A macadamia nut SNP molecular marker and its application

Abstract

The present invention belongs to the technical field of plant molecular breeding and germplasm identification, and specifically relates to a macadamia SNP molecular marker and its application. Specifically, the present invention uses resequencing data of 208 macadamia germplasms to identify and screen core SNP markers in the whole genome. Based on the core SNP markers, the DNA fingerprint map and phylogenetic tree of the above macadamia germplasm are constructed, which provides data reference for the management, utilization and new variety registration of macadamia germplasm resources, and therefore has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant molecular breeding and germplasm identification, and specifically relates to a macadamia nut SNP molecular marker and an application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Macadamia nuts (Macadamia spp.) are native to Australia and are a woody oil crop with important nutritional value. The fat content of macadamia kernels is about 75%, mainly monounsaturated fatty acids. The health benefits of macadamia nuts have been reported many times.

[0004] Initially, through a comprehensive evaluation of early introduced germplasm, some foreign excellent varieties were selected and dominated the cultivation of macadamia nuts in China. With the progress of domestic macadamia germplasm resource collection and breeding work, the number of macadamia germplasm resources and newly registered, approved (recognized) varieties is increasing. Considering the relative scarcity of channels for introducing macadamia nuts and collecting germplasm resources in China, different research institutions may have different names for the same germplasm they preserve, and different provinces may have repeated registrations or approvals for the same excellent strain. Effective and accurate identification of existing germplasm resources and varieties is crucial to the management of macadamia germplasm resources, the protection of variety rights and the healthy development of the industry.

[0005] Germplasm identification based on morphological characteristics is a traditional method, which has the disadvantages of being time-consuming, laborious and susceptible to environmental influences. In recent years, the use of DNA fingerprinting methods for plant identification has become more common, and it can serve as an effective supplement to traditional morphological identification. Various types of molecular markers can be used to construct DNA fingerprints, among which SSR markers and SNP markers are more recommended. In a study report on DNA fingerprinting of macadamia nuts, Li Zhiqiang et al. used 9 pairs of primers screened from 240 pairs of SSR primers to construct DNA fingerprints of 83 macadamia nut germplasm resources. Huang Jianting et al. developed 12 pairs of EST-SSR primers for the construction of DNA fingerprints of 51 macadamia nut germplasms. Compared with other molecular markers, SNP markers have the characteristics of high throughput, large number and wide distribution. SNP markers have been widely used in the construction of DNA fingerprints of plants such as Brassica napus, cigar tobacco, oil tea and tea trees. However, the inventors found that there is no research on the application of SNP markers to macadamia nut fingerprints. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a macadamia SNP molecular marker and its application. Specifically, the present invention uses the resequencing data of 208 macadamia germplasms to identify and screen core SNP markers in the whole genome. Based on the core SNP markers, the DNA fingerprint map and phylogenetic tree of the above macadamia germplasm are constructed to provide data reference for the management, utilization and new variety registration of macadamia germplasm resources. Based on the above research results, the present invention is completed.

[0007] Specifically, the present invention relates to the following technical solutions:

[0008] In a first aspect of the present invention, a macadamia nut SNP molecular marker is provided, and the site information of the SNP molecular marker is as follows (Table 1):

[0009] Table 1

[0010]

[0011]

[0012]

[0013]

[0014]

[0015]

[0016]

[0017]

[0018] The physical positions of the above SNP molecular markers are based on the Macadamia integrifolia genome (Accession number: GWHBAUK00000000) as the reference genome.

[0019] In a second aspect of the present invention, the use of the above-mentioned macadamia nut SNP molecular marker and / or a product for detecting the above-mentioned macadamia nut SNP molecular marker in any one or more of the following is provided:

[0020] 1) Analysis of genetic diversity of macadamia;

[0021] 2) Construction of macadamia phylogenetic tree and principal component analysis;

[0022] 3) Construction of macadamia genetic map;

[0023] 4) Identification of macadamia germplasm;

[0024] 5) Analysis of the genetic relationship of macadamia nuts;

[0025] 6) Macadamia functional gene mining;

[0026] 7) Molecular marker-assisted breeding of macadamia nuts.

