A molecular marker-assisted breeding method for high egg production performance of Leizhou black ducks

Abstract

The present invention discloses a molecular marker-assisted breeding method for high egg production performance of Leizhou black ducks. The present invention found that there are 8 SNP sites on intron 4 of the VEGFA gene of Leizhou black ducks, among which there is a mutation I4-14004G>T, and the initial egg weight of individuals with genotype GT is significantly higher than that of individuals with genotype GG. These 8 SNP sites constitute a total of 14 haplotypes with biological statistical significance. Among them, in terms of the age at first laying, H1H3 has significant differences with H1H6 and H2H5; in terms of the initial egg weight, H1H3 has significant differences with H4H6, H1H7 has significant differences with H2H3, H2H3 has significant differences with H4H6, H5H6, H3H5 has significant differences with H4H6; in terms of the number of eggs produced at 300 days of age, haplotype H1H6 has significant differences with H2H3 and H3H5, H2H3 has significant differences with H5H5, and H3H5 has significant differences with H5H5. The breeding method of the present invention can be used to screen out individuals of Leizhou black ducks with late laying age, high egg weight and high egg production, laying a theoretical foundation and providing a feasible method for establishing a high-yield line of Leizhou black ducks.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and genetic breeding, and particularly relates to a molecular marker-assisted breeding method for high-egg-producing Leizhou black ducks. Background Art

[0002] my country boasts a rich genetic resource for duck breeds. Numerous breeds have emerged across the country due to regional, economic, and cultural differences. Numerous literature reports indicate that local duck breeds possess unique genetic advantages. Utilizing these superior genetic resources is crucial for cultivating unique, high-quality breeds and enhancing the international recognition of Chinese duck brands. The Leizhou Black Duck, a local duck population widely distributed on the Leizhou Peninsula in Zhanjiang City, Guangdong Province, is a long-term free-range population maintained by farmers on the Leizhou Peninsula. They are small, produce large eggs, and are characterized by their tolerance to roughage, strong disease resistance, foraging ability, and adaptability to a wide range of environments. However, a lack of systematic breeding currently results in significant inter-individual variability, hindering large-scale production.

[0003] The applicant and its collaborating units have completed systematic breeding of Leizhou Black Duck populations, establishing high- and low-yield groups and a breeding core group, enabling them to implement technologies such as duckling breeding and specialized breeding. However, there is currently a lack of molecular markers and breeding methods for selecting Leizhou Black Ducks for their superior reproductive traits.

[0004] Vascular endothelial growth factor A (VEGFA), also known as vascular permeability factor (VPF), is a highly specific vascular endothelial cell growth factor that promotes vascular permeability, extracellular matrix degeneration, endothelial cell migration, proliferation and angiogenesis. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide a molecular marker related to the egg-laying performance of Leizhou black ducks and an auxiliary breeding method, so as to apply them to the actual breeding process of Leizhou black ducks and select a Leizhou black duck population with high egg-laying performance.

[0006] The first object of the present invention is to provide a molecular marker-assisted breeding method for high egg production and weight trait of Leizhou black duck, which comprises the following steps:

[0007] a. Blood was collected from the wing vein of Leizhou black ducks to extract genomic DNA;

[0008] b. Using the primer pair: upstream primer F: 5′-ATCATGAGAATTAAACCCCATCAGAGTCAG-3′ and downstream primer R: 5′-CTTGAATCCATTCTGGTAGGACACACAC-3′, PCR amplification was performed using the above genomic DNA as a template;

[0009] c. The PCR amplification product was purified and sequenced, and the sequencing results were compared with the VEGFA gene sequence shown in SEQ ID NO.1 to determine the genotype of the Leizhou black duck individual SNP site I4-14004 G>T (corresponding to Table 2).

[0010] Individuals with the GT genotype at this locus are selected for breeding, that is, Leizhou black duck individuals with the trait of high egg production weight.

[0011] A second object of the present invention is to provide a molecular marker-assisted breeding method for high egg production performance of Leizhou black ducks, which comprises the following steps:

[0012] a. Blood was collected from the wing vein of Leizhou black ducks to extract genomic DNA;

[0013] b. Using the primer pair: upstream primer F: 5′-ATCATGAGAATTAAACCCCATCAGAGTCAG-3′ and downstream primer R: 5′-CTTGAATCCATTCTGGTAGGACACACAC-3′, PCR amplification was performed using the above genomic DNA as a template;

[0014] c. The PCR amplification product was purified and sequenced, and the sequencing results were compared with the VEGFA gene sequence shown in SEQ ID NO.1 to obtain the genotype of the Leizhou black duck individual at the following 8 SNP sites: I4-13876 C>T, I4-13925 C>T, I4-13926 C>G, I4-14004 G>T, I4-14063 C>T, I4-14076 G>A, I4-14173 T>C, and I4-14175 T>C, and the haplotype combination of the Leizhou black duck individual was determined (corresponding to Tables 3 and 4);

[0015] d. For the trait of age at first laying, give priority to breeding individuals with the haplotype combination of H1H6 or H2H5, and screen out individuals with the haplotype combination of H1H3; for the trait of egg weight at first laying, give priority to breeding individuals with the haplotype combination of H1H3, H2H3, or H3H5, and screen out individuals with the haplotype combination of H4H6, H1H7, or H5H6; for the trait of egg number at 300 days of age, give priority to breeding individuals with the haplotype combination of H1H6 or H5H5, and screen out individuals with the haplotype combination of H2H3 or H3H5.

