Pinctada martensii molecular marker combination, chip and application thereof

Abstract

This invention discloses a molecular marker array, microarray, and its applications for Pinctada martensii. The molecular marker array comprises 31,053 molecular markers, the physical locations of which were determined by sequence alignment based on the Pinctada martensii reference genome CNP0002248. The SNP sites in the Pinctada martensii molecular marker array of this invention are highly representative, polymorphic, and versatile. Furthermore, it covers sites and functional sites significantly associated with traits such as shell length, shell width, shell height, shell weight, shell color, pearl color, and nacre thickness. It can be used for gene identification and functional analysis of important economic traits in Pinctada martensii, research on population genetic diversity, germplasm resource conservation, genetic improvement, and marker-assisted breeding, to cultivate new varieties with excellent economic traits and promote the healthy and sustainable development of the marine pearl industry.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a molecular marker combination, chip, and application of Pinctada martensii. Background Technology

[0002] Pinctada martensii (Pearl oyster) Pinctada fucata martensii Pinctada martensii, also known as the Hepu pearl oyster, is one of the main oyster species for cultivating seawater pearls. Its pearls, renowned as "South Sea pearls," have significant economic value. For a long time, overfishing, germplasm degradation, frequent diseases, and slow progress in selecting superior traits (such as growth rate, shell width coefficient, and nacre thickness) have severely hampered the sustainable development of the pearl farming industry. Therefore, it is urgent to conduct research on the genetic diversity of Pinctada martensii populations, protect germplasm resources, improve genetics, and use molecular marker-assisted breeding to cultivate new varieties with superior economic traits, thereby promoting the healthy and sustainable development of the seawater pearl industry. Summary of the Invention

[0003] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a molecular marker combination for Pinctada martensii.

[0004] The present invention also proposes a primer set and / or probe for detecting the above-mentioned molecular marker combination of Pinctada martensii.

[0005] The present invention also proposes a chip.

[0006] The present invention also proposes a reagent kit.

[0007] This invention also proposes an application of the above-mentioned molecular marker combination, primer set and / or probe, chip or reagent kit of Pinctada martensii.

[0008] This invention also proposes a method for distinguishing different Pinctada martensii species.

[0009] This invention also proposes a breeding method for Pinctada martensii.

[0010] According to one aspect of the present invention, a molecular marker combinatorial system for Pinctada martensii is proposed, comprising 31,053 molecular markers. The physical locations of the 31,053 molecular markers are determined by sequence alignment based on the Pinctada martensii reference genome CNP0002248, and the specific site information is shown in Table 1 below.

[0011] Table 1

[0012] In some embodiments of the present invention, the genotype information of the molecular marker is represented in the form of "ref / alt (reference base / variant base)".

[0013] In a second aspect of the invention, primer sets and / or probes for detecting the above-mentioned molecular marker combinations of Pinctada martensii are proposed.

[0014] In some embodiments of the present invention, the probe has a length of 90-110 bp.

[0015] In some embodiments of the present invention, the probe is approximately 100 bp in length.

[0016] In some embodiments of the present invention, the GC content of the probe is between 20% and 80%.

[0017] In some embodiments of the present invention, the number of homologous regions of the probe is less than 5.

[0018] In some embodiments of the present invention, the number of SNP variant sites on the probe is ≤5.

[0019] In some embodiments of the present invention, the probe has no SSR region and N region.

[0020] In a third aspect of the invention, a chip is provided that includes the aforementioned primer set and / or probes.

[0021] In a fourth aspect of the invention, a kit is provided comprising the primer set and / or probes described above.

[0022] In a fifth aspect of the invention, the use of the above-described molecular marker combinations, primer sets and / or probes, chips, or kits of *Pinctada martensii* is proposed in any of the following: C1) Identification of Pinctada martensii germplasm resources; C2) Assessment of genetic diversity in Pinctada martensii; C3) Construction of genetic map and QTL mapping of Pinctada martensii; C4) Genome-wide association study of Pinctada martensii; C5) Marker-assisted selection of Pinctada martensii; C6) Targeted improvement of Pinctada martensii; C7) Multigene aggregation breeding, whole-genome selection breeding, or intelligent design breeding of Pinctada martensii; C8) Genotyping of Pinctada martensii; C9) Cluster analysis and phylogenetic analysis of Pinctada martensii; C10) Gene identification and functional analysis of important economic traits of Pinctada martensii; C11) Population structure analysis of Pinctada martensii; C12) Germplasm resource exploration and utilization; C13) Molecular breeding of Pinctada martensii; C14) Distinguish between different varieties of Pinctada martensii or trace the origin of Pinctada martensii varieties.