[0027] The third aspect of the present invention is a method for constructing a DNA fingerprint of macadamia nuts, the method at least comprising: extracting genomic DNA of macadamia nuts to be tested, determining the genotype of the SNP site of the macadamia nuts to be tested based on the above-mentioned macadamia nut SNP molecular markers, and using qrencode to construct a 2D barcode fingerprint based on the macadamia nut SNP molecular markers.

[0028] A fourth aspect of the present invention provides a method for constructing a phylogenetic tree of macadamia germplasm resources, the method comprising: based on the above-mentioned macadamia SNP molecular markers, using IQ-TREE software, based on the maximum likelihood method, bootstrap repeated 1,000 times, to construct a phylogenetic tree of the macadamia germplasm to be tested.

[0029] A fifth aspect of the present invention provides a method for cluster analysis of macadamia germplasm resources, the method comprising: based on the above-mentioned macadamia SNP molecular markers, using EIGENSOFT software to perform principal component analysis to obtain the clustering of the macadamia germplasm to be tested.

[0030] Beneficial technical effects of one or more of the above technical solutions:

[0031] The above technical solution obtained 337 core SNP markers of macadamia nut by screening, and further evaluated its genetic diversity, constructed phylogenetic trees and DNA fingerprint maps, and performed principal component analysis on them; thereby effectively identifying macadamia nut germplasm, improving the efficiency of macadamia nut variety identification, and providing data reference for the management, utilization and new variety registration of macadamia nut germplasm resources, and has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the drawings required for use in the description of the embodiment will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0033] Figure 1 The figure shows the distribution of 337 core SNPs on macadamia chromosomes in the examples of the present invention.

[0034] Figure 24 genetic diversity index distribution diagrams of 337 SNP sites in the embodiment of the present invention.

[0035] Figure 3 Phylogenetic tree (A) and principal component analysis (B) of 208 macadamia germplasms based on 337 SNPs in the examples of the present invention.

[0036] Figure 4 This is the DNA fingerprint of 208 macadamia germplasms in the embodiment of the present invention, where each row represents a SNP site and each column represents a germplasm. The colors are yellow, green, blue and purple, respectively, missing data are displayed in gray, and heterozygous site data are displayed in white.

[0037] Figure 5 This is the distribution of SNP sites that differ between two qualities of 208 macadamia nuts in the examples of the present invention. DETAILED DESCRIPTION

[0038] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0039] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0040] The present invention is further described in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit the content thereof. If the specific experimental conditions are not specified in the examples, they are usually carried out under conventional conditions or under conditions recommended by the sales company; the materials and reagents used in the examples, unless otherwise specified, can be purchased through commercial channels.

[0041] In a typical embodiment of the present invention, a macadamia SNP molecular marker is provided, and the SNP molecular marker is as shown above, specifically a set of numbers SNP1-SNP337. The above SNP molecular marker is a core SNP marker of macadamia obtained by screening, which has high quality, strong representativeness, and high genetic variation, and is therefore very suitable for the construction of a DNA fingerprint of macadamia.

[0042] In another specific embodiment of the present invention, the use of the above-mentioned macadamia nut SNP molecular marker and / or the product for detecting the above-mentioned macadamia nut SNP molecular marker in any one or more of the following is provided:

[0043] 1) Analysis of genetic diversity of macadamia;

[0044] 2) Construction of macadamia phylogenetic tree and principal component analysis;

[0045] 3) Construction of macadamia genetic map;

[0046] 4) Identification of macadamia germplasm;

[0047] 5) Analysis of the genetic relationship of macadamia nuts;

[0048] 6) Macadamia functional gene mining;

[0049] 7) Molecular marker-assisted breeding of macadamia nuts.

[0050] In another specific embodiment of the present invention, the products for detecting macadamia nut SNP molecular markers include but are not limited to primers, probes, gene chips, reagents, detection devices and equipment used based on high-throughput sequencing, which are not specifically limited here.