[0016] Preferably, the PCR amplification has a total reaction system of 25 μL, which contains 11 μL of ddH2O, 0.5 μL of upstream and downstream primers, 12.5 μL of Green Taq Mix, and 0.5 μL of Leizhou black duck genomic DNA template.

[0017] Preferably, the PCR amplification reaction procedure is: pre-denaturation at 94°C for 4 min; 35 cycles of 94°C for 30 s, 53.4°C for 30 s, and 72°C for 20 s; and extension at 72°C for 10 min.

[0018] There are 8 SNP sites in intron 4 of VEGFA gene, such as Figures 2 to 9 As shown, the mutation sites are located at I4-13876C>T, I4-13925C>T, I4-13926C>G, I4-14004G>T, I4-14063C>T, I4-14076G>A, I4-14173T>C, and I4-14175T>C. The primer pair F: 5′-ATCATGAGAATTAAACCCCATCAGAGTCAG-3′ and R: 5′-CTTGAATCCATTCTGGTAGGACACACAC-3′ was used to amplify intron 4 of the VEGFA gene. The genotypes of haplotypes H1-H7 at the above eight SNPs are shown in Table 3.

[0019] The present invention can screen out individuals with low egg production by screening out haplotypes with differences, thereby screening out individuals with late egg production age, high egg production weight and high egg production of Leizhou black ducks, laying a theoretical foundation and providing a feasible method for establishing a high-yield line of Leizhou black ducks. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The electrophoresis bands are the result of VEGFA gene amplification using different primers; M is DL2000 DNA Marker, and 3, 4, and 6 are the results of VEGFA gene amplification using different exon primers. Band 4 amplified by primer 4 (upstream primer F: 5′-ATCATGAGAATTAAACCCCATCAGAGTCAG-3′ and downstream primer R: 5′-CTTGAATCCATTCTGGTAGGACACACAC-3′) is the target band with a size of 829 bp.

[0021] Figure 2 It is the sequencing map of 14-138763 genotypes of VEGFA gene.

[0022] Figure 3 It is the sequencing map of 14-139253 genotypes of VEGFA gene.

[0023] Figure 4It is the sequencing map of 14-139263 genotypes of VEGFA gene.

[0024] Figure 5 It is the sequencing map of 3 genotypes of VEGFA gene I4-140043.

[0025] Figure 6 It is the sequencing map of 140633 genotypes of VEGFA gene I4-1.

[0026] Figure 7 It is the sequencing map of 140763 genotypes of VEGFA gene I4-1.

[0027] Figure 8 It is the sequencing map of 141732 genotypes of VEGFA gene I4-14.

[0028] Figure 9 It is the sequencing map of 141752 genotypes of VEGFA gene I4-14.

[0029] Figure 10 It is a chain reaction of SNPs sites in the VEGFA gene of Leizhou black duck.

[0030] Figure 11 is a haplotype block linkage map. DETAILED DESCRIPTION

[0031] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0032] The main reagents: Green Taq Mix, whole genome DNA extraction kit, and Ultra GelRed were all purchased from Nanjing Novozymes Co., Ltd.; agarose was purchased from Bogreen Biotechnology Co., Ltd., and DL-2000Marker was purchased from Sangon Biotechnology Co., Ltd.

[0033] Main instruments: PCR amplification instrument and electronic balance were purchased from Shanghai Jingke; electrophoresis instrument was purchased from Beijing Liuyi Biotechnology Co., Ltd.; electrophoresis gel imager and spectrophotometer were purchased from Thermo.

[0034] Example 1: DNA pool construction and PCR amplification

[0035] 1. Test method

[0036] (1) Experimental animals

[0037] A total of 99 Leizhou black ducks were used in this experiment. They were an F4 generation basic group provided by Hengcheng Breeding and Farming Cooperative in Machee Town, Potou District, Zhanjiang City. The age at first laying, weight at first laying, weight at first laying eggs, and number of eggs laid at 300 days of age were recorded for each individual. More than 2 mL of blood was collected from the wing vein using a lithium heparin anticoagulant tube and placed in a -20°C refrigerator for subsequent genomic DNA extraction.

[0038] (2) Collection of blood genomic DNA and construction of DNA pool

[0039] Blood DNA was extracted using the Novozymes whole genome DNA extraction kit, and the DNA concentration and purity were detected using a spectrophotometer. After reaching the standard, the DNA was stored at -20°C. 40 DNA samples were randomly selected and mixed in equal amounts to prepare a DNA pool sample.