[0023] In some embodiments of the present invention, the different varieties of Pinctada martensii include at least one of the following: “Haixuan No. 1” Pinctada martensii, yellow-shell color-selected Pinctada martensii, black-shell color-selected Pinctada martensii, low-temperature resistant Pinctada martensii, and natural population Pinctada martensii from Beibu Gulf.

[0024] In some embodiments of the present invention, the application can be implemented through the following methods: S1. Genotyping of the samples to be tested is performed using at least one of the following: molecular marker combination of Pinctada martensii, primer set and / or probe, chip and kit, to obtain genotyping results; S2. Analyze the genotyping results obtained in step S1.

[0025] In a sixth aspect of the present invention, a method for distinguishing different species of Pinctada martensii is proposed, comprising the following steps: using one of the above-mentioned Pinctada martensii molecular marker combinations, primer sets and / or probes, chips and kits to distinguish different species of Pinctada martensii.

[0026] In some embodiments of the present invention, the different varieties of Pinctada martensii include at least one of the following: “Haixuan No. 1” Pinctada martensii, yellow-shell color-selected Pinctada martensii, black-shell color-selected Pinctada martensii, low-temperature resistant Pinctada martensii, and natural population Pinctada martensii from Beibu Gulf.

[0027] In some embodiments of the present invention, the detection is performed based on liquid-phase probe capture sequencing genotyping technology.

[0028] In a seventh aspect of the present invention, a breeding method for Pinctada martensii is proposed, comprising the following steps: using one of the above-mentioned Pinctada martensii molecular marker combination, primer set and / or probe, chip and kit to detect the DNA of Pinctada martensii to be tested, and selecting Pinctada martensii for subsequent breeding.

[0029] In some embodiments of the present invention, the detection is performed based on liquid-phase probe capture sequencing genotyping technology.

[0030] The present invention has at least the following beneficial effects: The molecular markers in the Pinctada martensii molecular marker combination of this invention are obtained from the whole genome data analysis of 968 Pinctada martensii materials from the core breeding population and multiple wild populations. The selected gene regions have a gene coverage rate of over 97.18%, which is highly representative, polymorphic, and versatile. At the same time, it covers loci and functional sites that are significantly associated with traits such as shell length, shell width, shell height, shell weight, shell color, pearl color, and nacre thickness. It can be used for gene identification and functional analysis of important economic traits of Pinctada martensii, research on the genetic diversity of Pinctada martensii populations, germplasm resource protection, genetic improvement, and molecular marker-assisted breeding, so as to cultivate new varieties with excellent economic traits and promote the healthy and sustainable development of the marine pearl industry.

[0031] The molecular marker combination of the present invention for Pinctada martensii was used to prepare a whole genome liquid chip of Pinctada martensii, which can be widely used in different application scenarios such as germplasm resource identification and genetic diversity assessment of different Pinctada martensii samples, genetic map construction and whole genome association analysis, mining, identification and functional analysis of important trait genes, and whole genome selection breeding. It is of great significance for improving the breeding efficiency of Pinctada martensii and promoting the high-quality development of the Pinctada martensii industry.

[0032] The genotyping method of this invention is based on precise localization sequencing genotyping technology using liquid-phase capture of the target region's genomic sequence. It can not only genotype the target locus but also detect all genetic variation sites within the target region and a certain surrounding range, outputting a large amount of information. Based on high-throughput sequencing technology, this detection method has high throughput, producing a large amount of data at once, capable of simultaneously covering the detection of nearly a thousand samples; it is also compatible with mainstream second-generation sequencing platforms such as Illumina and MGI, demonstrating broad platform adaptability. Attached Figure Description

