DNA fingerprint library of a water lily variety and its application

Abstract

The present invention belongs to the technical field of molecular biology, and particularly relates to a DNA fingerprint library of water lily varieties and its application. The DNA fingerprint library of water lily varieties provided by the present invention includes 45 SNP molecular markers, which can be used to distinguish different subgenus water lily varieties. Moreover, without designing primers, the genotype of the corresponding site of the water lily sample to be tested can be directly input into the corresponding analysis software to detect the subgenus type of the water lily sample to be tested. It has the advantages of simplicity, rapidity and low cost, and can be applied on a large scale in production.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and particularly relates to a DNA fingerprint library of water lily varieties and its application. Background Art

[0002] Water lilies are divided into two major categories according to ecological characteristics: hardy waterlily and tropical waterlily. According to geographical origin, the genus Nymphaea can be divided into subgenera such as pantemperate, pantropical, paleotropical, neotropical, and Australian. Water lily germplasm resources have extremely rich genetic diversity, but their phenotypes are very similar before flowering and it is difficult to distinguish them. For example, Blue Star and White Blue Star have extremely similar leaf shapes and colors, and can only be distinguished after flowering; even during the flowering period, a small number are difficult to distinguish, such as Egyptian White Water Lily and its variant Nymphaea lotus var. pubescens, both of which have pure white flowers and serrated leaves.

[0003] Molecular fingerprints can be used to accurately and quickly identify the genetic information of different germplasms. Therefore, when constructing a fingerprint library, it is not only required that the polymorphic sites of the markers be rich, but also that the detection method be easy to operate and highly repeatable. Although the SSR marker technology has advantages such as strong polymorphism, its disadvantages are also obvious. On the one hand, developing SSR markers requires a large number of tests and screenings after designing SSR primers based on reduced-representation genome sequencing or re-sequencing, etc. to finally determine the markers. On the other hand, the experimental throughput of SSR markers is limited and it is not convenient for large-scale operation. Using SNP sites as molecular markers can be directly combined with high-throughput sequencing technology to directly genotype the target SNPs through means such as reduced-representation sequencing, targeted sequencing, or re-sequencing, with high reliability and greater detection throughput. However, there is currently no record of SNP marker combinations for identifying the five subgenera of water lilies. Summary of the Invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art and provide a DNA fingerprint library of water lily varieties to accurately identify water lily germplasm resources.

[0005] The present invention provides a DNA fingerprint library of water lily varieties, and the DNA fingerprint library includes the following SNP molecular markers:

[0006]

[0007]

[0008] The present invention also provides the application of the DNA fingerprint library described in the above technical solution in identifying water lily varieties.

[0009] Preferably, the water lily is one or more of the water lilies of the subgenus Nymphaea of the paleotropical region, the subgenus Nymphaea of Australia, the subgenus Nymphaea of the neotropics, the subgenus Nymphaea of the wide temperate zone, and the subgenus Nymphaea of the wide tropics; the water lily sample to be tested includes tender water lily leaves.

[0010] The present invention also provides a DNA fingerprint of the water lily of the subgenus Nymphaea of the paleotropical region, and the DNA fingerprint includes the following SNP molecular markers:

[0011]

[0012]

[0013] The present invention also provides a DNA fingerprint of the water lily of the subgenus Nymphaea of Australia, and the DNA fingerprint includes the following SNP molecular markers:

[0014]

[0015]

[0016] The present invention also provides a DNA fingerprint of the water lily of the subgenus Nymphaea of the neotropics, and the DNA fingerprint includes the following SNP molecular markers:

[0017]

[0018]

[0019] The present invention also provides a DNA fingerprint of the water lily of the subgenus Nymphaea of the wide temperate zone, and the DNA fingerprint includes the following SNP molecular markers:

[0020]

[0021]

[0022] The present invention also provides a DNA fingerprint of the water lily of the subgenus Nymphaea of the wide tropics, and the DNA fingerprint includes the following SNP molecular markers:

[0023]

[0024]

[0025] The present invention provides a DNA fingerprint library of water lily varieties, including 45 SNP molecular markers, which can be used to distinguish different subgenus water lily varieties. Using the DNA fingerprint library provided by the present invention, without designing primers, directly inputting the genotypes of the corresponding sites of the water lily sample to be tested into the corresponding analysis software can detect the subgenus type of the water lily sample to be tested, which has the advantages of simplicity, rapidity, and low cost, and can be applied on a large scale in production. Description of the Drawings

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for use in the embodiments.

