Pseudo-ginseng genome breeding chip based on liquid phase hybridization capture technology and development method thereof

Abstract

The invention discloses a pseudo-ginseng genome breeding chip based on a liquid phase hybridization capture technology and a development method thereof. The development method comprises the following steps: S1, confirming a target area and screening sites; s2, designing an intelligent probe; s3, efficient capture detection and verification; and S4, application service. The invention provides a liquid phase chip technical solution which is low in cost, high in flexibility, high in precision, high in usability and concentrated in function. According to the technology, through a self-developed probe design strategy, the capture efficiency of a complex genome region is remarkably improved, and the technical indexes that the coverage degree is not lower than 97% and the uniformity is not lower than 90% are achieved. And by combining a DNBSEQ-T7 high-throughput sequencing platform, the overall sequencing throughput and data quality are effectively improved. Meanwhile, the scheme supports site customization design, capture sites can be flexibly configured according to different varieties, characters or breeding targets, and good expansibility and adaptability are achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of breeding chip, in particular to a Panax notoginseng genomic breeding chip based on liquid hybridization capture technology and a development method thereof. BACKGROUND

[0002] In genomic breeding and germplasm research, genotyping technology mainly relies on solid-phase chip, liquid-phase chip and whole genome sequencing (WGS). Solid-phase chip is based on microbead array hybridization technology, which has the advantages of maturity and stability, but has limitations such as fixed site, high cost and difficulty in covering complex genomic regions. While WGS can provide comprehensive data, the sequencing cost is high, and the data is redundant, which is not conducive to large-scale breeding applications. Liquid hybridization capture technology has wide marker coverage, including whole genome sites; high accuracy, based on NGS sequencing, which greatly avoids ambiguous results and provides high-depth coverage of target regions. In recent years, targeted capture sequencing technology has realized efficient enrichment of target regions through liquid hybridization probes, which has improved cost and flexibility, but still faces problems such as complex probe design and insufficient uniformity.

[0003] Moreover, in the prior art, the genotyping chip for Panax notoginseng has many limitations, mainly in the following aspects: first, there is no publicly released SNP / InDel chip for Panax notoginseng on the market, and it is urgent to develop an efficient breeding chip suitable for this species. Second, the sample size of existing research is limited, making it difficult to accurately locate multiple phenotypes related to saponin content and disease resistance, resulting in key functional genes (such as saponin synthesis-related genes and disease resistance genes) not being effectively included in the probe design, limiting the chip's functional coverage capability in actual breeding. Third, although whole genome sequencing (WGS) technology can provide comprehensive genetic information, it is costly, redundant and complex in analysis process, making it difficult to adapt to long-term application of large-scale breeding populations; compared with traditional targeted capture technology (such as liquid hybridization probe capture), it to some extent balances data depth and detection cost, but still faces problems such as long probe design cycle, poor capture effect on complex genomic regions, low capture uniformity (usually less than 80%), limited throughput, etc., making it difficult to meet the high-throughput and high-precision genotyping needs of Panax notoginseng complex genome. SUMMARY

[0004] The purpose of the present application is to provide a Panax notoginseng genomic breeding chip based on liquid hybridization capture technology and a development method thereof, which solves the problems of high cost, low throughput, insufficient target region coverage and poor adaptability to complex genomes in traditional genomic sequencing and breeding chip technology.

[0005] The above technical purpose of the present application is realized by the following technical scheme: A method for developing Panax notoginseng genomic breeding chips based on liquid-phase hybridization capture technology, comprising the following steps: S1, target region confirmation and site screening: integrate high-depth resequencing data, perform GWAS research on SNP and InDel related to different Panax notoginseng saponins and disease resistance related phenotypes based on GEMMA model and FastLMM model, and extract function gene sites positioned significantly, screen with strict standards of MAF≥0.05 and deletion rate<10%, and finally optimize about 2K SNP / Indel sites related to breeding traits; S2, intelligent probe design: the probe length is generally 100 nt, considering the capture efficiency and sample adaptability; the design considers the influence of genome specificity and GC / AT content on binding capacity, and avoids hairpin structure and dimer formation, and for complex regions, the capture efficiency is improved by density compensation; S3, high-efficiency capture detection and verification: combined with DNBSEQ-T7 high-throughput sequencing platform, the average sequencing depth of the target region is realized> 10X, and a quality control system covering the coverage and uniformity is established to ensure the accuracy and stability of the typing results.