[0033] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The 30K Pinctada martensii oyster in this embodiment of the invention. c GPS liquid-phase chip site chromosome density distribution map; Figure 2 As described in the embodiments of the present invention c Schematic diagram of GPS liquid phase chip process testing; Figure 3 This is a graph showing the sample detection rate results in an embodiment of the present invention; Figure 4 This is a graph showing the sample heterozygosity detection results in an embodiment of the present invention; Figure 5 This is a graph showing the sample detection rate results in an embodiment of the present invention; Figure 6 This is a graph showing the sample heterozygosity detection results in an embodiment of the present invention; Figure 7 This is a graph showing the average genotypic consistency rate of repeated samples in this embodiment of the invention. Figure 8 This is a clustering analysis diagram in an embodiment of the present invention; Figure 9 This is a PCA analysis diagram from an embodiment of the present invention. Detailed Implementation

[0034] The following will describe the concept and technical effects of the present invention clearly and completely with reference to the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0035] Example 1 This embodiment provides a molecular marker ensemble of Pinctada martensii. The Pinctada martensii molecular marker ensemble includes 31,053 molecular marker sites. The physical locations of these 31,053 molecular marker sites were determined based on sequence alignment from the Pinctada martensii reference genome CNP0002248 (https: / / db.cngb.org / search / project / CNP0002248 / ). Specific information about these 31,053 molecular marker sites is shown in Table 1 of the specification. The screening process is as follows: 1. Collection of whole-genome sequencing data of Pinctada martensii To obtain a rich variety of whole-genome SNPs and Indel sites, a total of 968 Pinctada martensii accessions were collected from the core breeding population and multiple wild populations and sent to Huazhi Biotechnology Co., Ltd. for sequencing to obtain whole-genome sequencing data.

[0036] 2. Variation detection in resequencing data of Pinctada martensii The alignment and variant detection of the resequencing data of *Pinctada martensii* were conducted by Huazhi Biotechnology Co., Ltd., and a total of 13,776,393 SNP sites and 4,483,286 InDel sites were screened.

[0037] 3. Site screening The site screening was carried out by Huazhi Biotechnology Co., Ltd., which calculated and statistically analyzed the MAF value, detection rate, heterozygosity rate and sequencing depth of the sites. The target site set was obtained based on the minimum allele frequency (MAF) ≥ 0.1, deletion rate < 0.1, heterozygosity rate ≤ 40%, and sequencing depth ≥ 10×.

[0038] Probes were designed for the selected target loci. The probe design principles were as follows: a. Probes were designed within a 100 bp range to the left and right of each target locus, with a probe length generally of 100 bp; b. GC content of the target region was calculated; c. The number of homologous regions of the target probe was calculated. The probe selection criteria were as follows: a. GC content of the probe was between 20% and 80%; b. Number of homologous regions < 5; c. SNP variant sites on the probe ≤ 5; d. No SSR or N regions on the probe; e. After selecting the probe sequences that finally met the requirements, based on the principle of uniform distribution, 30,602 highly specific SNP sites and 451 InDel sites were selected. At the same time, 5,675 sites and functional sites that were significantly associated with traits such as shell length, shell width, shell height, shell weight, total weight, and nacre thickness of Pinctada martensii were integrated. These sites can be used for marker-assisted breeding of important economic traits and for the discovery and utilization of key trait genes. After deduplication, 31,053 molecular marker sites were finally obtained. The site information is shown in Table 1 of the instruction manual.

[0039] Example 2 This embodiment provides a 30K liquid phase chip for Pinctada martensii (Millettia speciosa). c GPS liquid phase chip).

[0040] The 31,053 molecular marker sites screened in Example 1 were used, based on the probe design principles in Example 1, and employing Huazhi's independently developed (Genotyping by Pinpoint Sequencing of liguid captured target) c GPS-based liquid-phase chip technology was used to synthesize a 30K liquid-phase chip of *Pinctada martensii*. The chromosome density distribution map of the 30K liquid-phase chip loci in *Pinctada martensii* is shown below. Figure 1 As shown. c GPS is based on an optimized thermodynamic stability algorithm model. It designs probes for genomic sequences in different target regions, uses synthesized specific probes to capture and enrich multiple different target sequences located at different genomic positions through liquid-phase hybridization, and then constructs sequencing libraries and performs high-throughput sequencing on the captured and enriched target genomic sequences to obtain the genotypes of all SNP / InDel loci within the target region. c GPS liquid-phase chip technology is a highly efficient genotyping technology applicable to chip development and genotyping of different biological species. c A schematic diagram of the GPS liquid phase chip testing process is shown below. Figure 2 As shown.