[0027] Figure 1 It is a clustering analysis diagram of 68 water lily germplasm resources using 45 SNPs;

[0028] Figure 2 It is a principal component analysis diagram of 68 water lily germplasm resources using 45 SNPs;

[0029] Figure 3 It is a clustering analysis diagram of 32 water lily germplasm resources using 45 SNPs;

[0030] Among them, Ao.zhou represents the Australian subgenus, Gu.re represents the Paleotropical subgenus, Guang.re represents the Pantropical subgenus, Guang.wen represents the Pantemperate subgenus, and Xin.re represents the Neotropical subgenus. Specific embodiments

[0031] The present invention provides a DNA fingerprint library of water lily varieties, and the DNA fingerprint library includes the following SNP molecular markers:

[0032]

[0033]

[0034] The present invention collected a total of 100 water lily variety germplasms (see Table 1), took fresh leaves to extract genomic DNA respectively, and then carried out steps such as enzyme digestion, sequencing, quality control, genomic mapping and screening on the genomic DNA respectively to construct a DNA fingerprint library.

[0035] In the present invention, the restriction endonucleases used for enzyme digestion are preferably EcoR I and Hin1 II; the steps of sequencing preferably include: ligating the enzyme digestion fragments obtained by enzyme digestion with sequencing adapters with barcodes, selecting fragments with lengths in the range of 250 - 550 bp, and performing PE125 paired-end sequencing to obtain initial reads.

[0036] After the enzymatic digestion and sequencing are completed, the present invention preferably performs quality control on the initial reads obtained by sequencing to obtain high-quality reads; the quality control preferably includes: performing quality statistics on the initial reads obtained by sequencing and performing a first filtration; the first filtration parameters are as follows: 1) Use the Cutadapt software to remove reads containing adapters; 2) Use the Trimmomatic software to remove reads with a proportion of N greater than 10% and low-quality reads (the number of bases with a quality value Q≤10 accounts for more than 50% of the entire read).

[0037] After obtaining the high-quality reads, the present invention preferably performs genomic mapping on the high-quality reads to obtain high-quality SNPs; the genomic mapping includes: mapping the high-quality reads to the water lily reference genome (V1.0, http: / / eplant.njau.edu.cn / waterlily / download.php) and then performing variant detection; the mapping is preferably performed using the UnifiedGenotyper module of the software GATK (3.4-46); the variant detection is preferably filtered using VariantFiltration, and the filtration parameters are -Window 4, -filter "QD<4.0||FS>60.0||MQ<40.0", -G_filter "GQ<20".

[0038] After obtaining the high-quality SNPs, the present invention preferably screens the high-quality SNPs to obtain the DNA fingerprint library of the water lily variety; the screening preferably includes primary screening and secondary screening; the primary screening preferably includes: dividing all water lily variety germplasms into dataset 1 (68 copies) and dataset 2 (32 copies), and retaining the loci with a detection rate higher than 95% in dataset 1 and a minor allele frequency not lower than 0.05 at the same time; the secondary screening preferably includes: using dataset 2 to perform a second filtration on the results of the primary screening. The screening of the present invention preferably uses the plink software, and the parameters of the second filtration are --indep-pairwise 50 1 0.8.

[0039] The DNA fingerprint library of the water lily variety of the present invention can clearly divide 68 water lily germplasm resources into 5 subgroups: the widespread temperate subgenus (42 copies), the widespread tropical subgenus (20 copies), the paleotropical subgenus (3 copies), the neotropical subgenus (1 copy), and the Australian subgenus (2 copies). The classification results are completely consistent with the classification results based on phenotypic identification, indicating that these 45 SNPs can be used as molecular fingerprints for the classification of water lily germplasm resources.

