InDel molecular marker primer pair combination for constructing fresh soybean DNA fingerprint spectrum

Abstract

The invention provides a primer pair combination of an InDel molecular marker combination for constructing a DNA (Deoxyribonucleic Acid) fingerprint spectrum of fresh soybeans' Tonghuang No.1 '. The DNA fingerprint spectrum consists of 31 InDel molecular markers which are more than or equal to 30bp and are distributed in 15 chromosomes; and primer pairs corresponding to the 31 molecular markers are as shown in SEQ ID NO. 1 to 62. The invention also provides an application for identifying fresh soybeans' TonghuaoNo.1 '. Identifying true and false hybrids of the F1 filial generation of which the parents are fresh soybeans' Tonghuang No.1 'and' Williams 82 '; the method is used for soybean germplasm resource genetic diversity and soybean variety resource evolutionary tree analysis. The primer pair combination of the InDel molecular marker combination is used for solving the problems of genetic diversity analysis, identification and germplasm resource utilization.

Description

Technical Field

[0001] The invention belongs to the technical field of DNA fingerprints, and in particular relates to a primer pair combination of an InDel molecular marker combination for constructing a DNA fingerprint of fresh soybean "Tongsu No. 1" and an application thereof. Background Art

[0002] At present, the cultivated soybeans planted on a large scale are the result of long-term selection and systematic improvement based on wild soybeans. They can be divided into fresh-eating soybeans and grain-eating soybeans due to their different harvesting times. Among them, fresh-eating soybeans are harvested between the growth period R6 and R7 and eaten as fresh vegetables. They are soft in texture, delicious in flavor, rich in nutrition, and high in vitamins, sucrose and starch, but low in indigestible oligosaccharides and anti-nutrients.

[0003] Traditional breeding methods are mainly based on morphological markers for selection, while morphological characteristics are the result of the combined effects of genetics and the environment, and are easily affected by environmental changes, which has great limitations. With the development of molecular biology, molecular marker technology has become an important means of genetic improvement of crops. The combined use of morphological markers and molecular markers will play a huge role in the genetic improvement of crops and the utilization of germplasm resources. Therefore, genetic difference analysis of soybeans at the genome level will be of great significance to soybean population genetics research, marker-assisted breeding and gene identification.

[0004] In recent years, a number of edible soybean varieties have been developed through the combination of physiological breeding and molecular-assisted screening. The development of molecular markers to analyze the genetic diversity of edible soybeans is not only conducive to fully utilizing and tapping into excellent germplasm resources, but also has important guiding significance for broadening the genetic background and screening excellent varieties (lines) for application in edible soybean breeding and production. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a primer pair combination of an InDel molecular marker combination for constructing a DNA fingerprint map of fresh soybean "Tongsu No. 1" and its application, in view of the deficiencies of the above-mentioned prior art. The primer pair combination of the InDel molecular marker combination is used for genetic diversity analysis, identification and germplasm resource utilization.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: a primer pair combination of an InDel molecular marker combination for constructing a DNA fingerprint of fresh soybean "Tongsu No. 1", wherein the DNA fingerprint of the fresh soybean "Tongsu No. 1" is composed of 31 InDel molecular markers ≥30bp distributed in 15 chromosomes;

[0007] The nucleotide sequence of the first primer pair used to amplify the Gm02_687775_687827 molecular marker is shown in SEQ ID NO. 1-2;

[0008] The nucleotide sequence of the second primer pair used to amplify the Gm02_38671726_38671769 molecular marker is shown in SEQ ID NO. 3-4;

[0009] The nucleotide sequence of the third primer pair used to amplify the Gm02_46235870_46235916 molecular marker is shown in SEQ ID NOs. 5 to 6;

[0010] The nucleotide sequence of the fourth primer pair used to amplify the Gm02_48831944_48831999 molecular marker is shown in SEQ ID NOs. 7 to 8;

[0011] The nucleotide sequence of the fifth primer pair used to amplify the Gm02_50061608_50061609 molecular marker is shown in SEQ ID NOs. 9 to 10;

[0012] The nucleotide sequence of the sixth primer pair used to amplify the Gm03_3866902_3866939 molecular marker is shown in SEQ ID NOs. 11 to 12;

[0013] The nucleotide sequence of the seventh primer pair used to amplify the Gm03_40000161_40000198 molecular marker is shown in SEQ ID NOs. 13 to 14;

[0014] The nucleotide sequence of the eighth primer pair used to amplify the Gm04_1014450_1014481 molecular marker is shown in SEQ ID NOs. 15-16;

[0015] The nucleotide sequence of the ninth primer pair used to amplify the Gm04_47260900_47260937 molecular marker is shown in SEQ ID NOs. 17 to 18;

[0016] The nucleotide sequence of the 10th primer pair used to amplify the Gm07_20149006_20149007 molecular marker is shown in SEQ ID NOs. 19 to 20;

[0017] The nucleotide sequence of the 11th primer pair used to amplify the Gm08_44385355_44385399 molecular marker is shown in SEQ ID NOs. 21-22;

[0018] The nucleotide sequence of the 12th primer pair used to amplify the Gm09_12267638_12267680 molecular marker is shown in SEQ ID NOs. 23-24;