[0040] (3) Primer design

[0041] According to the genome sequence of chromosome 3 of the mallard VEGFA gene provided by NCBI (number: NC_051774.1), the primers of the VEGFA gene (whose sequence is shown in SEQ ID NO.1) were designed using the BLAST function on the NCBI website. The VEGFA gene has a total of 9 exons, and a total of 7 pairs of primers were designed (the primers were synthesized by Sangon Biotech Co., Ltd.) to amplify all 9 exons. Primer 4 included the segment of exons 4-6. Because the distance between exons 4-6 is very short, they were designed into the same primer. Then, these seven pairs of primers were used to perform PCR to verify the primer specificity. The results showed that only primers 3, 4, and 6 had primer specificity ( Figure 1 ), the PCR products of these three primer pairs were sent to Sangon sequencing, and it was found that the PCR product of primer 4 contained the most mutation sites. Therefore, primer 4 with the most mutation sites was selected to continue the subsequent experiments. The specific sequence of primer 4 is:

[0042] The upstream primer F: 5′-ATCATGAGAATTAAACCCCATCAGAGTCAG-3′ and the downstream primer R: 5′-CTTGAATCCATTCTGGTAGGACACACAC-3′, which corresponds to an amplified product length of 829 bp.

[0043] (4) PCR amplification and verification of target genes

[0044] The total PCR reaction volume (25 μL) consisted of 11 μL ddH₂O, 0.5 μL each of upstream and downstream primers, 12.5 μL Green Taq Mix, and 0.5 μL of Leizhou black duck genomic DNA template. The reaction procedure was as follows: initial denaturation at 94°C for 4 min; 35 cycles of 94°C for 30 s, 53.4°C for 30 s, and 72°C for 20 s; and extension at 72°C for 10 min. The product was verified by 1% agarose gel electrophoresis for the presence of the desired band and then sent to Sangon Biotech Co., Ltd. for sequencing.

[0045] 2. Experimental results

[0046] The PCR amplified VEGFA gene product of Leizhou black duck was verified by 1% agarose gel electrophoresis. Figure 1 Band 4 is shown, and the product amplification has good specificity, no other bands, and the band is clear, which can be directly used for sequencing.

[0047] After sequencing, it was found that the mutation sites in the PCR product of primer 4 were all present in introns. A total of 8 SNP sites were found in intron 4 of the VEGFA gene, such as Figures 2 to 9 The SNP sites are respectively located at I4-13876 C>T, I4-13925 C>T, I4-13926 C>G, I4-14004 G>T, I4-14063 C>T, I4-14076 G>A, I4-14173 T>C, and I4-14175T>C; wherein, for example, the SNP site I4-13876 C>T indicates that the SNP site is at the 13876th base on the VEGFA gene sequence as shown in SEQ ID NO.1, and the same applies to other sites.

[0048] Example 2: Population genetics analysis of 8 SNP sites in the VEGFA gene

[0049] 1. Experimental methods

[0050] SeqMan software was used to perform sequence alignment and count the SNP sites and genotypes of each individual, and the allele gene frequency, genotype frequency, genetic heterozygosity (H), effective number of alleles (Ne), polymorphism information content (PIC) and chi-square test value χ2 of each individual SNP site were calculated.

[0051] 2. Experimental results

[0052] The results of population genetic analysis of the eight SNPs in the VEGFA gene are shown in Table 1. The chi-square value (χ2) was less than 5.99, indicating a p-value greater than 0.05. The PIC was between 0.25 and 0.5, indicating moderate polymorphism.

[0053] Table 1 Population genetic characteristics analysis of 8 SNP sites in VEGFA gene

[0054]

[0055] Note: A χ2 value less than 5.99 indicates a p-value greater than 0.05. A PIC between 0.25 and 0.5 indicates moderate polymorphism.

[0056] Example 3: Correlation analysis between VEGFA gene SNPs and egg production performance of Leizhou black ducks

[0057] 1. Experimental methods

[0058] The association analysis between the egg-laying traits of Leizhou black duck and genotype and haplotype was performed using the multiple comparison analysis of variance in one-way variance of SPSS 22.0 software. When P < 0.05, it indicated a significant difference, and when P < 0.01, it indicated an extremely significant difference.

[0059] 2. Experimental results

[0060] The results are shown in Table 2. The first egg weight of individuals with GT genotype of I4-14004 G>T was significantly higher than that of individuals with GG genotype (P<0.05).

[0061] Table 2 Significance analysis of VEGFA gene SNP sites and egg-laying performance of Leizhou black ducks

[0062]

[0063] Example 4: Correlation analysis between 8 SNP sites and egg production performance of Leizhou black ducks

[0064] 1. Experimental methods

[0065] HaploView 4.2 software was used to perform linkage analysis on the eight SNP loci.

[0066] 2. Experimental results

[0067] The results of the SNPs linkage reaction of the VEGFA gene of Leizhou black duck are shown in Figure 10 , haplotype domain linkage map see Figure 11 The haplotype frequencies of VEGFA gene are shown in Table 3, and the haplotype combinations with significant differences in Leizhou black duck are shown in Table 4. The results show that there is strong linkage inheritance in sites 1 to 8, and the 8 sites form two structural domains, constituting 14 haplotypes with statistical significance (the number of individuals is greater than 3).

