InDel marker of chestnut plant and application of InDel marker

Abstract

The invention provides an InDel marker of a chestnut plant and application of the InDel marker, and belongs to the technical field of molecular markers. The nucleotide sequences of the InDel marker are as shown in SEQ ID NO.1-SEQ ID NO.76. The InDel molecular marker is developed through a high-throughput genome sequencing data technology, and detection of the InDel genotype can be accurately, simply and conveniently achieved through a GBTS technology; the method can lay a foundation for specific identification of chestnut plant germplasm resources, pedigree analysis / hybrid identification, population genetic analysis, genetic linkage map construction, agronomic trait gene positioning, molecular marker-assisted selective breeding and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular markers, and in particular relates to an InDel marker of Castanea plants and an application thereof. Background Art

[0002] Chestnut is native to China and is a unique economic forest tree species in China with a wide distribution. Since chestnut is propagated by seed and grafting, a large seed variation population has been formed in nature, which contains many valuable special resources and excellent types. These germplasm resources are of great significance to production cultivation, new variety breeding and processed product development, and the accurate identification and diversity analysis of these germplasm resources are the basic premise for their utilization and protection. With the popularization of high-throughput sequencing technology in recent years, a new generation of SNP and InDel markers have been developed. Among them, InDel markers are called the most promising DNA molecular markers. They are a special type of di-allelic genetic markers with good stability and easy detection. Therefore, InDel markers are more suitable as fingerprint molecular markers. The development and maturity of genotyping by target sequencing (GBTS) technology based on multiplex PCR makes the detection of InDel marker genotypes simpler and more efficient. At present, InDel markers have been used in the specific identification of germplasm resources of crops such as sorghum, mulberry, and apple, but no relevant reports have been seen in chestnuts. Summary of the invention

[0003] In view of this, the object of the present invention is to provide an InDel marker for Castanea plants and application thereof.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides an InDel marker of Castanea plants, wherein the nucleotide sequence of the InDel marker is shown as SEQ ID NO.1 to SEQ ID NO.76.

[0006] The present invention provides a probe combination for detecting the above-mentioned InDel markers, and the nucleotide sequences of the probe combination are shown in SEQ ID NO.77 to SEQ ID NO.228.

[0007] Preferably, the 5' end of each probe sequence of the probe combination is labeled with biotin.

[0008] The present invention also provides a detection system for detecting the above-mentioned InDel marker, wherein the detection system comprises streptavidin-coated magnetic beads and the above-mentioned probe combination.

[0009] The present invention also provides an application of the above-mentioned probe combination or detection system in identifying Castanea germplasm resources.

[0010] The present invention also provides an application of the above-mentioned probe combination or detection system in identifying the genotype of InDel markers of Castanea germplasm resources, wherein the InDel markers are InDel markers having nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.76.

[0011] The present invention also provides an application of the above-mentioned probe combination or detection system in pedigree analysis of Castanea germplasm resources and / or hybrid identification.

[0012] The present invention also provides an application of the above-mentioned probe combination or detection system in the systematic development relationship analysis of Castanea germplasm resources.

[0013] The present invention also provides an application of the above-mentioned probe combination or detection system in the construction of genetic map and gene positioning of Castanea plants.

[0014] Preferably, the Castanea plant includes one or more of Castanea mollissima, Castanea henryi, Castanea henryi and Castanea japonica.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention provides an InDel marker of Castanea plants and its application. The present invention develops an InDel molecular marker through high-throughput genome sequencing data technology, and can accurately and easily detect the InDel genotype through GBTS technology, which can lay a foundation for the specific identification of Castanea plant germplasm resources, pedigree analysis / hybrid identification, population genetic analysis, genetic linkage map construction, agronomic trait gene positioning and molecular marker-assisted selection breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The distribution of 76 InDel markers in the chestnut genome, Chr01-Chr12 represent the chromosome names in the chestnut genome; the round-headed rectangle represents the chromosome; the number on the left side of the round-headed rectangle represents the physical position of the InDel marker on the chromosome (Mb), and the string on the right side represents the corresponding marker name;