[0019] The nucleotide sequence of the 13th primer pair used to amplify the Gm09_33743573_33743612 molecular marker is shown in SEQ ID NOs. 25-26;

[0020] The nucleotide sequence of the 14th primer pair used to amplify the Gm09_37146920_37146952 molecular marker is shown in SEQ ID NOs. 27-28;

[0021] The nucleotide sequence of the 15th primer pair used to amplify the Gm10_21414835_21414878 molecular marker is shown in SEQ ID NOs. 29-30;

[0022] The nucleotide sequence of the 16th primer pair used to amplify the Gm11_9027110_9027111 molecular marker is shown in SEQ ID NOs. 31-32;

[0023] The nucleotide sequence of the 17th primer pair used to amplify the Gm13_28033266_28033267 molecular marker is shown in SEQ ID NOs. 33-34;

[0024] The nucleotide sequence of the 18th primer pair used to amplify the Gm13_32370867_32370901 molecular marker is shown in SEQ ID NOs. 35-36;

[0025] The nucleotide sequence of the 19th primer pair used to amplify the Gm13_34120611_34120657 molecular marker is shown in SEQ ID NOs. 37-38;

[0026] The nucleotide sequence of the 20th primer pair used to amplify the Gm13_42180045_42180091 molecular marker is shown in SEQ ID NOs. 39 to 40;

[0027] The nucleotide sequence of the 21st primer pair used to amplify the Gm14_7027282_7027313 molecular marker is shown in SEQ ID NOs. 41-42;

[0028] The nucleotide sequence of the 22nd primer pair used to amplify the Gm16_3283288_3283319 molecular marker is shown in SEQ ID NOs. 43-44;

[0029] The nucleotide sequence of the 23rd primer pair used to amplify the Gm16_32569599_32569630 molecular marker is shown in SEQ ID NOs. 45-46;

[0030] The nucleotide sequence of the 24th primer pair used to amplify the Gm16_35889588_35889638 molecular marker is shown in SEQ ID NOs. 47-48;

[0031] The nucleotide sequence of the 25th primer pair used to amplify the Gm16_37504818_37504854 molecular marker is shown in SEQ ID NOs. 49-50;

[0032] The nucleotide sequence of the 26th primer pair used to amplify the Gm17_36063299_36063330 molecular marker is shown in SEQ ID NOs. 51-52;

[0033] The nucleotide sequence of the 27th primer pair used to amplify the Gm17_39292061_39292062 molecular marker is shown in SEQ ID NOs. 53-54;

[0034] The nucleotide sequence of the 28th primer pair used to amplify the Gm17_39474344_39474381 molecular marker is shown in SEQ ID NOs. 55-56;

[0035] The nucleotide sequence of the 29th primer pair used to amplify the Gm18_2089656_2089688 molecular marker is shown in SEQ ID NOs. 57-58;

[0036] The nucleotide sequence of the 30th primer pair used to amplify the Gm19_47148869_47148912 molecular marker is shown in SEQ ID NOs. 59 to 60;

[0037] The nucleotide sequence of the 31st primer pair used to amplify the Gm20_47615536_47615570 molecular marker is shown in SEQ ID NOs. 61-62.

[0038] The present invention also provides the use of the primer pair combination of the above-mentioned InDel molecular marker combination for constructing the DNA fingerprint of the fresh soybean "Tongsu No. 1", and the primer pair combination of the InDel molecular marker combination is used to identify the fresh soybean "Tongsu No. 1".

[0039] The present invention also provides an application of the primer pair combination of the InDel molecular marker combination for constructing the DNA fingerprint of the fresh soybean "Tongsu No. 1", characterized in that the primer pair combination of the InDel molecular marker combination is used to identify true and false hybrids of the F1 hybrid offspring whose parents are the fresh soybean "Tongsu No. 1" and "Williams 82".

[0040] Preferably, if the PCR amplification band of the F1 hybrid offspring is not completely the same as the female parent of the two parents "Williams 82" and "Tongsu No. 1", it is a true F1 hybrid;

[0041] If the PCR amplification band of the F1 hybrid offspring is completely identical to the female parent of the two parents "Williams 82" and "Tongsu No. 1", it is a false hybrid.

[0042] The present invention also provides an application of the primer pair combination of the above-mentioned InDel molecular marker combination for constructing the DNA fingerprint of the fresh soybean "Tongsu No. 1", and the primer pair combination of the InDel molecular marker combination is used for genetic diversity of soybean germplasm resources and evolutionary tree analysis of soybean variety resources.

[0043] The fingerprint map of the fresh soybean "Tongsu No. 1" based on whole genome resequencing of the present invention can be used for genetic diversity analysis, identification and germplasm resource utilization.

[0044] The primer pair combination based on 31 InDel molecular markers of the present invention is not only suitable for the accurate identification of the authenticity of soybean varieties, but can also be widely used in related fields such as soybean molecular marker-assisted breeding.

[0045] The specific method is:

[0046] DNA extraction, library construction and whole genome resequencing of fresh soybean "Tongsu No. 1" samples:

[0047] S1. Use a DNA extraction kit (Hi-DNAsecure Plant Kit, Tiangen DP350) to extract DNA from fresh young soybean leaves.