[0040] The present invention also provides the application of the DNA fingerprint library described in the above technical solution in identifying water lilies; the water lilies preferably include one or more of the water lilies of the subgenus Nymphaea in the paleotropical region, the subgenus Nymphaea in Australia, the subgenus Nymphaea in the neotropics, the subgenus Nymphaea in the wide temperate zone, and the subgenus Nymphaea in the wide tropics, and more preferably include the water lilies of the subgenus Nymphaea in Australia, the subgenus Nymphaea in the neotropics, the subgenus Nymphaea in the wide temperate zone, and the subgenus Nymphaea in the wide tropics. In the present invention, the water lily sample to be tested preferably includes fresh and tender leaves of water lilies.

[0041] The present invention also provides a DNA fingerprint of the water lily of the subgenus Nymphaea in the paleotropical region, and the DNA fingerprint includes the following SNP molecular markers:

[0042]

[0043]

[0044] The present invention also provides a DNA fingerprint of the water lily of the subgenus Nymphaea in Australia, and the DNA fingerprint includes the following SNP molecular markers:

[0045]

[0046]

[0047] The present invention also provides a DNA fingerprint of the water lily of the subgenus Nymphaea in the neotropics, and the DNA fingerprint includes the following SNP molecular markers:

[0048]

[0049]

[0050] The present invention also provides a DNA fingerprint of the water lily of the subgenus Nymphaea in the wide temperate zone, and the DNA fingerprint includes the following SNP molecular markers:

[0051] SNP Chromosome Position Nucleotide SNP Chromosome Position Nucleotide SNP1 1 425008 A SNP2 1 4524705 A / C SNP3 1 4524738 C / G SNP4 1 4524741 C / G SNP5 1 4524750 A / G SNP6 1 17880730 A / G SNP7 1 30104824 C / T SNP8 1 30104869 C / T SNP9 2 33706489 C SNP10 2 34205107 G SNP11 3 3789860 T / C SNP12 3 3789893 T SNP13 3 3789897 C / G SNP14 3 3789940 G SNP15 3 8886074 G SNP16 3 8886253 A SNP17 3 12046890 A / C SNP18 3 12046916 C / G SNP19 3 44886244 G SNP20 3 44886247 C SNP21 5 2137861 C / T SNP22 5 2137873 C / T SNP23 5 2137894 G SNP24 5 32759182 A / G SNP25 6 28986690 A / C SNP26 6 28986708 A / C SNP27 6 28986723 A / G SNP28 6 28986751 T / G SNP29 6 28986753 C SNP30 6 28986759 G SNP31 6 28986807 C / G SNP32 8 28372626 C / G SNP33 8 28372633 A SNP34 8 28372690 G SNP35 8 28372710 A / G SNP36 12 8325394 C SNP37 14 13317845 T SNP38 14 15165528 C / T SNP39 14 15165543 C / T SNP40 14 15165612 A / G SNP41 14 15165708 C / T SNP42 14 15165720 C / T SNP43 14 15165726 A SNP44 14 15165756 C / T SNP45 14 15165771 T 。

[0052] The present invention also provides a DNA fingerprint of the water lily of the subgenus Nymphaea in the wide tropics, and the DNA fingerprint includes the following SNP molecular markers:

[0053] SNP Chromosome Position Nucleotide SNP Chromosome Position Nucleotide SNP1 1 425008 A / G SNP2 1 4524705 C SNP3 1 4524738 C / G SNP4 1 4524741 C SNP5 1 4524750 A / G SNP6 1 17880730 G SNP7 1 30104824 C / T SNP8 1 30104869 C / T SNP9 2 33706489 C / T SNP10 2 34205107 G SNP11 3 3789860 T SNP12 3 3789893 T SNP13 3 3789897 G SNP14 3 3789940 T SNP15 3 8886074 T / G SNP16 3 8886253 A / C SNP17 3 12046890 A / C SNP18 3 12046916 G SNP19 3 44886244 T SNP20 3 44886247 A / C SNP21 5 2137861 C SNP22 5 2137873 C SNP23 5 2137894 G / C SNP24 5 32759182 A / G SNP25 6 28986690 A SNP26 6 28986708 C SNP27 6 28986723 G SNP28 6 28986751 T SNP29 6 28986753 T / G SNP30 6 28986759 G SNP31 6 28986807 C / G SNP32 8 28372626 C SNP33 8 28372633 A / G SNP34 8 28372690 G SNP35 8 28372710 G SNP36 12 8325394 C SNP37 14 13317845 T SNP38 14 15165528 T SNP39 14 15165543 C SNP40 14 15165612 A SNP41 14 15165708 C / T SNP42 14 15165720 T SNP43 14 15165726 A SNP44 14 15165756 C / T SNP45 14 15165771 T 。