[0018] Figure 2 is the relative position of the probe, InDel variant site, and gDNA library reads;

[0019] Figure 3 This is the flowchart of GenoBaits based on targeted genotyping by sequencing (GBTS) technology;

[0020] Figure 4Schematic diagram of genotype discrimination for sequencing reads;

[0021] Figure 5 This is the clustering tree of Castanea germplasm resources. DETAILED DESCRIPTION

[0022] The present invention provides an InDel marker of Castanea plants, wherein the nucleotide sequence of the InDel marker is shown as SEQ ID NO.1 to SEQ ID NO.76.

[0023] The present invention provides a probe combination for detecting the above-mentioned InDel markers, and the nucleotide sequences of the probe combination are shown in SEQ ID NO.77 to SEQ ID NO.228.

[0024] In the present invention, the 5' end of each probe sequence of the probe combination is labeled with biotin.

[0025] The present invention also provides a detection system for detecting the above-mentioned InDel marker, wherein the detection system comprises streptavidin-coated magnetic beads and the above-mentioned probe combination.

[0026] The present invention also provides an application of the above-mentioned probe combination or detection system in identifying Castanea germplasm resources.

[0027] The present invention also provides an application of the above-mentioned probe combination or detection system in identifying the genotype of InDel markers of Castanea germplasm resources, wherein the InDel markers are InDel markers having nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.76.

[0028] The present invention also provides an application of the above-mentioned probe combination or detection system in pedigree analysis of Castanea germplasm resources and / or hybrid identification.

[0029] The present invention also provides an application of the above-mentioned probe combination or detection system in the systematic development relationship analysis of Castanea germplasm resources.

[0030] The present invention also provides an application of the above-mentioned probe combination or detection system in the construction of genetic map and gene positioning of Castanea plants.

[0031] The present invention also provides a method for determining the genotype of InDel-marked germplasm resources of Castanea plants, comprising the following steps: extracting genomic DNA from leaves of Castanea plants, constructing a gDNA library, labeling the 5' end of each probe in a probe combination (designed according to the principle of DNA complementarity) with biotin, obtaining a biotin-labeled probe combination, and then hybridizing and capturing an InDel-marked region in a genome with a nucleotide sequence such as SEQ ID NO.1 to SEQ ID NO.76 to obtain a hybridized probe combination, and then using streptavidin-coated magnetic beads to adsorb targets hybridized with the hybridized probe combination, and obtaining a sequencing library by amplification; and after sequencing, counting the number of different types of reads covering each InDel-marked region, and then analyzing the genotype of the InDel-marked region.

[0032] The criteria for determining sequencing read types are:

[0033] (S1) The read that completely covers the InDel sequence is judged as the insertion type and recorded as read ins ;

[0034] (S2) Reads that cover both the InDel sequence and the upstream sequence or both the InDel sequence and the downstream sequence are considered to be insertion type and are recorded as reads. ins ;

[0035] (S3) covers both the upstream and downstream sequences of InDel, but cannot cover the read of the InDel sequence. It is judged as a deletion type and recorded as read del ;

[0036] The threshold for determining whether a read covers an InDel sequence or upstream and downstream sequences is 20 bp;

[0037] The method for determining the genotype of the InDel marker is to calculate the read count of each InDel site. ins and read del The ratio of the number of reads covering the marker to the total number of reads is classified according to the following threshold standards:

[0038] (1) When the total number of reads is <100, it is considered that the genotype is not detected at this site and is recorded as “-”;

[0039] (2) Total number of reads ≥ 100 and read ins Quantity>read del When delIf the number of reads is greater than 10% of the total number of reads, the genotype of the marker is considered to be heterozygous and is recorded as "H"; del If the number is ≤ 10% of the total number of reads, the genotype of the site is considered to be insertion, which is recorded as "I";

[0040] (3) Total number of reads ≥ 100 and read ins Quantity<read del When ins If the number of reads is greater than 10% of the total number of reads, the genotype of the marker is considered to be heterozygous and is recorded as "H"; ins If the number is ≤ 10% of the total number of reads, the genotype of the site is considered to be deletion, denoted as "D".