[0068] Table 3 Haplotype frequencies of VEGFA genes

[0069]

[0070] Table 4 Haplotype combinations with significant differences in Leizhou black duck

[0071]

[0072] SPSS 22.0 one-way ANOVA was used to analyze the correlation between 14 haplotypes and the egg-laying performance of Leizhou black ducks. The results are shown in Table 5. Among them, in terms of age at first laying, H1H3 had significant differences with H1H6 and H2H5 (P < 0.05); in terms of egg weight at first laying, H1H3 had significant differences with H4H6, H1H7 had significant differences with H2H3, H2H3 had significant differences with H4H6 and H5H6, and H3H5 had significant differences with H4H6 (P < 0.05); in terms of number of eggs laid at 300 days, haplotype H1H6 had significant differences with H2H3 and H3H5, H2H3 had significant differences with H5H5, and H3H5 had significant differences with H5H5 (P < 0.05); there were no significant differences between other haplotypes and the egg-laying traits of Leizhou black ducks (P > 0.05). The significance of this experimental result is that the above-mentioned markers with significant advantages in one / multiple egg-laying performances can be used to screen out individuals of Leizhou black ducks with late laying age, high egg weight and high egg production, laying a theoretical foundation and providing a feasible method for establishing a high-yield line of Leizhou black ducks.

[0073] For example, as shown in Table 5, H1H6 and H2H3 showed significant differences in the number of eggs laid at 300 days of age, and H1H6 and H1H3 also showed significant differences in the age at first laying. Therefore, H1H6 is the optimal haplotype, which can be used to select a Leizhou black duck strain with a low first laying age and high egg production. The specific operation is to collect blood samples from a common Leizhou black duck population, extract genomic DNA, and amplify the corresponding segment of the VEGFA gene using primer 4. After sequencing the sequence of this segment, individuals with the H1H6 genotype are retained, thus selecting a Leizhou black duck strain with a low first laying age and high egg production.

[0074] Table 5 Correlation analysis between haplotypes and reproductive traits of Leizhou black duck

[0075]

[0076]