[0054] In order to further illustrate the present invention, the following describes in detail a DNA fingerprint library of a water lily variety and its application provided by the present invention in conjunction with the drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0055] Example 1

[0056] 1. Experimental materials

[0057] A total of 100 accessions of water lily germplasm resources from 5 different subgenera were selected, specifically: subgenus Pantropical (60 accessions), subgenus Pantemperate (27 accessions), subgenus Paleotropical (6 accessions), subgenus Neotropical (3 accessions), and subgenus Australian (4 accessions). Among them, 68 accessions (test samples) were randomly selected for the construction of molecular fingerprint maps, including subgenus Pantemperate (42 accessions), subgenus Pantropical (20 accessions), subgenus Paleotropical (3 accessions), subgenus Neotropical (1 accession), and subgenus Australian (2 accessions), and the remaining 32 accessions (validation samples) were used for the verification of fingerprint maps. The material list is shown in Table 1. All materials were planted in the aquatic pool of Wuhan Academy of Landscape Sciences, and fresh leaves were taken to extract genomic DNA for subsequent experiments.

[0058] Table 1 Water lily germplasm resources used

[0059]

[0060]

[0061]

[0062]

[0063] 2. Enzyme digestion and sequencing

[0064] Two restriction endonucleases (EcoR I and Hin1 II) were used to fully digest 100 DNA samples, and the digestion conditions were referred to the instruction manual of the restriction endonuclease. After digestion, sequencing adapters with barcodes were added, the samples were mixed, a small fragment library (250 - 550 bp) was constructed, and paired-end 125 sequencing was performed.

[0065] 3. Reads quality control

[0066] The raw clean reads were strictly filtered, and the obtained high-quality reads were used for subsequent analysis. The filtering steps are as follows: 1) Remove reads containing adapters; 2) Remove reads with a proportion of N greater than 10%; 3) Remove low-quality reads (the number of bases with quality value Q ≤ 10 accounts for more than 50% of the entire read).

[0067] 4. Genome mapping and SNP calling

[0068] After mapping the high-quality reads of all 100 samples to the water lily reference genome (V1.0, http: / / eplant.njau.edu.cn / waterlily / download.php), variant detection was performed. The genome mapping was carried out using the alignment software BWA (0.7.12) with the mem algorithm, and the alignment parameters were -k32 -M. Variant detection was performed using the UnifiedGenotyper module of the software GATK (3.4-46). The processed alignment files were used for variant detection of multiple samples, and the detected variants were filtered using VariantFiltration. The filtration parameters were -Window 4, -filter "QD<4.0||FS>60.0||MQ<40.0", -G_filter "GQ<20", resulting in a total of 2,411,988 high-quality SNPs.

[0069] 5. Core SNP Screening

[0070] According to the "test samples" and "validation samples", the above 2,411,988 SNP datasets were divided into two parts according to the samples. One was the dataset of 68 water lily germplasm resources (SNP.set1), which was used for fingerprint SNP screening; the other was the dataset of 32 water lily germplasm resources (SNP.set2). Core SNPs were screened based on SNP.set1 as candidate SNPs for constructing the fingerprint map. First, the plink software was used for screening, and only the loci with a detection rate higher than 95% in all samples (i.e., the parameter -geno 0.05) were retained, and at the same time, the minor allele frequency was not less than 0.05 (i.e., the parameter -maf 0.05); then, the plink software was used to filter the linkage disequilibrium loci, and the filtration parameter was --indep-pairwise 50 1 0.8. Finally, 45 SNP loci (Table 2) were obtained, and they were used as molecular fingerprints to distinguish and identify water lily germplasm resources and analyze genetic diversity. Finally, the genotypes of the above 45 candidate positions were filtered from SNP.set2 for fingerprint map verification, as shown in Table 2.