[0048] S2. Use fluorescent dye (Quant-iT PicoGreen dsDNA Assay Kit) to detect the total amount of DNA; use 0.8% agarose gel electrophoresis to detect the quality of DNA extraction, and use an ultraviolet spectrophotometer to quantify the DNA.

[0049] S3. Prepare the sequencing library using the standard library construction process of Illumina's TruSeq DNA PCR-free prep kit.

[0050] S4. First, perform quality inspection on the library on Agilent Bioanalyzer, and then perform high-throughput sequencing on the machine after gradient dilution of qualified sequencing libraries (Index sequences cannot be repeated).

[0051] Analyze the data of Tongsu No. 1 resequencing and construct fingerprint map:

[0052] S1. Further process the data to remove 3' end adapter contamination, use sliding window method for quality filtering, and generate high-quality sequences.

[0053] S2. The resequencing data were aligned with the Williams whole genome sequence using the bwa (0.7.17-r1188) mem program, and duplicates were removed using the “MarkDuplicates” program in the Picard software package. The GATK program was used to screen and identify SNP and InDel variant sites. CNVnator 0.2.7 was used to identify and detect CNV variants. BreakDancer 1.1 software was used to detect SV variants in the genome.

[0054] S3. Use ANNOVAR software to annotate SNP and InDel sites.

[0055] S4. Based on the analysis results of SNP, InDel, CNV and SV, the reference sequence was used as a ruler and the R circlize software was used to display them on a circular diagram to visually compare the variation analysis results.

[0056] Application of fingerprint map constructed from resequencing data of Tongsu No. 1:

[0057] S1. Screen the whole genome of "Tongsu No. 1" for 31 homozygous InDel variants ≥ 30bp distributed in 15 chromosomes, design primers for InDel molecular markers, and perform e-PCR amplification on the genome sequences of "Williams 82" and "Tongsu No. 1" through the Primer Check plug-in. If the 31 InDel molecular marker PCR amplification bands of its F1 hybrid offspring are only completely identical with the female parent of the two parents, it is a false hybrid; if it is not completely identical with the female parent of the two parents, it can be determined to be a true F1 hybrid.

[0058] S2. Use 14 InDel molecular markers with good specificity to identify the genetic diversity of 29 soybean germplasm resources such as Jidou 17 through PCR amplification or e-PCR amplification based on whole genome sequences, and construct an evolutionary tree for evolutionary analysis.

[0059] Compared with the prior art, the present invention has the following advantages:

[0060] 1. The present invention realizes the identification and screening of SNP, InDel, CNV, and SV markers across the entire genome based on the resequencing data analysis method, with higher density and larger quantity, and more options for developing specific molecular markers, laying a foundation for the classification, identification, and utilization of soybean germplasm resources.

[0061] 2. The present invention integrates data quality control, InDel screening, and primer design to develop a specific InDel molecular marker for the fresh soybean "Tongsu No. 1". The authenticity of the hybrid F1 generation and multiple soybean germplasm resources can be identified through different molecular marker combinations. This method has good repeatability, fast and easy operation, and can identify more soybean germplasm materials as needed.

[0062] 3. The primer set constructed by the present invention shows a high amplification efficiency. The product bands obtained by PCR or e-PCR amplification are clear and have high size resolution, and can show significant polymorphism among different soybean varieties. It not only improves the efficiency of variety identification, but also provides a strong molecular basis for soybean genetic research and breeding.

[0063] 4. The soybean DNA fingerprint and specific molecular markers constructed by the present invention can be identified at the soybean seedling stage, which is of great significance for early rapid identification, not only improving work efficiency, but also helping to shorten the variety approval cycle and accelerate the promotion and application of new varieties. The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 It is the original resequencing data statistics and quality control chart of the "Tongsu No. 1" sample in Example 3 of the present invention.

[0065] Figure 2 This is the sequencing depth and coverage diagram of the "Tongsu No. 1" sample of Example 3 of the present invention.

[0066] Figure 3 This is the density distribution diagram of SNPs on chromosomes of the "Tongsu No. 1" sample of Example 3 of the present invention.

[0067] Figure 4 It is the number of different types of SNPs in the coding region of the "Tongsu No. 1" sample of Example 3 of the present invention and the number of SNPs in different regions of the genome.

[0068] Figure 5 This is the statistic of the mutation base preference of the "Tongsu No. 1" sample of Example 3 of the present invention.

[0069] Figure 6This is the SNP mutation spectrum analysis of the "Tongsu No. 1" sample of Example 3 of the present invention.

[0070] Figure 7 This is the InDel length distribution diagram of the "Tongsu No. 1" sample of Example 3 of the present invention.

[0071] Figure 8 It is the number of different types of Indels in the coding region of the "Tongsu No. 1" sample of Example 3 of the present invention and the number of Indels in different regions of the genome.

[0072] Fig. 9 It is the SV length distribution of the "Tongsu No. 1" sample of Example 3 of the present invention.

[0073] Fig.10 It is a circos diagram of the SNP, InDel, CNV and SV variation results of the "Tongsu No. 1" sample of Example 3 of the present invention.

[0074] Fig.11 It is the e-PCR amplification band of "Williams 82" in Example 4 of the present invention based on 31 InDel molecular markers.