[0051] In another specific embodiment of the present invention, in said 3), constructing the macadamia nut genetic map is specifically constructing a macadamia nut DNA fingerprint map.

[0052] In another specific embodiment of the present invention, in said 4), the identification of macadamia germplasm at least includes the identification of macadamia varieties.

[0053] In another specific embodiment of the present invention, a method for constructing a DNA fingerprint of macadamia nuts is provided, the method at least comprising: extracting genomic DNA of macadamia nuts to be tested, determining the genotype of the SNP site of macadamia nuts to be tested based on the above-mentioned macadamia nut SNP molecular markers, and using qrencode to construct a 2D barcode fingerprint based on the macadamia nut SNP molecular markers.

[0054] In another specific embodiment of the present invention, a method for constructing a phylogenetic tree of macadamia germplasm resources is provided, the method comprising: based on the above-mentioned macadamia SNP molecular markers, using IQ-TREE software, based on the maximum likelihood method, bootstrap repeated 1,000 times, to construct a phylogenetic tree of the macadamia germplasm to be tested.

[0055] In another specific embodiment of the present invention, a method for clustering analysis of macadamia germplasm resources is provided, the method comprising: based on the above-mentioned macadamia SNP molecular markers, using EIGENSOFT software to perform principal component analysis to obtain the clustering situation of the macadamia germplasm to be tested.

[0056] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0057] Example

[0058] 1. Materials

[0059] A total of 208 macadamia germplasm resources were collected in the experiment (Table 2), all of which were stored in the Jinghong macadamia germplasm resource nursery of the Ministry of Agriculture and Rural Affairs (Xishuangbanna, Yunnan, China; 22°0′N, 100°46′E). Among them, 49 germplasms were introduced from the United States, 48 ​​germplasms were introduced from Australia, and 111 germplasms were obtained through breeding in China. Among the above 111 germplasms, 29, 77 and 5 germplasms were obtained through hybrid breeding, seed selection and mutation breeding, respectively. The above materials were divided into American germplasm (A1), Australian germplasm (B2) and Chinese germplasm (C3) according to their sources.

[0060] Table 2

[0061]

[0062]

[0063] 2. Methods

[0064] 2.1 Extraction of genomic DNA

[0065] Fresh leaves of 208 accessions were collected, frozen in liquid nitrogen and stored in an ultra-low temperature refrigerator at -80°C. Genomic DNA was extracted using a kit (TaKaRa MiniBEST). 1% agarose gel electrophoresis and Qubit 3.0 nucleic acid protein quantifier were used to detect the quality and concentration of DNA.

[0066] 2.2 Library construction and whole genome resequencing

[0067] The DNA was fragmented by ultrasonic shearing, and then the library was established through the steps of fragment size selection, DNA end repair, 3′ end A addition, sequencing adapter connection, PCR amplification and purification, and finally the library was sequenced using the DNBSEQ-T7 platform. The original sequencing sequence obtained by sequencing was filtered, and the main steps were as follows: (1) removing the reads with adapters; (2) filtering the reads with N content exceeding 10%; (3) removing the reads with bases with a quality value below 10 exceeding 50%. The filtered sequencing sequences were aligned to the macadamia reference genome (Accession number: GWHBAUK00000000, https: / / ngdc.cncb.ac.cn / gwh / Assembly / 18686 / show) using BWA software. The sequencing depth, genome coverage and other information of each sample were counted.

[0068] 2.3 Detection and screening of SNP mutations

[0069] According to the positioning results, samtools (v1.9) was used to filter redundant reads to ensure the accuracy of the detection results. Then, the local haplotype assembly algorithm of GATK was used to detect SNP variations. Each sample first generated gVCF, and then the population joint-genotype was performed. Finally, the SNP variation site set was filtered and obtained. The SNP site set was strictly filtered to ensure its reliability. The main filtering parameters were as follows: (1) SNPs within 5 bp near INDEL and adjacent INDELs within 10 bp were filtered out; (2) the number of variants within the 5 bp window did not exceed 2; (3) QUAL quality values ​​below 30 were filtered out; (4) QD values ​​below 2.0 were filtered out; (5) MQ values ​​below 40 were filtered out; (6) FS values ​​above 60 were filtered out; (7) Other variation filtering parameters were processed using the default values ​​officially specified by GATK. SNP sites with minor allele frequency greater than 5% and data missing rate less than 20% were further filtered. Based on the reference genome, snpEff software (3.6c) was used for SNP annotation.