[0041] Example 3 This embodiment provides a method for genotyping Pinctada martensii samples using the 30K liquid phase chip from Embodiment 2. The steps are as follows: 1. Extraction and detection of genomic DNA from Pinctada martensii Samples from *Pinctada martensii* were selected as validation samples for the 30K liquid chromatography-mass spectrometry (LC-MS) chip development system. Genomic DNA was extracted from the tissue using a magnetic bead method. The integrity and purity of the genomic DNA were analyzed by 1% agarose gel electrophoresis, and the concentration was accurately quantified using Qubit.

[0042] 2. c GPS Experimental Procedure (1) Take 200ng of qualified gDNA, digest the genomic DNA with fragmentation enzyme, repair the digestion ends, add an A base to the 3' end, ligate the sequencing adapter and DNA fragment with T4 ligase, purify the ligation product; perform PCR amplification on the purified ligation product, and then screen the amplified product to obtain the library. (2) After adding the probe and hybridization reagent to the library and incubating, the target region is captured, followed by a round of PCR amplification to construct the desired library. c GPS sequencing libraries were prepared and then high-throughput sequencing was performed using the BGI sequencer with the PE150 sequencing strategy. (3) Regarding the above c GPS sequencing libraries were analyzed, including variant site analysis, to obtain the raw VCF variant result file. The raw data from high-throughput sequencing underwent quality control filtering, and FASTP software was used to remove adapter fragments and low-quality reads, resulting in high-quality CleanReads. BWA software was used to align the obtained CleanReads with a reference genome and sort them to obtain the sorted BAM file. GATK software was used to analyze the variant sites in the sequencing results to obtain the genotyping results for the target locus. A mutation read support ratio ≥0.8 or ≤0.2 indicates a homozygous genotype, while a mutation read support ratio between 0.2 and 0.8 indicates a heterozygous genotype.

[0043] Example 4 This embodiment provides the application of the 30K liquid phase chip in Pinctada martensii genotyping. Genotyping was performed on 10 Pinctada martensii samples from different populations (including 3 duplicate samples; additionally, the samples were different from the 968 Pinctada martensii materials in Example 1), as well as 3 non-Pinctada martensii samples from Crassula ovata, Pinctada martensii, and Pinctada macrocarpa (see Example 3 for specific operating methods).

[0044] Table 2

[0045] The results are shown in Table 2 and Figure 3-4 As shown, the detection rates of the 13 Pinctada martensii samples (including 3 duplicates) ranged from 95.00% to 97.18%, with an average detection rate of 96.56%. The detection rates of the 3 other species ranged from 2.28% to 8.08% (due to significant differences between genomes). The heterozygosity of the 13 Pinctada martensii samples ranged from 16.19% to 23.10%, with an average heterozygosity of 21.39%. Evaluation of the genotyping performance of the Pinctada martensii 30K liquid phase chip showed that the Pinctada martensii 30K liquid phase chip prepared in Example 2 of this invention exhibited good detection stability, high target site detection rate, and accurate and reliable genotyping results, capable of distinguishing different populations of Pinctada martensii.

[0046] Further genotyping was performed on 91 samples of Pinctada martensii from different populations (including 4 duplicate samples), and the results are as follows: Figure 5-6 As shown, the detection rate of the 91 Pinctada martensii samples ranged from 95.16% to 98.71%, with an average detection rate of 97.03%. The heterozygosity ranged from 15.82% to 30.43%, with an average heterozygosity of 21.45%. Seven technical replicates were set up for chip validation. The genotypic consistency rate of the replicated samples is shown in the figure. Figure 7 As shown in the figure, the genotypic consistency rate of the duplicated samples ranged from 98.14% to 98.59%, with an average consistency rate of 98.39%.

[0047] Example 5 This embodiment provides the application of the 30K liquid phase chip of Pinctada martensii in population cluster analysis of Pinctada martensii. Using the 30K liquid phase chip of Pinctada martensii from Example 2, genotyping was performed on 87 samples (of known species) of different Pinctada martensii varieties (samples different from the 968 Pinctada martensii materials in Example 1) (specific operation method is described in Example 3), obtaining genotyping results. Population structure analysis was performed on the 87 samples, and the genetic distance matrix was calculated using the IBS method in Plink software, followed by cluster analysis and phylogenetic tree construction.