[0006] Further preferably, S1 specifically comprises the following steps: S1.1, first collect and analyze high-depth (average 11.7X) whole-genome resequencing data of 3 different populations, a total of nearly 932 samples, perform GWAS mining, and screen out potential important genetic sites; S1.2, functionally annotate the above SNP sites, and extract function sites with potential biological significance; S1.3, combine the results of GWAS analysis of SNP and InDel based on GEMMA model and FastLMM model, and obtain the final 2K chip sites; S1.4, comprehensively screening about 2K function genes and mutation sites of key economic traits related to saponin content and disease resistance, and developing liquid-phase capture chip for genomic selection breeding.

[0007] Further preferably, S2 specifically comprises the following steps: S2.1, probe length: the probe length can be flexibly selected between 80-120 nt, and for special samples such as ancient DNA, the length can be adjusted according to the sample characteristics to balance the binding efficiency and capture effect; S2.2, probe layer: refers to the average number of times the target region is covered by probes, for animal and plant genomes, single-layer design is recommended to reduce redundancy and non-specific capture; S2.3, probe specificity: that is, the uniqueness of the probe in the whole genome; when designing, the probe should be avoided to be arranged in the low complexity region; if the functional hotspot is just located in the region, the adjacent sequence background should be comprehensively evaluated, the probe should be arranged carefully to ensure the reliability of the capture; S2.4, probe binding capacity: the optimal capture region GC content is about 50%; in the high GC region, although the probe binding capacity is strong, the DNA fragments are also easy to self-bind, which leads to the decline of the competition of the probe capture; and the high AT region has weak binding capacity, which also affects the capture efficiency; therefore, in the extreme GC / AT content region, the probe density should be appropriately increased to make up for the insufficient efficiency; S2.5, probe preparation and amplification: the probe needs to be amplified in quantity through PCR after high-throughput synthesis; due to the influence of different GC contents on the amplification efficiency, the amplification conditions should be optimized to avoid amplification bias and the decline of the capture uniformity; S2.6, probe secondary structure optimization: in the design, the probe itself forming a hairpin structure or the probes forming a dimer should be avoided as much as possible, and these structures will interfere with the binding of the probe and the target DNA; the probe hybridization characteristics are reasonably utilized to optimize the coverage, uniformity and specificity on the premise of ensuring the overall capture capacity; in addition, each probe can effectively capture the adjacent fragments within a certain range on both sides of the target sequence matched with the probe at 100%, and the coverage capacity of the actual capture region is improved.

[0008] Further preferably, in S2.1, the sample has a length of 100 nt.

[0009] Further preferably, in S3, the accuracy and specificity of the typing are comprehensively ensured by performing systematic detection on the deletion rate, secondary allele frequency and the like of the typing data of the sample chip site.

[0010] The application also provides a Panax notoginseng genomic breeding chip based on a liquid-phase hybridization capture technology.

[0011] In summary, the application has the following beneficial effects: The application provides a low-cost, high-flexibility, high-precision, high-usage and function-concentrated liquid-phase chip technology solution. The technology significantly improves the capture efficiency of a complex genomic region through a self-developed probe design strategy, and reaches the technical indexes of a coverage degree of not less than 97% and a uniformity of not less than 90%. In combination with the DNBSEQ-T7 high-throughput sequencing platform, the overall sequencing throughput and data quality are effectively improved. Meanwhile, the solution supports site customization design, can flexibly configure the capture site according to different varieties, traits or breeding targets, and has good expansibility and adaptability. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1is one of the saponin content phenotypes in the application - 3 total saponin content phenotype SNP_GWAS result figure; Figure 2 is one of the saponin content phenotypes in the application - 3 total saponin content phenotype InDel_GWAS result figure; Figure 3 is one of the disease resistance related phenotypes in the application - disease resistance phenotype SNP_GWAS result figure of GEMMA model; Figure 4 is one of the disease resistance related phenotypes in the application - disease resistance phenotype SNP_GWAS result figure of FastLMM model; Figure 5 is one of the disease resistance related phenotypes in the application - disease resistance phenotype InDel_GWAS result figure of FastLMM model; Figure 6 is the capture effect diagram in the application; Figure 7 is the chip site statistics diagram in the application; Figure 8 is the site distribution diagram on the chromosome in the application. DETAILED DESCRIPTION

[0013] The application will be further described in detail below in combination with the drawings.

[0014] Embodiment, a ginseng genome breeding chip development method based on liquid phase hybridization capture technology, comprising the following steps: S1, target region confirmation and site screening: integrate high-depth resequencing data, perform GWAS research on SNP and InDel of different ginseng saponin and disease resistance related phenotypes based on GEMMA model and FastLMM model, and extract the function gene sites positioned significantly, adopt strict standards of MAF≥0.05 and deletion rate<10% for screening, and finally optimize and configure about 2K SNP / Indel sites related to breeding traits; S1.1, first, high-depth (average 11.7X) whole genome resequencing data of nearly 932 samples of 3 different populations were collected and analyzed, GWAS mining was carried out, and potential important genetic sites were screened out; S1.2, function annotation was performed on the above SNP sites, and function sites with potential biological significance were extracted; S1.3, the results of joint GWAS analysis of SNP and InDel based on GEMMA model and FastLMM model were screened out, and the final 2K chip sites were obtained; S1.4, Based on the above information, about 2K functional genes and mutation sites related to saponin content and disease resistance were finally screened out, and a liquid capture chip SQ2K for genomic selection breeding was developed.