[0077] Note: The same letters in different capital letters in the same column indicate significant differences (P < 0.05). No comparison is made between different letters in the same column. Sequence Listing <110> Guangdong Ocean University <120> A molecular marker-assisted breeding method for high egg production performance of Leizhou black ducks <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 23013 <212> DNA <213> Green-headed mallard (Anas platyrhynchos) <400> 1 tcggcggagc gtctctcccc gcggccgcgc cgagcagttg cagagcgccg gcgagcgggc 60 ggtggagcgg gcgctgcgct gcgctgcgcg gagccgccgc tggagtgcgc ggagccgccg 120 ctgccgccga gccggcgggg gacagcgcgc cgcgggagca ccgaagccct ccccggccac 180 cgggacctgc gcttggctcg ccgccttgct ttatcctccc gtttaattga tttttttttt 240 tttcccctag actttggatt tttatttttt ttttttctcc ctttccttca aagccccctc 300 cctcccccct tttttccccc tcttctccgc cccggttgcc tccctctcca gccagtttgg 360 attttttttt tttctttttt tttttttttt tccaccccct tctgattttt taaagcggtg 420 gacggccgag cggcggcggg ctcagcccct tccatccatc ccccccggcg gggggaggca 480 gcagcagcag cagcagccgc cgccgcgaag aagaagaaga agaggcgagg tggggggggg 540 tgggagtgcg cgcaggaaaa gagatgcatt gaaacttttc cccgcaaact tcggcgtgag 600 tgagtgcgcg 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gccgtgcgga gttcaaaata cctagctgga aaaacaaata 1500 gagatcaaag aggacaaaat ggatcctgaa tttcttccag attgattttt ttttttttt 1560 ttcaaaatgc atatctcctg caccgagatt acaaaacccc tcggtttttt cccttctctc 1620 cttctcggt cacttttact tttgccttat tgtcattcct gagcgattag aaaccgactc 1680 ttcctccttg tctttctctc cttaaaaaaaaa aaaaaaaaag gagagagaaa aagaaaggca 1740 gctttctgtg caaacctgtc ctataaatct gttttcaaaa gagtcaagag ggctgtgttg 1800 ccaagagata ttaaggggtt tagacagagt gcccacgtga aagtggctgt atgctaggcg 1860 gcgtggatta tgttttaagg gctatgagga gctagttctc ccctgccatt cctccccagc 1920 tctcggcgag aatccgggcc agaccatctc ccacagtggt gaacataaac gtcgcttgca 1980 aactaatcag ttatcacat tgtttgttca gcaccgatgc tggaaggtaa ctccccaatt 2040 aggagccatt tcagctgtaa ccgaagtcat gcaggcactg cacgctttgt ttctagcctg 2100 ttatatctcc actttctcca gtgacagccg cacgttaggc tgtgctcgct gggaaccggg 2160 gctgagctct cattagagcg tgtcacggag cagctccgtg cagcctgcct gccagatttg 2220 gctcgtggga cgattctttc cgctctccaa gagtttttcc accgtcacct aaaacagggc 2280 ttctttgcct cccagctcct caggaaactt gcagagcagc gtctgttccc tggctgtagg 2340 ttagacaact ccgtgtctag tgataatacc cttcaaccct ccccttgctc cttctcccct 2400 ccccttcctc ttccccggag atgaagctgc ggatcaggta gggtccaggt ctgcctgtcc 2460 tcgggagtcg cgtgaaagcc tcaacgcttc cttttctccc aggcaggagg atggtgctgc 2520 tgagaaaaga cagagggtaa tggttggggg ggggggaaaa ggagcaggga aaaattccgg 2580 ggtggcgtgg gaaatgtggg gtggtgggaa gtgttgcggg gctaattgca cgagagatcg 2640 cgagatgtgc agccagccgg gtgatgcggg ccatataagt gctcagacgg acagacagtg 2700 aggtctgtcc ttcagttctg tccttatttg tccgttggtc gcctgggcac cacggtgatg 2760 ggagcgttgg aaatgccagc agatctcacg taggcagctt gacgatggcg ctgggcactg 2820 gcagagagcg tggctcttca cgccggggcc aggggttttg tgctttggtg ataatattta 2880 tctgcagcgt taatcagact gtcgtgctga ataaaaccta tctagctagc caggcgcgct 2940 ggagagaatt aaagaaaagc cctggcaaga ggctgagctg gggaggcagg ggccagcgcg 3000 tgtcaggctg ctttcctgaa ggcgagggag gagggacgca ggaatcacgg tgctcccccg 3060 gggaaggcag cagggggatg gctcctgacc gagggagctg gtgtggttgc tcctgcagcg 3120 aggctgggac agcggggaat tgtgtcccgg cccgggctgg gtggcgctga tgccacaggt 3180 gtctgcctgt gcctgtggca ccagccctgc cgtgcaggag agggagggag aggtggtttt 3240 gcagtgccac cctcccagct gttggccggc gggcgtgtag ccctgcaggg tgctggaggt 3300 tgcgtttttg tccccttcca ctccctggtt tcctagagaa taggggaagg cagctccttc 3360 cccatcttcc tttgtccggg ggtctcagcg gcggcatggt ggcgggtccg tgctggtggg 3420 ggtctgggag ctcatccctt cccaccccgc gccgtggagg gacctggcta ttaccagctg 3480 gattgccaca gggaacaggc agcgatcaga cacggaggga gggagagcga gcctgtgacc 3540 agactgctaa tgatcacaca cccaccagcc gcgctgcctt cggcgtgcct ctgcgccagg 3600 ccccgctggg agcccccaga gcctgcctcc cgagcccccc cggcagccag gaccccacat 3660 gggtccccgt caccttcccc ggacccgctg