[0071] Table 2 Information of 45 SNP Loci in the SNP Molecular Fingerprint Map of Water Lily

[0072]

[0073]

[0074] 6. Classification and Genetic Diversity Analysis of Water Lily Germplasm Resources

[0075] (1) Construct a phylogenetic tree for 68 test samples using the above 45 SNPs. Use the R package phylotools to convert the locus information of 68 samples into a fasta format file, perform sequence alignment using the ClustalW module of the MEGA software, and then construct a phylogenetic tree using the neighbor-joining methods, specifically as shown in Figure 1 as follows.

[0076] According to Figure 1 it can be seen that these 45 SNPs can clearly divide 68 water lily germplasm resources into 5 subgroups: the subgenus Euryale (42), the subgenus Nymphaea (20), the subgenus Archaeonymphaea (3), the subgenus Neotropaea (1), and the subgenus Brachyceras (2). This classification result is completely consistent with the classification result based on phenotypic identification, indicating that these 45 SNPs can be used as molecular fingerprints for the classification of water lily germplasm resources.

[0077] (2) Use the plink software and this set of SNP markers to perform PCA analysis on water lily germplasm resources. Among them, the principal component analysis (PCA) is based on the degree of SNP differences between individuals, and individuals are clustered into different subgroups according to the morphological and structural characteristics of stems, leaves, and flowers. The results are as shown in Figure 2 .

[0078] According to Figure 2 it can be seen that these 68 germplasm resources can be separated by the two principal components with the highest variance explanation rate (PC1: 31.9%; PC2: 22.7%).

[0079] Example 2

[0080] To verify the reliability of using the 45 SNPs in Example 1 as molecular fingerprints for the classification of water lily germplasm resources. Use the dataset SNP.set2 (i.e., the 32 water lily germplasm resource materials in Example 1) to perform cluster analysis on 32 water lily germplasm resource materials. The specific steps are the same as those in step 6 of Example 1. The results are as shown in Figure 3 .

[0081] According to Figure 3 it can be seen that using the 45 SNP molecular markers obtained in Example 1, 32 germplasm resource materials can be divided into 5 subgenera: the subgenus Euryale (18), the subgenus Nymphaea (7), the subgenus Archaeonymphaea (3), the subgenus Neotropaea (2), and the subgenus Brachyceras (2). This classification result is completely consistent with the actual subgenus types of the 32 germplasm resource materials, indicating that these 45 SNPs are a set of molecular fingerprint maps that can be used for the classification research of water lily germplasm resources.

[0082] Although the above embodiments have described the present invention in detail, they are only some embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of a DNA fingerprint library of a water lily variety in the identification of water lily varieties, wherein the water lily variety is one or more of the water lilies of the subgenus Archaeotropaea, subgenus Australia, subgenus Neotropaea, subgenus Pantemperata, and subgenus Pantropica, and the DNA fingerprint library includes the following SNP molecular markers: 。 2. The application according to claim 1, wherein The water lily sample to be tested is the tender leaf of the water lily.

3. Application of the DNA fingerprint of the water lily of the subgenus Archaeotropaea in the identification of the water lily varieties of the subgenus Archaeotropaea, wherein the DNA fingerprint includes the following SNP molecular markers: 。 4. Application of the DNA fingerprint of the water lily of the subgenus Australia in the identification of the water lily varieties of the subgenus Australia, wherein the DNA fingerprint includes the following SNP molecular markers:

5. Application of the DNA fingerprint of the water lily of the subgenus Neotropaea in the identification of the water lily varieties of the subgenus Neotropaea, wherein the DNA fingerprint includes the following SNP molecular markers:

6. Application of the DNA fingerprint of the water lily of the subgenus Pantemperata in the identification of the water lily varieties of the subgenus Pantemperata, wherein the DNA fingerprint includes the following SNP molecular markers:

7. Application of the DNA fingerprint of the water lily of the subgenus Pantropica in the identification of the water lily varieties of the subgenus Pantropica, wherein the DNA fingerprint includes the following SNP molecular markers:

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