[0041] The present invention develops a set of stable co-dominant InDel markers and a genotype detection method based on GBTS, laying a foundation for molecular identification, pedigree analysis and population genetic analysis of Castanea germplasm resources.

[0042] In the present invention, the Castanea plant includes one or more of Castanea mollissima, Castanea henryi, and Castanea chinensis. As an embodiment, the Castanea mollissima includes Qianxi short-branch, Chang'an Mingjian chestnut, Fengkai oil chestnut, Yunfeng, Huanglipu, Yanshan Zaofeng (SQY095), Yanshan Zaofeng (YSZF), Yanli (CL18), Yanli (SQY097), Yanlong (SQY089), Yanlong (YL), Yanbao (CL54), Yanbao (SQY090), Yanxi (SQY087), Yanxi (YX), Qianxi One or more of Zaohong (CL11), Qianxi Zaohong (SQY071), Shuofeng (SQY020), Funing No. 16 (UN06), Yinfeng (CL09), Beijing No. 8 (CL37), Huaixiang (SQY062), Xingtang 1 (SJZ01), Xingtang 2 (SJZ02), Xingtang 3 (SJZ03), Xingtang 4 (SJZ04), Huangpeng (SQY056), Jinping (SQY057), Likang (SQY058), and Hongli No. 1 (SQY059). The Castanea chinensis includes one or more of Zhahua 2 (GZ04), Maijiamao Li 1 (GZ05), and Maijiamao Li 2 (GZ06). The Castanea henryi includes one or more of Huali No. 1 (HuN03), Daguo Castanea henryi (HuN10), Castanea henryi 1 (HuN31), and Castanea henryi 2 (HuN32). The Japanese chestnut includes one or more of Gaocheng (LN07), Liaodan No. 15 (LN12), Liaodan No. 58 (LN13), Liaodan No. 61 (LN14), Liaoli No. 15 (LN16) and Tu 13 chestnut (LN20).

[0043] In the present invention, the InDel marker technology, as a high-throughput DNA fingerprint marker, can better reveal the polymorphism in the genome, and therefore has a more prominent advantage in germplasm resource identification, and can be applied to genetic map construction and gene positioning, germplasm resource specific identification, pedigree analysis and genetic structure analysis.

[0044] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0045] 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.

[0046] Example 1

[0047] 1. Mining InDel variant sites through genome resequencing

[0048] 1.1 Genome resequencing

[0049] Five chestnut varieties of Castanea, including 'Qianxi Short Branch' (North China variety group), 'Chang'an Mingjianli' (Northwest variety group), 'Fengkai Youli' (Southeast variety group), 'Yunfeng' (Southwest variety group), and 'Huanglipu' (middle and lower reaches of the Yangtze River variety group), as well as Castanea henryi 'August Fragrance' and Japanese chestnut 'Liaodan 15', were selected as representative varieties. Young leaves were collected and genomic DNA was extracted using the Plant / Seed DNA Miniprep Kit TD619-200 (Jianshi Biotechnology (Beijing) Co., Ltd.), gDNA library was constructed, and genome resequencing was performed. The sequencing platform was DNBSEQ-T10×4, and the sequencing depth of each sample was 15X.

[0050] 1.2 InDel variant site mining

[0051] The genome resequencing data obtained in 1.1 were compared with the chestnut reference genome (Sun Y, Lu Z, Zhu X, MaH. Genomic basis of homoploid hybrid speciation within chestnut trees. Nature Communications, 2020, 11: 3375), and all 44,355 InDel variation sites in the genome were mined using the Delly software. 120 InDel sites that were evenly distributed on the 12 chromosomes and whose genotypes were not completely consistent in the above 7 resequencing samples were selected; the accuracy of these variation sites was determined by the Sanger sequencing method, and finally 76 InDel sites were obtained for marker development. The sequences of these 76 InDel sites are shown in Table 1, and their distribution in the chestnut genome is shown in Figure 1As shown, the distance between adjacent markers is 4.4-15.6 Mb.