[0075] Fig.12 It is the e-PCR amplification band of "Tongsu No. 1" of Example 4 of the present invention based on 31 InDel molecular markers.

[0076] Fig.13 This is the genetic diversity analysis of soybean germplasm resources such as 29 soybean variety resources based on 14 InDel molecular markers in Example 4 of the present invention.

[0077] Fig.14 This is the evolutionary tree analysis of 29 soybean variety resources based on 14 InDel molecular markers in Example 4 of the present invention. DETAILED DESCRIPTION

[0078] The fresh soybean "Tongsu No. 1" in the present invention is commercially available, and "Williams 82" is provided by the germplasm bank of the Institute of Crop Germplasm Resources of Shandong Academy of Agricultural Sciences.

[0079] Example 1

[0080] This example is about DNA extraction, library construction and sequencing of fresh soybean "Tongsu No. 1" leaf samples.

[0081] 1. Sample DNA extraction, purification and quality inspection

[0082] (1) DNA extraction from samples: DNA from fresh young soybean leaves was extracted using a DNA extraction kit (Hi-DNAsecure Plant Kit, Tiangen DP350). The main extraction process was as follows: about 100 mg of fresh plant tissue was taken and ground thoroughly with liquid nitrogen; GP1 was preheated at 65°C in advance and then mercaptoethanol was added to make the final concentration 0.1%; the ground tissue powder was quickly transferred to 700 μl of GP1 preheated buffer, inverted to mix, and placed in a 65°C water bath for 20 min. The centrifuge tube was inverted 2 to 3 times; 700 μl of chloroform was added, inverted to mix thoroughly, and centrifuged at 12,000 rpm for 5 min; the upper aqueous phase was transferred to a new centrifuge tube, 700 μl of buffer GP2 was added, and after thorough mixing, the tube was transferred to the adsorption column CB3, centrifuged at 12,000 rpm for 30 sec, and the waste liquid was discarded; 500 μl of buffer G was added to the adsorption column CB3 D, centrifuge at 12000rpm for 30sec, discard the waste liquid, and place the adsorption column CB3 in the collection tube; add 600μl of rinsing solution PW to the adsorption column CB3, centrifuge at 12000rpm for 30sec, discard the waste liquid, and place the adsorption column CB3 in the collection tube; put the adsorption column CB3 back into the collection tube, centrifuge at 12000rpm for 2min, discard the waste liquid; leave the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinsing solution in the adsorption material. Note: Anhydrous ethanol needs to be added to buffer GD and rinsing solution PW before use.

[0083] (2) Sample DNA quality inspection: The total amount of DNA was detected using fluorescent dye (Quant-iT PicoGreen dsDNA Assay Kit); the quality of DNA extraction was detected by 0.8% agarose gel electrophoresis, and the DNA was quantified using an ultraviolet spectrophotometer; the DNA dilution working solution was stored at 4°C, and the storage solution was stored at -20°C.

[0084] 2. Second-generation library preparation

[0085] The sequencing library was prepared using the standard library construction process of Illumina's TruSeq DNA PCR-free prep kit. The main experimental process is as follows:

[0086] (1) First, the extracted DNA was randomly sheared by ultrasound and the sequence ends were repaired. The protruding bases at the 5' end of the DNA sequence were removed by EndRepair Mix2 in the kit, and a phosphate group was added to fill the missing bases at the 3' end.

[0087] (2) Adding an A base to the 3' end of the DNA sequence to prevent the DNA fragment from self-ligating and to ensure that the target sequence can be connected to the sequencing adapter (there is a protruding T base at the 3' end of the sequencing adapter).

[0088] (3) A sequencing adapter containing a library-specific tag (i.e., Index sequence) is added to the 5' end of the sequence so that the DNA molecule can be fixed on the Flow Cell.

[0089] (4) BECKMAN AMPure XP Beads were used to remove the self-ligated fragments by magnetic bead screening and purify the library system after adding the adapter.

[0090] (5) PCR amplification is performed on the DNA fragments connected to the adapter to enrich the sequencing library template, and the library enrichment product is purified again using BECKMAN AMPure XP Beads.

[0091] (6) The library was subjected to final fragment selection and purification by 2% agarose gel electrophoresis. The insert fragment of the library was ∼450 bp.

[0092] 3. Perform high-throughput sequencing on the machine

[0093] (1) Before sequencing, the library was quality checked on an Agilent Bioanalyzer using the Agilent High Sensitivity DNA Kit. A qualified library had only a single peak and no adapter dimers. The library was quantified using the Quant-iT PicoGreen dsDNA Assay Kit on the Promega QuantiFluor fluorescence quantification system. The concentration of a qualified library should be above 2 nM.

[0094] (2) After the qualified sequencing library (Index sequence cannot be repeated) is diluted in a gradient manner, it is mixed in the corresponding proportion according to the required sequencing amount, the double-stranded DNA library is denatured into single-stranded DNA using NaOH, and 2×150bp double-end sequencing is performed using a Nova Seq sequencer.

[0095] Example 2

[0096] This example is to analyze the data of the resequencing of the fresh soybean "Tongsu No. 1".