[0070] 2.4 Screening of core SNP markers and construction of DNA fingerprints

[0071] The detected SNP sites were screened to obtain core markers based on the following principles: (1) the markers are evenly distributed on the genome; (2) there are no missing sites in the markers, that is, the site integrity is 100%; (3) sites with minor allele fraction (MAF) less than 20% are discarded; (4) sites with polymorphic information content (PIC) less than 0.35 are discarded; (5) Hardy-Weinberg test, retaining sites with p-value greater than 0.01; (6) there are no other site mutations 100 bp before and after the selected markers. qrencode was used to construct a 2D barcode fingerprint map of the selected core markers. At the same time, based on the selected core SNPs, IQ-TREE software was used to construct a phylogenetic tree of various germplasms based on the maximum likelihood method and 1,000 bootstrap repetitions. Principal component analysis was performed using EIGENSOFT software to obtain the clustering of germplasms.

[0072] 3. Results

[0073] 3.1 Evaluation of whole-genome resequencing

[0074] The whole genome resequencing of 208 macadamia germplasm materials generated a total of 1110.83Gb of effective data, with a Q30 of 90.87%. The alignment rate with the reference genome was 90.53% to 99.63%, with an average alignment rate of 99.01%; the average sequencing depth and coverage distribution were 6× and 92.30%. The above data show that the sequencing data is of high quality and the results are reliable.

[0075] 3.2 Identification and screening of SNPs

[0076] A total of 458,205,696 SNPs were identified using resequencing technology. Statistical analysis showed that the main variation type of SNPs was conversion, with a total of 305,637,172 SNPs, and the conversion / transversion ratio was 1.99; the number of homozygous SNPs and heterozygous SNPs were 262,858,296 and 195,347,400, respectively, and the proportion of heterozygous SNP types was 18.41% to 92.01%; for each material, the proportion of SNPs located in the intergenic region was 26.66% to 57.90%, and the proportion of SNPs located in the endogenous region was 26.66% to 57.90%. The proportion of intronic regions was 20.80% to 40.28%, the proportion of upstream regions of genes was 11.33% to 21.77%, the proportion of downstream regions of genes was 10.24% to 19.70%, the proportion within 5′-UTR of genes was 0.58% to 1.09%, and the proportion within 3′-UTR of genes was 0.92% to 1.72%; the synonymous mutations and non-synonymous mutations SNPs contained in the coding regions were 344,600 and 404,104, respectively.

[0077] The identified SNPs were further screened to obtain core markers, and finally, a total of 337 SNP markers were obtained. The location information and variation types of the above 337 SNPs are shown in Table 1. Among them, 29 SNPs are located in the upstream region of the gene, 56 are located in the downstream region of the gene, 104 are located in the intergenic region, 1 is located in the gene, and 117 are located in the intron region. The number of SNPs with synonymous coding mutations and non-synonymous coding mutations is 13 and 5 respectively. The number of SNPs in the 5'UTR and 3'UTR of the gene is 1 and 9 respectively. There are 2 SNPs belonging to mutations in the splicing site region. The 337 SNP sites are evenly distributed on each chromosome ( Figure 1 ). Among them, the number of SNPs on chromosome 12 is the largest, which is 39. The number of SNPs on chromosome 14 is the smallest, which is 12.