[0052] Table 1 Sequences of InDel sites

[0053]

[0054]

[0055]

[0056] Example 2

[0057] 1. Construct the InDel site genotype detection system described in Example 1

[0058] The detection probes for the InDel variant sites described in Example 1 (see Table 1) were designed using the multiplex PCR-based targeted sequencing genotyping (GBTS) technology. Two probes (probe L and probe R) were designed for each InDel variant site. Probe L and probe R needed to cover the breakpoints at the left and right ends of the InDel sequence, respectively (see Figure 2 ) to improve the capture efficiency of InDel sites and flanking sequences. The probe sequence of each InDel marker is shown in Table 2. The 5' end of each probe is labeled with biotin so that it can be captured by streptavidin-coated magnetic beads (such as Figure 3 All probe sequences were synthesized and biotin-labeled by Sangon Biotechnology (Shanghai) Co., Ltd.

[0059] Table 2 Probe sequences for InDel marker genotype detection

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066] The genomic DNA of chestnut leaves was extracted using a plant / seed DNA small-amount extraction kit TD619-200 (Jianshi Biotechnology (Beijing) Co., Ltd.), and the concentration of the DNA solution was finally adjusted to 100 ng / μL; the DNA was sheared into 300-500 bp fragments using Covaris S220, and the gDNA library was constructed and purified according to the instruction manual of the GenoBaits library construction kit (Article No. GP000105, purchased from Shijiazhuang Boridi Biotechnology Co., Ltd.), and the 5' end of each probe in the probe combination (designed according to the principle of DNA complementarity) with nucleotide sequences such as SEQ ID NO. 77-208 was labeled with biotin, and the obtained biotin-labeled probe combination was hybridized with the InDel labeled region of the nucleotide sequence in the genome such as SEQ ID NO. 1-76 to obtain a hybridized probe combination, and then the hybridized probe combination was molecularly adsorbed with streptavidin-coated magnetic beads to capture the target hybridized with the probe, and finally the captured target sequence was eluted; the second-generation sequencing library was obtained by amplification (see Figure 2 and Figure 3 ), DNBSEQ-T10×4 sequencer was used for second-generation sequencing, and the sequencing depth was 2000X. The bioinformatics analysis software Genome Analysis Toolkit (GATK) was used to count the number of different types of reads covering each InDel marker region in the sequencing data, and then analyze the genotype of the InDel marker.

[0067] The criteria for determining sequencing read types are as follows: Figure 4 As shown, specifically:

[0068] (S1) The read that completely covers the InDel sequence is judged as the insertion type and recorded as read ins (like Figure 4 read1 in );

[0069] (S2) Reads that cover both the InDel sequence and the upstream sequence or both the InDel sequence and the downstream sequence are considered to be insertion type and are recorded as reads. ins (like Figure 4 read2 and read3 in );

[0070] (S3) covers both the upstream and downstream sequences of InDel, but cannot cover the read of the InDel sequence. It is judged as a deletion type and recorded as read del (like Figure 4 read4 in ).

[0071] The threshold for determining whether a read covers an InDel sequence or upstream and downstream sequences is 20 bp.

[0072] The method for determining the genotype of the InDel marker is to calculate the read count of each InDel site. ins and read del The ratio of the number of reads covering the marker to the total number of reads is classified according to the following threshold standards:

[0073] (1) When the total number of reads is <100, it is considered that the genotype is not detected at this site and is recorded as “-”;

[0074] (2) Total number of reads ≥ 100 and read ins Quantity>read del When del If the number of reads is greater than 10% of the total number of reads, the genotype of the marker is considered to be heterozygous and is recorded as "H"; del If the number is ≤ 10% of the total number of reads, the genotype of the site is considered to be insertion, which is recorded as "I";

[0075] (3) Total number of reads ≥ 100 and read ins Quantity<read del When ins If the number of reads is greater than 10% of the total number of reads, the genotype of the marker is considered to be heterozygous and is recorded as "H"; ins If the number is ≤ 10% of the total number of reads, the genotype of the site is considered to be deletion, denoted as "D".