[0097] 1. Obtain high-quality resequencing data

[0098] In order to ensure the quality of subsequent information analysis, it is necessary to further filter the offline data, that is, filter the original offline data to generate high-quality sequences. The data filtering standards mainly include:

[0099] (1) Use AdapterRemoval (version 2) to remove 3' end adapter contamination.

[0100] (2) The sliding window method is used for quality filtering, with the window size set to 5 bp and the step size set to 1 bp. Each time a base is moved forward, 5 bases are taken to calculate the average Q value of the window. If the Q value of the last base is ≤2, only the bases before this position are retained; if the average Q value of the window is ≤20, only the second to last base and the bases before this position are retained.

[0101] (3) If the length of any read in the paired-end sequence is ≤50 bp, the paired-end read is removed.

[0102] 2. Compare the resequencing data with the whole genome sequence

[0103] (1) The bwamem program (0.7.17-r1188) was used to align the high-quality data obtained after filtering to the reference genome. The alignment parameters were all based on the default parameters of bwamem.

[0104] (2) Picard 1.107 software was used to sort the sam files and convert them into bam files, and the "FixMateInformation" command was used to ensure the consistency of all paired-end reads information. The generation of sequencing data involves library amplification and cluster formation, and these two steps are prone to produce some duplicates.

[0105] (3) Use "MarkDuplicates" in the Picard software package to remove duplicates, that is, if multiple paired reads have the same chromosome coordinates after alignment, only the paired reads with the highest score are retained. Reads near InDel are most prone to mapping errors. In order to minimize SNPs caused by mapping errors, reads near InDel need to be re-aligned to improve the accuracy of SNP Calling. The IndelRealigner command in the GATK program is used to re-align all reads near InDel to improve the accuracy of SNP prediction.

[0106] 3.SNP detection and annotation

[0107] GATK software is used to detect SNP. The specific steps are as follows:

[0108] (1) Realignment using known InDel information. This is done in two steps: the first step is to use the RealignerTargetCreator command in the GAT K software package to output a file containing all possible InDels; the second step is to use the IndelRealigner command to realign all reads near InDels to improve the accuracy of SNP prediction.

[0109] (2) The UnifiedGenotyper program was used to obtain the SNP sites of the samples, with stand_call_conf set to 30 and stand_emit_conf set to 10. In order to ensure the reliability of the SNP sites, the obtained SNP sites were further filtered, and the filtering criteria were: Fisher test of strand bias (FS) ≤ 60; HaplotypeScore ≤ 13.0; MappingQuality (MQ) ≥ 40; Quality Dept h (QD) ≥ 2; ReadPosRankSum ≥ -8.0; MQRankSum> -12.5; DP ≥ 4 reads.

[0110] (3) ANNOVAR software was used to annotate SNP sites.

[0111] 4. InDel detection and annotation

[0112] (1) The UnifiedGenotyper program in the GenomeAnalysisTK v3.8 software package was used to obtain all mutation points of the sample. The stand_call_conf was set to 30, the stand_emit_conf was set to 10, and Select Variants was used to extract InDel mutation information.

[0113] (2) In order to ensure the reliability of the InDel results, the InDel sites were further filtered according to the following filtering criteria: Fisher Test of Strand Bias (FS) ≤ 200; Read depth (DP) > 4; Quality Depth (QD) ≥ 2; ReadPosRankSum ≥ -20.

[0114] (3) ANNOVAR software was used to annotate INDEL sites.

[0115] 5. CNV detection and annotation

[0116] CNVnator 0.2.7 was used to detect copy number variation (CNV) in the whole genome, with bin_size set to 100. The results obtained by CNVnator were screened to remove the multi-copy regions in the genome, and the Read Depth value was set (the Read Depth of the normal chromosome region was defined as 1), and the regions with RD values ​​≤ 0.05 or ≥ 1.8 were retained.

[0117] 6.SV detection and annotation

[0118] BreakDancer 1.1 software was used to detect chromosome structural variations in the genome. The principle of this software for detecting chromosome structural variations is based on pair-end mapping, which uses the abnormal length of the pair end read insert to perform detection.

[0119] Example 3

[0120] This embodiment is to construct a fingerprint map based on the resequencing data of the fresh soybean "Tongsu No. 1".

[0121] 1. Quality assessment results of resequencing raw data

[0122] Statistics and quality control of the original resequencing data of the "Tongsu No. 1" sample ( Figure 1 ), GC content was 39.28%, the percentage of ambiguous bases (N) was 0.01%, the percentage of bases with base recognition accuracy above 99% (Q20) was 98.33%, and the percentage of bases with base recognition accuracy above 99.9% (Q30) was 95.78%. The average quality value of single bases of sequencing reads at the R1 and R2 ends ranged from 35 to 40, and the average estimated error rate was less than 0.016%. There was no AT and GC separation in the sequencing data, and the number of AT and GC was basically consistent. The above results showed that the sequencing results were of good quality.

[0123] 2. Filtering and preprocessing results of resequencing raw data

[0124] The resequencing raw data were further filtered to generate high-quality sequences. The number of high-quality reads was 172796170, the percentage of high-quality reads to the original reads was 96.57%, the number of high-quality read bases was 25808869698, the percentage of high-quality read bases to the total number of original bases was 95.52%, and the sequencing depth was 27.08×.