[0015] S2, Intelligent probe design: The probe length is generally 100 nt, which takes into account the capture efficiency and sample adaptability; the design takes into account the influence of genome specificity and GC / AT content on binding capacity, and avoids the formation of hairpin structure and dimer; for complex regions, the capture efficiency is improved by density compensation to improve coverage (≥97%) and uniformity (≥90%); S2.1, Probe length: The probe length can be flexibly selected between 80-120 nt, and in this embodiment, the sample uses 100 nt length. For special samples such as ancient DNA, the length can be adjusted according to the characteristics of the sample to balance the binding efficiency and capture effect; S2.2, Probe layer: It refers to the number of times the target region is covered by the probe on average. For animal and plant genomes, single-layer design is recommended to reduce redundancy and non-specific capture; S2.3, Probe specificity: It refers to the unique matching of the probe in the whole genome; the higher the specificity, the better the capture efficiency. When designing, the probe should be avoided to be placed in the low complexity region; if the functional hotspot is located in such a region, the adjacent sequence background should be comprehensively evaluated, and the probe should be carefully placed to ensure the reliability of capture; S2.4, Probe binding capacity: The optimal capture region GC content is about 50%; in high GC region, although the probe binding capacity is strong, the DNA fragments are also easy to self-bind, which leads to the decline of the competition of probe capture; while in high AT region, the binding capacity is weak, which also affects the capture efficiency; therefore, in the extreme GC / AT content region, the probe density should be appropriately increased to make up for the insufficient efficiency; S2.5, Probe preparation and amplification: After high-throughput synthesis, the probe needs to be amplified in quantity by PCR; due to the influence of different GC contents on amplification efficiency, the amplification conditions should be optimized to avoid amplification bias and decrease in capture uniformity; S2.6, Probe secondary structure optimization: In the design, the probe itself forming hairpin structure or the probe forming dimer should be avoided as much as possible, which will interfere with the binding of the target DNA; the probe hybridization characteristics are reasonably utilized to optimize the coverage, uniformity and specificity on the premise of ensuring the overall capture capacity; in addition, each probe can effectively capture the adjacent fragments within a certain range on both sides of the 100% matched target sequence, which improves the coverage capacity of the actual capture region.

[0016] S3, High-efficiency capture, detection and verification: combined with the DNBSEQ-T7 high-throughput sequencing platform, achieve an average of >10X sequencing depth in the target region, and establish a quality control system covering coverage and uniformity, to ensure the accuracy and stability of the typing results; through the systematic detection of sample chip site typing data on indicators such as deletion rate and secondary allele frequency, the accuracy and specificity of typing are comprehensively guaranteed.

[0017] The "SQ2K" chip proposed in the application is the first molecular breeding chip specially designed for Panax notoginseng breeding. The chip is developed based on liquid-phase hybridization capture technology and has significant advantages in flexibility, accuracy, cost-effectiveness and application range, filling the technical gap in this field and providing an efficient and reliable tool for Panax notoginseng molecular breeding. Its advantages are as follows: a. High cost performance: relying on a high-throughput synthesis platform to prepare liquid-phase probes, significantly reducing costs and shortening delivery cycles.

[0018] b. Flexible customization: supports flexible customization of sites and probes, not limited by fixed sites, covers about 2K sites, and covers multiple trait-related functional sites related to saponin content and disease resistance.

[0019] c. Rich Index combination: more than 2000 Indexes can be provided to adapt to large-scale sample parallel sequencing.

[0020] d. Diversified solutions: covering the whole process from library construction, capture to data analysis, meeting the needs of different breeding scenarios.

[0021] e. Wide applicability: widely used in variety identification, functional gene mining, population evolution, whole-genome association analysis, whole-genome selection analysis, fingerprint construction, eQTL, TWAS and genetic map analysis, for germplasm resource genotype identification, GS, genetic relationship and variety protection.

[0022] Experimental verification The chip designed based on 3 populations, 932 high-depth resequencing samples and various information collected has been verified. The SQ2K chip designed by the scheme in item 5 covers key economic traits related to saponin content and disease resistance, and successfully develops a liquid-phase capture chip for genomic selection breeding, and the GWAS results are as shown in Figures 1 to 5 , and the capture effect is as shown in Figure 6 .