[0048] The results are as follows Figure 8 As shown in the figure, the 30K liquid phase chip of Pinctada martensii prepared in Example 2 can effectively classify Pinctada martensii from different sources, and the classification results are consistent with the actual results.

[0049] Example 6 This embodiment demonstrates the application of the 30K liquid phase chip of *Pinctada martensii* in the structural analysis of *Pinctada martensii* populations. Using the 30K liquid phase chip from Example 2, principal component analysis was performed on 87 *Pinctada martensii* samples (of known species, and different from the 968 samples in Example 1) using PLINK software (V 1.9). Specific operational methods are described in Example 3, and the results are shown in [example description missing]. Figure 9 In the scatter plot, each point represents a sample, and points of the same color represent the same population. The closer two samples are in the plot, the more similar their genetic backgrounds; individuals with similar genetic backgrounds will cluster together. The results show that principal component analysis (PCA) can accurately classify Pinctada martensii samples into three categories, consistent with actual results.

[0050] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A molecular marker combination for Pinctada martensii, characterized in that, It includes 31,053 molecular markers. The physical locations of these 31,053 molecular markers were determined by sequence alignment based on the pearl oyster reference genome CNP0002248. The specific site information is shown in Table 1 of the specification.

2. Primer sets and / or probes for detecting the molecular marker combination of Pinctada martensii as described in claim 1.

3. The primer set and / or probe according to claim 2, characterized in that, The probe has a length of 90-110 bp; And / or, the GC content of the probe is between 20% and 80%; And / or, the number of homologous regions of the probe is <5; And / or, the number of SNP variant sites on the probe is ≤5; And / or, the probe has no SSR region and N region.

4. A chip, characterized in that, The chip includes the primer set and / or probe as described in claim 2 or 3; Preferably, the chip comprises a liquid phase chip.

5. A reagent kit, characterized in that, The kit contains the primer set and / or probe as described in claim 2 or 3.

6. The use of the molecular marker combination of *Pinctada martensii* according to claim 1, the primer set and / or probe according to claim 2 or 3, the chip according to claim 4, or the kit according to claim 5 in any of the following: C1) Identification of Pinctada martensii germplasm resources; C2) Assessment of genetic diversity in Pinctada martensii; C3) Construction of genetic map and QTL mapping of Pinctada martensii; C4) Genome-wide association study of Pinctada martensii; C5) Marker-assisted selection of Pinctada martensii; C6) Targeted improvement of Pinctada martensii; C7) Multigene aggregation breeding, whole-genome selection breeding, or intelligent design breeding of Pinctada martensii; C8) Genotyping of Pinctada martensii; C9) Cluster analysis and phylogenetic analysis of Pinctada martensii; C10) Gene identification and functional analysis of important economic traits of Pinctada martensii; C11) Population structure analysis of Pinctada martensii; C12) Germplasm resource exploration and utilization; C13) Molecular breeding of Pinctada martensii; C14) Distinguish between different varieties of Pinctada martensii or trace the origin of Pinctada martensii varieties.

7. The application according to claim 6, characterized in that, The different varieties of Pinctada martensii include at least one of the following: "Haixuan No. 1" Pinctada martensii, yellow-shell color-selected Pinctada martensii, black-shell color-selected Pinctada martensii, low-temperature resistant Pinctada martensii, and natural population Pinctada martensii from Beibu Gulf.

8. A method for distinguishing different varieties of Pinctada martensii, characterized in that, The method includes the following steps: using the molecular marker combination of Pinctada martensii as described in claim 1, the primer set and / or probe as described in claim 2 or 3, the chip as described in claim 4, or the kit as described in claim 5 to distinguish different species of Pinctada martensii.

9. The method according to claim 8, characterized in that, The different varieties of Pinctada martensii include at least one of the following: "Haixuan No. 1" Pinctada martensii, yellow-shell color-selected Pinctada martensii, black-shell color-selected Pinctada martensii, low-temperature resistant Pinctada martensii, and natural population Pinctada martensii from Beibu Gulf.

10. A breeding method for Pinctada martensii, characterized in that, The process includes the following steps: using the molecular marker combination of Pinctada martensii as described in claim 1, the primer set and / or probe as described in claim 2 or 3, the chip as described in claim 4, or the kit as described in claim 5 to detect the DNA of the Pinctada martensii to be tested, and selecting Pinctada martensii for subsequent breeding.

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