[0076] 2. Detection and application of InDel marker genotypes in Castanea germplasm resources

[0077] Young leaves of germplasm resources were collected, genomic DNA was extracted, and a gDNA library was constructed; the genotypes of 76 InDel markers in the germplasm resources were identified by the InDel marker genotype detection system constructed above.

[0078] Example 3

[0079] 1. Construction of InDel genotype database of Castanea germplasm resources

[0080] The present invention collected 70 Castanea germplasm resources, including 57 varieties (lines) of chestnut, 4 varieties of Castanea henryi, 6 varieties of Japanese chestnut and 3 varieties of Castanea chinensis; the 57 chestnut varieties (lines) included 25 varieties of the North China variety group, 4 varieties of the Northwest variety group, 11 varieties of the Southwest variety group, 2 varieties of the Southeast variety group, 5 varieties of the Yangtze River Basin variety group and 10 hybrid superior lines (see Table 3). The young leaves of the germplasm resources were collected, and the genomic DNA was extracted using the Plant / Seed DNA Mini-Extraction Kit TD619-200 (Jianshi Biotechnology (Beijing) Co., Ltd.), and the concentration of the DNA solution was finally adjusted to 100 ng / μL for InDel genotype detection.

[0081] Table 3 Castanea germplasm resources used to construct the InDel genotype database

[0082]

[0083]

[0084] First, the 76 InDel markers developed above were divided into 8 groups according to the order shown in Table 4; the genotypes of the InDel markers of each germplasm resource were concatenated in the order in the InDel genotype database of Castanea germplasm resources, with spaces between the groups.

[0085] Table 4 Arrangement order of InDel markers

[0086]

[0087] The marker developed in Example 1 and the genotype detection method in Example 2 were used to perform InDel marker genotype detection on 70 germplasm resources. The detection results of each germplasm resource were organized into a row, with the first column being the sample ID and the second column being the genotype combination of the InDel marker. Finally, a database containing 70 Castanea germplasm resources and 76 InDel marker genotypes was constructed, and the database construction results are shown in Table 5.

[0088] Table 5 InDel genotype combination database of Castanea germplasm resources

[0089]

[0090]

[0091] 2. Specific identification of Castanea germplasm resources

[0092] The specificity of the genotype combinations of the above 70 Castanea germplasm resources was analyzed using CMIPAT software (registration number: 2024SR1209846). The results showed that there were 64 specific genotype combinations in the database. Six pairs of replicate samples shared six specific genotype combinations, including two 'Qianxi Zaohong' (SQY071 and CL11), two 'Yanshan Zaofeng' (SQY095 and YSZF), two 'Yanbao' (SQY090 and CL54), two 'Yanlong' (SQY089 and YL), two 'Yanli' (SQY097 and CL18), and two 'Yanxi' (SQY087 and YX); the remaining 58 germplasm resources had specific genotype combinations. Since the genetic background of the repeated samples is exactly the same, the result of the CMIPAT software analysis is consistent with the result of the InDel genotype combination database of Castanea germplasm resources in Table 5, indicating that the InDel markers developed in the present invention have good stability and can be used for the specific identification of Castanea germplasm resources.

[0093] 3. Pedigree analysis and hybrid identification of Castanea germplasm resources