tgggcccgag gaaacgctgg gaggaggagg 3720 agggctcctt tgggagcagg ggaaagggga gcccgtgctg gggtcttgga ggggctctct 3780 gattcccgag cggccccgaa tcagggtctc ttgtccgtcc tgctccgtgt cagaggtgcc 3840 tgcgccgtgc tggcagctcg cgggattact gatctccgtc cctaggtaca atggggaagg 3900 agtgcagtgg ggtccccccg ccgcttaagt cgaaaccgct gagaagaatt tccagcgcct 3960 aagacgaaaa gtgccttaac tcctgtccca gagcccctct caaagtggat ggattgtttg 4020 ttccagtcct aaacagttac aaagctcttt taatgaggct gcacagatta gggggagggg 4080 aagggagaga aagagtcctg gtggtggtgg tggtgagggg agtgatggct ggagaggaat 4140 gacttgatta accttttctc tcccttcaga gaggaacttg cttcctactg gcacttggag 4200 gagaaggggg acggggaagg tgcagataca gatggtgtga ggcaggttta tttgctcagg 4260 agggttttac cccacttcag gacgtgcatt cgctgtgacc tgcaaagggc ccaaaaagga 4320 gcttcccctg ctcagcccca ttctccgggg gcatttcccc tgctctcgag cctggggctt 4380 gcttccacac ctctgtgaaa tgctgaaaag ctatcggaaa caaattgtgt tacagagctg 4440 ttgggtaatt ccgaaagaca gcagaggaga gcgtgacctt caggctccca ccgaagggca 4500 cgctgggcct gcttgtccct tggaaggctt ttggcttccc atcgcccttt atgatatgta 4560 gcagcctggt ctcaagtgag gcagaacccc ctgcctgctc tgttgtttga gggaacgggc 4620 tcgcagggca ttagccacag gtgaaaggca ctgccaaaag ggcagagagg tttcttggta 4680 tagcaggcag acagcgtgtt actgatagtt ctgggaaacc tcttgggtga ctgctgggag 4740 ctcccgttcg agcgctgcag agctctgcca ggggggagga acagcaacct cgtccaccct 4800 gctcctggca ggctccagta ctgatcccgt ttcccaccct acgggtagaa ttgcaggtct 4860 aggtctaggt gagagctggg agggtgctct ggctcccgcg gcggtgcatt gggcagggag 4920 aggtgctggc cacgcgcagg tgagcagtga aagtgtgcgg ctcactcctg cttcccaaca 4980 cccatgcacc acgtgtgcgt gtttgtgtgt gcccaagcac ggaggcaggg ggcttggcag 5040 caggctgggc cgcctcacct tgggaacggg cagaaagacc ctcaaggtgg ctcagagctg 5100 atttcgctta gtccatcacg gtcactcggt gtgcgaaccg taaatccctc tgcgaaaggt 5160 cttaggagca ttgtgacttg gataggtcgg cctggctcgt gatcttcttc ccgtgctatg 5220 acatgaatgg aggcttttct tttgacccgt agatcttcgg gccctgtgcg tctttggaag 5280 gccgctgcaa cctctgctgc ctccattttg tttgtgctgt tgcagtgacc ttcacattc 5340 aggagcaaaa ttgttcccag gaacctgctt tttaagctgt tatcacggcg ctgaggttag 5400 gcacacaggc tttcctcccc agggacataa cagatccacg cgtggggtgg ctcaggtctg 5460 ggctgctgtc ttattcccag gtacagaagg acgtcggttg aaatgagaag caaggagttc 5520 ggtcaggggg accttagaca aagggagctg tgcacagaca caaaggaatg gctgggagag 5580 gggcttcgcc ggctctcttt cctctcctt tgaaaagctg acacttcagg ccgctcggaa 5640 ggtggtgctg ccctgtgcac agggctgcta gaagactata ggtggcagat tctcgttgtg 5700 agctcgaggt ggactttgcc tgctcaattc agtcccgtct ccagctgtgc gatcagaggg 5760 ccttttccta agcccaaata cagcacgttg ctgatgaaac agtgctctgc atgtttgcat 5820 tgccgctctc tttttgcacc tctgctgaaa ggcagcaggt tccggggtgg atggcgtcac 5880 gcaggtggca acataccttg cagaagtgaa gaaacccaag cttttatttt tttttttttt 5940 ggttttggtt cggaacagaa cagcaggggg aaacctgact tggctggaaa gttacgtcag 6000 agctaaaacc ctcacgcaga gcaggaagag tggagttttt gtttcacctg gaattaaaaa 6060 aagacttcct cagtgaagcg gcgtaacacc gctccatccc agtctggggg tgggttccct 6120 attgctgctc ccgcctgcgt cagcagtttg gaaatgcccc ttgtcccaag aggacagcag 6180 gcgcgaatgc agagctcggg gcctcttttt gtcccaggga agcagtgcag gggctgggga 6240 cgggcccgtt acgtgacagt gccagcgcag agggacactg gggggggatc gcagcagcta 6300 gccgccctga cagcgggcca gatagcgaca cctctgcggc cactgctgct tggtgacaga 6360 catcggtgac attgagcggg gcgcgatgcc ctttggcgcc tgacagccct tctccccgct 6420 cctcctgggg cagattcacc tttatggcgg ggaaggggag gcctgaagga gaaaaggcag 6480 gcctggcgct gctctggagg tggcagcggg cccacggctt gccaagcccc acggcttagc 6540 gcgggccagc gggagaaggt cacccgcctt gttttcctct ccgtgccctc gtctgctcag 6600 tgctttttgc ccccagccgc tgcaaacccc tcgggggggc tccttccaag ccctcgggcc 6660 ccttcccctg cctgctttcc tctccctttt cccttccccg gccccgttct ccctgcgggg 6720 ccgaggatgg ggagcgggat ggggaacaaa agggctctgt gcccagcccg gcgtgggggg 6780 ctccgcgtgg tgaccgcttt tctcttgtct ttgcagttgt ccaaggctgc tcctgccctg 6840 ggggatgggg agcggaaacc caacgaaggt aagagccggg tggcttcccc ggggggctcg 6900 ggggaaggcg gccggggccc ctgggagcgg ccaacaccgc cacctcccgg ggagccgcag 6960 cgggagcggg