[0125] 3. Comparison of resequencing data with whole genome sequences

[0126] (1) Overview of comparison results

[0127] From the sequence alignment results, we can see that the total number of reads is 172796170, the number of reads aligned to the reference genome is 172015034, the percentage of reads aligned to the reference genome is 99.55% of the total number of reads, the number of repeated sequence reads is 36610129, and the percentage of repeated sequence reads to the total number of reads is 21.19%.

[0128] From the statistics of sequencing depth and coverage, we can know that ( Figure 2 ), with an average coverage depth of 27.8×, and the percentages of sites covered by at least 1, 4, 10, and 20 sequences in the reference genome were 93.04%, 85.35%, 71.06%, and 38.70%, respectively.

[0129] (2) SNP detection, analysis and annotation

[0130] The number of SNPs counted by sliding window showed that the total number of SNP sites was 1744120, the number of heterozygous genotypes was 354610, and the number of homozygous genotypes was 1389510. The number of transitions between purine and purine or between pyrimidine and pyrimidine was 1150576, the number of transversions between purine and pyrimidine was 593544, and the ratio of transition / transversion was 1.93.

[0131] Statistical analysis of the distribution of SNPs on each chromosome ( Figure 3 ), there are 1720673 SNP sites distributed in 20 nuclear chromosomes and chloroplast and mitochondrial genomes, among which there are 92186, 93083, 105015, 72168, 52741, 100165, 68067, 52508, 74465, 85153, 42687, 43660, 90144, 59929, 149728, 114377, 62241, 172572, 120913, 68781 SNP sites in chromosome1-20, respectively. The distribution densities were 628.43, 541.46, 447.10, 709.50, 801.55, 508.62, 660.37, 899.43, 679.15, 606.42, 928.71, 951.24, 501.70, 832.54, 359.01, 333.21, 670.63, 337.75, 424.05, and 695.63 / bp, respectively. There were 45 SNP sites in the chloroplast and chromosome genomes, with distribution densities of 8945.73 and 3382.62 / bp, respectively.

[0132] According to the SNP annotation results ( Figure 4 ) showed that a total of 71,135 SNP sites were detected in the exon region, of which 30,793 were synonymous mutations, 38,288 were non-synonymous mutations, 835 SNPs resulted in the acquisition of stop codons, 155 SNPs resulted in the loss of stop codons, and 1,064 sites of unknown function due to errors in the gene structure annotation database used for annotation. There were 391, 3605, 1873, 6, 2, 1724, 188028, 1284767, 17844, 18951, 43, 78470, 75944, and 4942 SNP sites located in the splicing junction 2bp region, noncoding ncRNA region, exon region of noncoding ncRNA region, splicing junction 2bp region of noncoding nc RNA, intron region of noncoding ncRNA region, intron region, intergenic region, 5'UTR region, 3'UTR region, region between 5'UTR and 3'UTR, region 1kb upstream of transcription start site, region 1kb downstream of transcription stop site, and regions upstream and downstream of transcription start site, respectively.

[0133] According to the statistical results of SNP mutation base preference ( Figure 5 ) It can be seen that compared with the base sequences before and after the SNP mutation site, after the SNP mutation occurs, the A and T bases decrease, and the G and C bases increase.

[0134] The results of SNP mutation spectrum analysis ( Figure 6 ) shows that genomic SNP mutations are divided into 6 categories: T:A>C:G, C:G>T:A, T:A>G:C, T:A>A:T, C:G>G:C, C:G>A:T, among which Ts transition mutations T:A>C:G and C:G>T:A are the main SNP mutation types, accounting for 65.97%, and the other four Tv transversion mutations account for 34.03%.

[0135] (3) InDel detection, analysis and annotation

[0136] After filtering, a total of 232,213 InDel sites were obtained, including 28,149 heterozygous sites, 204,064 homozygous sites, 113,091 Insertion sites, and 119,122 Deletion sites. Figure 7 It can be seen that the proportions of Insertion sites and Deletion sites of different lengths are basically the same.

[0137] Annotate the sites ( Figure 8), there are 2986 InDel sites distributed in the exon region, 652 frameshift deletions lead to changes in the reading frame of protein-coding genes (length is a non-integer multiple of 3), 521 frameshift insertions lead to changes in the reading frame of protein-coding genes (length is a non-integer multiple of 3), 900 non-frameshift deletions do not change the reading frame of protein-coding genes (length is an integer multiple of 3), 810 non-frameshift insertions do not change the reading frame of protein-coding genes (length is an integer multiple of 3), 39 InDels lead to the acquisition of stop codons, 5 InDels lead to the loss of stop codons, 143 are located in the splicing junction 2bp region, 565 are located in the non-coding RNA region, 276 are located in the exon region of the non-coding region, and 1 is located in the splicing junction of the non-coding RNA. 2 bp regions, 288 were located in the intronic regions of the noncoding regions, 36,503 were located in the intronic regions, 149,916 were located in the intergenic regions, 5,835 were located in the 5'UTR region, 4,857 were located in the 3'UTR region, 4 were located in the region between the 5'UTR and 3'UTR, 15,025 were located in the region 1 kb upstream of the transcription start site, 15,277 were located in the region 1 kb downstream of the transcription stop site, and 1,102 were located in the regions both upstream and downstream of the transcription start site.