[0094] The InDel markers developed by the present invention follow the Mendelian inheritance law between parents and offspring, so the genotype of each InDel marker follows the following law between parents and F1 hybrids in pedigree analysis: when the genotype of the F1 sample is D, the genotype combination of the parent pair is D×D, D×H, H×D or H×H; when the genotype of the F1 sample is I, the genotype combination of the parent pair can be I×I, I×H, H×I or H×H; when the genotype of the F1 sample is H, the genotype combination of the parent pair is D×I, I×D, D×H, I×H, H×D, H×I or H×H. Based on this law, each InDel marker of each hybrid can be compared one by one by CMIPAT software to see whether it conforms to the above-mentioned inheritance law, and the confidence of the possible parent pair of the F1 sample (the number of markers conforming to the law / the total number of markers) can be calculated. The database contains 8 hybrid superior lines, of which A34, A35 and A36 are hybrid offspring of 'Yanshan Zaofeng'×'Yanli', B2 and B4 are hybrid offspring of 'Yanlong'×'Shuofeng', and C1, C2 and C5 are hybrid offspring of 'Yanbao'×"Funing No. 16". According to the genotype combination based on InDel markers in Example 1, the CMIPAT software was used to predict the possible parents and parent pairs of these 8 hybrids in the database. The results showed that the optimal parent combination of these hybrids was completely consistent with the known parent combination, and the confidence level was ≥96.05%, as shown in Table 6. This shows that the InDel markers developed by the present invention can be used for pedigree analysis of Castanea germplasm resources, and can also be used for identification and analysis of the purity of hybrid offspring, that is, to calculate the proportion of true hybrids in a specific hybrid population.

[0095] Table 6 InDel genotype combination database of Castanea germplasm resources

[0096]

[0097] 4. Analysis of phylogenetic relationships of Castanea germplasm resources

[0098] After removing the F1 hybrids and duplicate samples from the above database, the remaining 56 germplasms were reorganized into genotype format according to the assignment method of D = 1, H = 2, I = 3, and the genotypes of each marker were separated by tabs. Then, the hclust() function in R language was used to construct the phylogenetic tree of these germplasm resources.

[0099] like Figure 5 As shown, the results show that the cluster analysis of the InDel markers developed in Example 1 of the present invention can cluster the various varieties of Japanese chestnut, Castanea henryi and Castanea mollissima in specific branches, and indicate that the relationship between Japanese chestnut and Castanea mollissima is farther than that between Castanea henryi. This indicates that the InDel markers developed in the present invention can be used for phylogenetic relationship analysis of Castanea germplasm resources.

[0100] 5. Construction of genetic map and gene localization of Castanea

[0101] As an important genetic marker, InDel markers have been widely used in the construction of crop linkage maps.

[0102] The present invention uses Mapchat software to draw a physical map of the 76 InDel molecular markers developed in Example 1 on the chestnut chromosome ( Figure 1 ); By detecting the InDel genotype of each individual in a large-scale F1 population and calculating the genetic linkage relationship between InDel markers, the genetic linkage map of chestnut can be further drawn, which can lay the foundation for the genetic positioning of chestnut agronomic traits.

[0103] 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. An InDel marker for Castanea, characterized in that: The nucleotide sequences of the InDel markers are shown in SEQ ID NO.1 to SEQ ID NO.

76.

2. A probe combination for detecting the InDel marker of claim 1, characterized in that: The nucleotide sequences of the probe combination are shown in SEQ ID NO.77 to SEQ ID NO.

228.

3. The probe combination according to claim 2, characterized in that: The 5' end of each probe sequence of the probe combination is labeled with biotin.

4. A detection system for detecting the InDel marker according to claim 1, characterized in that: The detection system comprises streptavidin-coated magnetic beads and the probe combination according to claim 2 or 3.

5. Use of the probe combination according to claim 2 or 3 or the detection system according to claim 4 in identifying Castanea germplasm resources.

6. Use of the probe combination according to claim 2 or 3 or the detection system according to claim 4 in identifying the genotype of InDel markers of Castanea germplasm resources, characterized in that: The InDel marker is the InDel marker according to claim 1.

7. Use of the probe combination according to claim 2 or 3 or the detection system according to claim 4 in pedigree analysis of Castanea germplasm resources and / or hybrid identification.

8. Use of the probe combination according to claim 2 or 3 or the detection system according to claim 4 in the analysis of phylogenetic relationships of Castanea germplasm resources.

9. Use of the probe combination according to claim 2 or 3 or the detection system according to claim 4 in the construction of genetic maps and gene localization of Castanea plants.

10. The use according to any one of claims 5 to 9, characterized in that: The Castanea plant includes one or more of Castanea mollissima, Castanea henryi ... and Castanea japanensis.

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