[0078] 3.3 Genetic diversity assessment using core markers

[0079] Genetic diversity analysis was performed on the screened core SNP markers, and the genetic diversity indicators are shown in Table 3. The observed heterozygosity ranged from 0.375 to 0.577, with an average of 0.433. The expected heterozygosity ranged from 0.453 to 0.500, with an average of 0.481. The observed heterozygosity was less than the expected heterozygosity, indicating the absence of heterozygotes. The Nei's genetic diversity index ranged from 0.454 to 0.501, and the Shnnon's index ranged from 0.645 to 0.693. The average values ​​of the Nei's genetic diversity index and the Shnnon's index were 0.482 and 0.674, respectively. The average value of the minor allele frequency was 0.412. The minimum value of the polymorphic information content was 0.350, the maximum value was 0.375, and the average value was 0.365. The distribution of the observed heterozygosity, minor allele frequency, Nei genetic diversity, and polymorphic information content of the 337 SNP loci is shown in Table 3. Figure 2 Genetic diversity assessment showed that the core markers had high genetic variation and were suitable for the construction of macadamia DNA fingerprint.

[0080] Table 3 Genetic diversity assessment of 337 SNP loci

[0081]

[0082]

[0083] 3.4 Phylogenetic tree construction and principal component analysis

[0084] Using 337 core SNP markers, phylogenetic tree construction and principal component analysis were performed on the tested materials ( Figure 3). The phylogenetic tree results showed that the germplasm introduced from the United States (A1) showed a closer relationship than the germplasm introduced from Australia (B2) and the germplasm collected in China (C3). In the principal component analysis, some germplasms in C3 also showed obvious clustering. The results of phylogenetic and principal component analysis using 337 SNPs were consistent.

[0085] 3.5 Construction of DNA fingerprint

[0086] Using a set of 337 core SNP loci, a DNA fingerprint of 208 macadamia germplasm resources was constructed ( Figure 4 ). Compare the number of SNP sites with differences between two types of DNA ( Figure 5 ), among which, the minimum number of SNP sites with differences between two kinds of plasmids was 49, the number of sites with differences between two kinds of plasmids was less than 100 was 28, the proportion of sites with differences between 100-200 was 14.09%, the proportion of sites with differences greater than 200 was 85.78%, and the maximum number of sites with differences was 259. 2D barcode fingerprints were constructed for the selected core markers using qrencode.

[0087] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of products for detecting macadamia SNP molecular markers in any one or more of the following: 1) Analysis of genetic diversity of macadamia; 2) Construction of macadamia phylogenetic tree and principal component analysis; 3) Construction of macadamia genetic map; 4) Macadamia germplasm identification; 5) Analysis of macadamia nut genetic relationships; The site information of the SNP molecular marker is as follows: The physical location of the above SNP molecular markers is based on macadamia ( Macadamia integrifolia ) genome (Accession number: GWHBAUK00000000) is used as the reference genome.

2. The use according to claim 1, characterized in that: The products for detecting macadamia nut SNP molecular markers include primers, probes, gene chips, reagents, detection devices and equipment used based on high-throughput sequencing.

3. The use according to claim 1, characterized in that: In the application 3), constructing the macadamia genetic map is specifically constructing a macadamia DNA fingerprint map.

4. The use according to claim 1, characterized in that: In the application 4), the identification of macadamia germplasm at least includes the identification of macadamia varieties.

5. The use according to claim 1, characterized in that: In the application 3), the method for constructing a DNA fingerprint of macadamia nuts at least includes: extracting genomic DNA of macadamia nuts to be tested, determining the genotype of the SNP site of the macadamia nuts to be tested based on the macadamia nut SNP molecular markers, and constructing a 2D barcode fingerprint based on the macadamia nut SNP molecular markers using qrencode.

6. The use according to claim 1, characterized in that: In the application 2), the method for constructing a phylogenetic tree of macadamia germplasm resources includes: based on macadamia SNP molecular markers, based on the maximum likelihood method, and bootstrap repeated 1,000 times, constructing a phylogenetic tree of the macadamia germplasm to be tested.

7. The method according to claim 6, characterized in that The method was performed using IQ-TREE software.

8. The use according to claim 1, characterized in that The method for cluster analysis of macadamia germplasm resources includes: based on macadamia SNP molecular markers and principal component analysis, obtaining the clustering situation of the macadamia germplasm to be tested.

9. The method according to claim 8, characterized in that The method was performed using EIGENSOFT software.

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