[0138] (4) CNV and SV detection and analysis

[0139] After identification, it was found that the total number of CNV variations was 8732, of which 739 were copy number deletions and 7993 were copy number gains.

[0140] After testing, it was found that the total number of SV mutations was 5749, including 1926 deletions (DEL), 178 inversions (INV), 2255 intra-chromosomal translocations (ITX), and 1390 inter-chromosomal translocations (CTX). Fig. 9 ), 201-300bp and >1000bp were the largest, both greater than 1000.

[0141] (5) Draw genomic SNP, InDel, CNV, and SV site variation maps

[0142] According to the analysis results of SNP, InDel, CNV and SV, the reference sequence was used as the ruler and displayed on a circular diagram using R circlize software ( Fig.10 ), the distribution of the variation analysis results can be compared visually.

[0143] Example 4

[0144] This embodiment is an application of a fingerprint map constructed from resequencing data.

[0145] 1. Identify the authenticity of soybean F1 hybrid seeds

[0146] Thirty-one homozygous InDel variants ≥30 bp distributed in 15 chromosomes were screened in the whole genome of “Williams 82” and “Tongsu 1” (Table 1), and primers were designed for InDel molecular markers using the Batch Target Region Primer Design plug-in of TBtools software (Table 2).

[0147] Based on 31 InDel molecular markers, the genome sequences of "Williams 82" and "Tongsu No. 1" were amplified by e-PCR using the Primer Check plug-in ( Fig.11 , Fig.12 ), and obtain the fingerprints of "Williams 82" and "Tongsu No. 1" based on 31 InDel molecular markers. Amplify the fingerprints of the F1 hybrid offspring based on 31 InDel molecular markers to be identified. If the PCR amplification bands of the 31 InDel molecular markers of the F1 hybrid offspring are only completely the same as the female parent of the two parents "Williams 82" and "Tongsu No. 1", it is a false hybrid; if the PCR amplification bands of the 31 InDel molecular markers of the F1 hybrid offspring are not completely the same as the female parent of the two parents "Williams 82" and "Tongsu No. 1", it can be determined to be a true F1 hybrid.

[0148] Table 1 Information of 31 InDel molecular markers

[0149]

[0150]

[0151] Table 231 InDel molecular marker PCR amplification primer information

[0152]

[0153]

[0154] 2. Conduct genetic diversity analysis of soybean germplasm resources

[0155] Use Gm02_48831944_48831999, Gm02_50061608_50061609, Gm03_3866902_3866939, Gm04_1014450_1014481, Gm07_20149006_20149007, Gm08_44385355_44385399, Gm09_12267638_12267680, Gm10_21 Fourteen InDel molecular markers, including 414835_21414878, Gm13_28033266_28033267, Gm13_32370867_32370901, Gm13_42180045_42180091, Gm16_35889588_35889638, Gm17_39474344_39474381, and Gm18_2089656_2089688, were identified by PCR amplification or e-PCR amplification based on whole genome (SoyOmics, https: / / ngdc.cncb.ac.cn / soyomics / index; LIS, https: / / www.legumeinfo.org / collections / glycine / ) sequence was used to identify Jidou 17, Keshan 1, Qihuang 34, 58-161, Amsoy, Fengdihuang, Handou 5, Genetic diversity of 29 soybean germplasm resources including Heihe 43, Jindou 23, Juxuan 23, PI_398296, PI_548362, PI_562565, PI_578357, PI483463, Zhonghuang 13, Shisheng Changye, Zihua 4, Yudou 22, Mancangjin, Huaxia 3, Tiefeng 18, Wandou 28, Zhonghuang 35, Hwangkeum, Hefeng 25, Fiskeby III, Tongshan Tianedan, Wenfeng 7, etc. Fig.13 ), and used the R language ggtree package to analyze the germplasm resource evolution and construct an evolutionary tree ( Fig.14 ).