gggctgcggc cgccgccatt agccccgccg gggtcggggt cgggatcggg 7020 atcaggattg ggatcggaat tgggactggg atcgggattg ggatcgggat cggggcgtgc 7080 ggcagggccg ggacgtgcag cggggccggc ccagcctgct gcgcgcttcc cataactccg 7140 gggagaactg cgtgcgtgcg ctggtgcaca cacacacgct catttatata tatatatata 7200 tatatttata tatatattta tgcgcgcaca caaagagctg agtggctgcg agcagcctcc 7260 taggaattcg ccacagtgcc gtaggaaggg atttgtcatc tcagccgcct gcctcctccc 7320 cggaggcgcc gctggtgccc gggcggccgg ggctgatccc cgaacaaagg ccggagccgt 7380 gccccgcagc cccggcaccg ccagcagtgg ctctggggac agaggggtgg caatgcaagg 7440 ggacttctag cctcgtcccc cagctcgggt tattgctgtg tgcagagcca ggcttggaaa 7500 aagcaccctt ttactccgct gtgagcaggg gtaaagggtt caagggctgg aggccgtatg 7560 ggttctgctt aatctgatgc tggcagcgta gtagggatgg gaccaggtga tcttggaggt 7620 cttctccagc cttaatggtt ttgcgaaacg aggtggtggc tgtgcagccg cagcgattct 7680 gtgctgccca aaggccaggc tgtggccatg tcctcggccc tgtgggtttg tgcgagcagg 7740 ccaggagggc agtcagccac caggctgctc aggggtgcct cctgccagcc ccctgcccgg 7800 tagagccctg tgccctgcag caggtgggct tcccgtccga agtcctctgc tcacaagtgg 7860 gggctgtgga cttggggggcc ttcaggccga ggtgatcatc aatcagcttc tgttctgacc 7920 gtgttttccg cattatcttg gcagcagggc aagcccctca ctggagtgac tggctctgtc 7980 ggaggtctcg cagggtcccc ccatgggctg tgacctccag cgttattcca gcgggaggtc 8040 agcagagggc tctagcagtg tgggcccaga ttggcttttt acattgcact cttacttcaa 8100 tttcttgtaa ctgtactgct atgaccggct gcaaggagcc agagatttgt ggcaggcagc 8160 gtttgggcta ggtgtccatt ttgcgtggcc gaaatttccc cgtttgctgg cgggctgccg 8220 cgtgttgcgc ttgacccttg ctcctgctga agtcaacaga agaaggttca gtgactccag 8280 agggatcaag atggagcctg ctgtgaggct tattttggta accagtccct ctgctgttgg 8340 taggaagc tctagggtcc tggactgtgc cgtttgttgt gtctgaccac gttcccgggg 8400 agggaagagt tgaacagcat ttaattccct gtcacaggaa ggttgatggg agcaggggag 8460 aagttggtct cgcaacgtga gcagtgggac tccagtgcta ttgccttgat gctattcctg 8520 gctctgctat gcaaatacat ctgcgagcta gaagtcagta ctcttggcat taccttcctg 8580 gctctgcttc tgtcctgctg tatgaccttg gcggttctct ctctgtacct cagtttcccc 8640 tcggtgaaga ggagtcgatg atgtgcctct gttgtacaag gacaatcctg cagtgccttc 8700 tgctgatgct cctggggtct ttggggtggg tgcgtaaggt gactttgtgt ctctggccat 8760 tttgtgcttg ctctgagatg tcaagctgca acaggttttt atttgggatg gacttaactc 8820 cggttggagc ctgtaatttt aacttcagcc cctaccttt tgtgctcgag agagccttcc 8880 acatgttttg tggcacggga agttgtagag gagttagaca aacactcttt gtttgttcca 8940 cgagaaaaga ttttctggta taaataaatg aaaccaccag cacttgtggc tgcactttta 9000 ttttaagag aacagtgtgt ctgtattgct gctaagccat tgacggggag ggaggcagaa 9060 tgatcacaga cctactcctt ttctagtcta gcttgttccc actcgactgc tcaaaatttg 9120 gacttaatcc tgcctgctgt ggccatgtct gcctaaagag ttattgtggg ccctggctga 9180 gttctaacct tatttccctc tccattcaca aagaagaacc tctgactcaa atgtggaggt 9240 gttttacaga gtaggggtgt gggttaaatg cgggattaac ttttaacatg ggctgaggcc 9300 caaattgcag gaagggagca ggctaagcgg tgttagtcct tcagtgacct tcctatgttt 9360 ccccatgaaa gacagacgtc cttccgtcct gccacagcca atggaaagga agcagggact 9420 gaatcagccc ggcaaagtgc ggaatatgac tcccaggaca taggagtaag tgtagataca 9480 gtagcagggg cagggctttg ttgtgaggat gactgtacct cagtcatgtg ctccggtttg 9540 ggaactggct gtgcaaatgg actgcggagc gccgcacagc ctccgtgcga atgctctgtg 9600 tccagtggca tcgaggcaac gcgcctttca tggcagggca ttctgactgg cagtcggata 9660 aagagtccct gttccctctt ccgaggctga ttcagacttt ggaaaatcct cctacgttaa 9720 agtaaaggca aaaactccag cgctgataac gggaggcgtt tggtcctgtc agatctccca 9780 gatctcaagt atagttatca gggtgcagcc ataaaagtaa aggctccgca aaataaatca 9840 aatgtgctgt tccatggcca atctgtagcc cgcattccct ggtgtaatct agcactgtgg 9900 actcgaatca gtttcacttc agttttgtgc ctggtttcgg tttctgcctc tttatgtgct 9960 ccagagggat tcagtgagca gcagttgccc tgcacaggta actatcgctt tccctccctc 10020 ttttcctccc tccttcccct tgcagtgggt ctggaagcag tcttttaccc agcgctgtgc 10080 ttgtattgcc ggagttcccg gatcctttct gctggcacag cgtgaataat cagcagcgca 10140 gttcagcggc tgtcactgtg cctgcagaat ttcccacata ttaaattgtt ccacttgttg 10200 gagcacaggc agggactaag gtcactctga aggggagcag