[0023] The application can be widely applied to variety identification, functional gene mining, population evolution, whole genome correlation analysis, whole genome selection analysis, fingerprint construction, eQTL, TWAS and genetic map analysis, and is used for genotypic identification of germplasm resources, GS, genetic relationship and variety protection.

[0024] Result statistics: Part of the chip site is shown in the following table: The chip site is shown in Figure 7 The distribution of the site on the chromosome is shown in Figure 8 .

[0025] The embodiment is only an explanation of the application, and is not a limitation of the application, and those skilled in the art can make modifications to the embodiment without creative contribution according to the needs after reading the specification, but as long as it is within the scope of the claims of the application, it is protected by the patent law.

Claims

1. A method for developing Panax notoginseng genomic breeding chip based on liquid-phase hybridization capture technology, characterized in that, Comprise the following steps: S1, target area confirmation and site screening: integrate high-depth resequencing data, perform GWAS research on different notoginsenosides and disease resistance related phenotypes based on GEMMA model and FastLMM model, and extract significant positioning functional gene sites, adopt strict standards of MAF≥0.05 and deletion rate<10% for screening, and finally optimize about 2K SNP / Indel sites related to breeding traits; S2, intelligent probe design: the probe length is generally 100nt, which balances capture efficiency and sample adaptability; the design considers the influence of genome specificity and GC / AT content on binding capacity, and avoids hairpin structure and dimer formation, and for complex regions, the capture efficiency is improved through density compensation; S3, high-efficiency capture detection and verification: combined with DNBSEQ-T7 high-throughput sequencing platform, realize the average>10X sequencing depth of the target region, and establish the quality control system of coverage and uniformity to ensure the accuracy and stability of the typing results.

2. The method of developing Panax notoginseng genomic breeding chip based on liquid hybridization capture technology according to claim 1, characterized in that, The S1 specifically comprises the following steps: S1.1, first collect and analyze high-depth (average 11.7X) whole genome resequencing data of 3 different populations, a total of nearly 932 samples, carry out GWAS mining, and screen out potential important genetic sites; S1.2, functionally annotate the above SNP sites, and extract functional sites with potential biological significance; S1.3, combine the results of GWAS analysis of SNP and InDel based on GEMMA model and FastLMM model, and obtain the final 2K chip sites; S1.4, comprehensively screen about 2K functional genes and mutation sites of saponin content related, disease resistance related key economic traits, and develop liquid capture chip for genome selection breeding.

3. The method according to claim 2, wherein the method is characterized by, The S2 specifically comprises the following steps: S2.1, probe length: the probe length can be flexibly selected between 80-120 nt, and for special samples such as ancient DNA, the length can be adjusted according to the sample characteristics to balance the binding efficiency and capture effect; S2.2, probe layer: refers to the average number of times the target region is covered by probes, for animal and plant genomes, single-layer design is recommended to reduce redundancy and non-specific capture; S2.3, probe specificity: the uniqueness of the probe in the whole genome; when designing, the probe should be avoided to be placed in the low complexity region; if the functional hotspot is located in such a region, the adjacent sequence background should be comprehensively evaluated, and the probe should be carefully placed to ensure the reliability of capture; S2.4, probe binding capacity: the optimal capture region GC content is about 50%; in high GC region, although the probe binding capacity is strong, the DNA fragments are also easy to self-bind, which leads to the decline of the competition of probe capture; and in high AT region, the binding capacity is weak, which also affects the capture efficiency; therefore, in the extreme GC / AT content region, the probe density should be appropriately increased to make up for the insufficient efficiency; S2.5, probe preparation and amplification: after high-throughput synthesis, the probes need to be amplified by PCR; due to the influence of different GC contents on amplification efficiency, the amplification conditions should be optimized to avoid amplification bias and reduce the uniformity of capture; S2.6, secondary structure optimization of probes: in the design, the formation of hairpin structure by the probes themselves or the formation of dimers between the probes should be avoided as much as possible, which will interfere with the binding of the probes to the target DNA; the hybridization characteristics of the probes should be reasonably utilized to optimize the coverage, uniformity and specificity under the premise of ensuring the overall capture ability; in addition, each probe can effectively capture the adjacent fragments within a certain range on both sides of the target sequence with 100% match, thereby improving the coverage ability of the actual capture region.

4. The method according to claim 3, wherein the method is characterized by: In the S2.1, the sample has a length of 100 nt.

5. The method of Panax notoginseng genomic breeding chip development based on liquid-phase hybridization capture technology according to claim 4, characterized in that: In the S3, the accuracy and specificity of the typing are comprehensively guaranteed by detecting the missing rate, secondary allele frequency and other indicators of the typing data of the sample chip site.

6. A Panax notoginseng genomic breeding chip prepared by the method of claim 1-5.

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