[0156] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A primer pair combination of InDel molecular marker combination for constructing DNA fingerprint of fresh soybean "Tongsu No. 1", characterized in that: The DNA fingerprint of the fresh soybean "Tongsu No. 1" consists of 31 InDel molecular markers of ≥30 bp distributed in 15 chromosomes; The nucleotide sequence of the first primer pair used to amplify the Gm02_687775_687827 molecular marker is shown in SEQ ID NO. 1-2; The nucleotide sequence of the second primer pair used to amplify the Gm02_38671726_38671769 molecular marker is shown in SEQ ID NO. 3-4; The nucleotide sequence of the third primer pair used to amplify the Gm02_46235870_46235916 molecular marker is shown in SEQ ID NOs. 5 to 6; The nucleotide sequence of the fourth primer pair used to amplify the Gm02_48831944_48831999 molecular marker is shown in SEQ ID NOs. 7 to 8; The nucleotide sequence of the fifth primer pair used to amplify the Gm02_50061608_50061609 molecular marker is shown in SEQ ID NOs. 9 to 10; The nucleotide sequence of the sixth primer pair used to amplify the Gm03_3866902_3866939 molecular marker is shown in SEQ ID NOs. 11 to 12; The nucleotide sequence of the seventh primer pair used to amplify the Gm03_40000161_40000198 molecular marker is shown in SEQ ID NOs. 13-14; The nucleotide sequence of the eighth primer pair used to amplify the Gm04_1014450_1014481 molecular marker is shown in SEQ ID NOs. 15-16; The nucleotide sequence of the ninth primer pair used to amplify the Gm04_47260900_47260937 molecular marker is shown in SEQ ID NOs. 17 to 18; The nucleotide sequence of the 10th primer pair used to amplify the Gm07_20149006_20149007 molecular marker is shown in SEQ ID NOs. 19 to 20; The nucleotide sequence of the 11th primer pair used to amplify the Gm08_44385355_44385399 molecular marker is shown in SEQ ID NOs. 21-22; The nucleotide sequence of the 12th primer pair used to amplify the Gm09_12267638_12267680 molecular marker is shown in SEQ ID NOs. 23-24; The nucleotide sequence of the 13th primer pair used to amplify the Gm09_33743573_33743612 molecular marker is shown in SEQ ID NOs. 25-26; The nucleotide sequence of the 14th primer pair used to amplify the Gm09_37146920_37146952 molecular marker is shown in SEQ ID NOs. 27-28; The nucleotide sequence of the 15th primer pair used to amplify the Gm10_21414835_21414878 molecular marker is shown in SEQ ID NOs. 29-30; The nucleotide sequence of the 16th primer pair used to amplify the Gm11_9027110_9027111 molecular marker is shown in SEQ ID NOs. 31-32; The nucleotide sequence of the 17th primer pair used to amplify the Gm13_28033266_28033267 molecular marker is shown in SEQ ID NOs. 33-34; The nucleotide sequence of the 18th primer pair used to amplify the Gm13_32370867_32370901 molecular marker is shown in SEQ ID NOs. 35-36; The nucleotide sequence of the 19th primer pair used to amplify the Gm13_34120611_34120657 molecular marker is shown in SEQ ID NOs. 37-38; The nucleotide sequence of the 20th primer pair used to amplify the Gm13_42180045_42180091 molecular marker is shown in SEQ ID NOs. 39 to 40; The nucleotide sequence of the 21st primer pair used to amplify the Gm14_7027282_7027313 molecular marker is shown in SEQ ID NOs. 41-42; The nucleotide sequence of the 22nd primer pair used to amplify the Gm16_3283288_3283319 molecular marker is shown in SEQ ID NOs. 43-44; The nucleotide sequence of the 23rd primer pair used to amplify the Gm16_32569599_32569630 molecular marker is shown in SEQ ID NOs. 45-46; The nucleotide sequence of the 24th primer pair used to amplify the Gm16_35889588_35889638 molecular marker is shown in SEQ ID NOs. 47-48; The nucleotide sequence of the 25th primer pair used to amplify the Gm16_37504818_37504854 molecular marker is shown in SEQ ID NOs. 49-50; The nucleotide sequence of the 26th primer pair used to amplify the Gm17_36063299_36063330 molecular marker is shown in SEQ ID NOs. 51-52; The nucleotide sequence of the 27th primer pair used to amplify the Gm17_39292061_39292062 molecular marker is shown in SEQ ID NOs. 53-54; The nucleotide sequence of the 28th primer pair used to amplify the Gm17_39474344_39474381 molecular marker is shown in SEQ ID NOs. 55-56; The nucleotide sequence of the 29th primer pair used to amplify the Gm18_2089656_2089688 molecular marker is shown in SEQ ID NOs. 57-58; The nucleotide sequence of the 30th primer pair used to amplify the Gm19_47148869_47148912 molecular marker is shown in SEQ ID NOs. 59 to 60; The nucleotide sequence of the 31st primer pair used to amplify the Gm20_47615536_47615570 molecular marker is shown in SEQ ID NOs. 61-62.

2. An application of a primer pair combination of an InDel molecular marker combination for constructing a DNA fingerprint of fresh soybean "Tongsu No. 1" as claimed in claim 1, characterized in that: The primer pair combination of the InDel molecular marker combination is used to identify the fresh soybean "Tongsu No. 1".

3. An application of a primer pair combination of an InDel molecular marker combination for constructing a DNA fingerprint of fresh soybean "Tongsu No. 1" as claimed in claim 1, characterized in that: The primer pair combination of the InDel molecular marker combination is used to identify true and false hybrids of F1 hybrid offspring whose parents are fresh soybean "Tongsu No. 1" and "Williams 82".

4. The use according to claim 3, characterized in that: If the PCR amplification band of the F1 hybrid offspring is not completely the same as the female parent of the two parents "Williams 82" and "Tongsu No. 1", it is a true F1 hybrid; If the PCR amplification band of the F1 hybrid offspring is completely identical to the female parent of the two parents "Williams 82" and "Tongsu No. 1", it is a pseudo-hybrid.

5. An application of a primer pair combination of an InDel molecular marker combination for constructing a DNA fingerprint of fresh soybean "Tongsu No. 1" as claimed in claim 1, characterized in that: The primer pair combination of the InDel molecular marker combination is used for genetic diversity of soybean germplasm resources and evolutionary tree analysis of soybean variety resources.

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