of the agarose gel electrophoresis experiment are: Prepare 3% agarose gel; perform electrophoresis at 130V constant voltage for 30 minutes to separate PCR products; finally, take photos and observe on a gel imaging system.

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