gaaggaggag gtagcacaga 10260 caagatcgga ggacaggcag gaggcacgct gctgctgggg gaggagggaa gaggaaggga 10320 agggacaggg aacagaggcg tgctgtcttt taggctagga ataccaacaa gcagtccatg 10380 cctgccaggc tacatttcct tttcacaagc cactttctct tttaatagct gctcacgctc 10440 tctgacactt caaactccct ggatgggagc tcctcccctc ttgtacccgc tctcagcact 10500 gtggcactgt cagctctgca tgacctagaa ttaactttca acaattaact gcgggctgtg 10560 agctccgagc cctgcagctt ttgcattttt gtggcaaagt aacctttatg ccagtttgtc 10620 acaagttcag ggcgctttag aaacgcttac cagagaccat ttttccatga tgcaagcagt 10680 ttgcgcttat cactccgagt tatcaacctc ggtgctgtag gactctgcat actgtacagg 10740 tggatcatta tagcgcataa cagcacgggg caggcactat ctctcggtgc acacgcttat 10800 cctctgctcc gtttgagaca acttcccttt gctgcctgca tgctggtgga gctgggaggc 10860 ctggagggct ccaggtactg gctgtttgca gaggtgggcg agcagctggg ttttgtgcgt 10920 gtgtacttta tctaccctat ctctcatctc tttaaatctc tccaaccctt gaccaagcag 10980 caagacgaag ctttggcaag aggctgggaa acattgagaa acgcttgtga aatcagcgca 11040 ggtcgcaagc agttagcgtt tgctccattc agagggaagg caggggataa tggcagatcc 11100 accccgttcc ccctcccaca ttgccttgtt gtggttcagg ggccaagtgt gtggtctggg 11160 taatcctggg ggtgctgtgg gctcgctctg agtcttccag gtgctggctg gcttccttgc 11220 tctgggagag tggtcggtgc agagacaggg aggctgcgat gtgtagcagg tgctgtcggt 11280 ctcctggagg tggatgctgt gtgtgggcaa aagctgtagc tggcggggca aacgaagcca 11340 catggatgca ctgttcacag ctgtctcacc actctcaggg ctgtctctgc gctgaggtct 11400 atagagagga tgcttctgtc tctggcttat gcccctgcct cctaaaacct ctgcttttga 11460 cagaaggtaa aagtgaaggg ccttaatcca gagcgcaggt catctgcagc agcctcaggt 11520 gctttttggc gcggctcctt tgcttagagg cagcatggct gagagtatgg gggacggcaa 11580 ggggacaagg cacctgtggg atgcatcgtg ctgccaggga gggccagtcc aaagagcgaa 11640 gctctccggt ccatcttgtc tcagtcccga agcagcagga ggaagggcca tctccttgtc 11700 ttcccctgct gatttggggt gtgggtggga gggagagagg gagggtgggt gagtagggca 11760 gagacgaagg acataggcct gcaagactgt tagtgaagag catagggtag atgtgttgca 11820 actgtctggc tcgttttaag gggcaaagca ctagacattc ttgagctaag tttgcattaa 11880 cttattccct ccattcctcc ctgtagttat caaattcctg gaagtctacg agcgcagttt 11940 ctgcaggaca attgagaccc tggtggacat tttccaggag taccccgatg aggtggagta 12000 catattcaag ccatcctgtg tgcctctgat gagatgtgcg ggttgctgcg gcgatgaggg 12060 cctagaatgt gtccctgtgg atgtgtacaa cgtcacgatg gaggtgaggg cccaacttgc 12120 gagctggcat gcttgggtgg aggaggcaag tcacccgtca cagccgcccc aagagataca gaagctagga gcaggcagag gagcatttct gcaaaaagga gcacttcgcg tgtagagaat gcagaggagc atttctgatg ctgttatctc tgaggccccg gagagacgc tccccagacc 12300 cttgctctac cttaaccctg catttggtcc acacagctgt tgctctgaag gcagctgagt agggcagtag ggaaaagcct gtcagtactc tctcccggag ggcatgcgag gtcagtcttt accaacagag caggggattt gggtcattag ctacactggc acagcagggg tggaaagggt ctctaagaaa cacactgctt cagtgtcaag tggaaaagcc tttctgcctc tgggcgtcct 12540. cctcctttgt ttggaggctg ctgaattcag ttcttggaag agacagggac tggccgtgat agcaaaggct gggcacacgg ggaagataca gactctgctt agagagcatt gtcactgccc ttgcctgcca gcctcagttc cagcttgcac ccaccgtggt gctagtgtcc attaccctta 12720 gcctgtagct ccctcaccta cttttgtccc tcagcctctt gaaagggcac tgtaaatatt gccatcagtt tgtgatatgg aggagagacg tgcaggactg cgtgactacc agccagtgct 12840 tttcacttgt gacctcggct gtgtttgaac tctggcagag aacctctcgg tgtccttcca tggttcccct gtactgcttt tccacttgca tgctccagcc tgggccagag cctactagca 12960 cggtgtaact aaggacagtt tccttcttac cattccttgc tgaattacgt gttttggctg gcatgcctgc atgcctggga agggtcagaa gcacttcagg agatccgtgg tgcaaatgct agaactcgtg gtccagccag gttctgggtt gagagactgt aacagcacat gcctggcttc ccatataact tccctgcagg cacacggagc taggggctgg gcgaagcctg gctctgcagg 13200 ggtgttttaa gcacagagca ccagatttgt tctgtgtgtc atctcgccga gaaggtgacg tgtcagccgg gaggagtgg tggcttaggg aacagcagtt agacatggag ctttcgcttt gggcctttgc tttagactga aacgtgcctc tctctggggag caggccgaat gctccgttac tgtcttgcta gtattgccca gggacggctc agcgagaatc ttactgtggt gttaacgccc 13440 tctcccatgc ctgtctccaa ggcagtccac cggtgctggc accttgcatg catgagcaaa cagagctttg gcacgagcct ggtgttgccg cttgcttgtg atcaaagggc cttctctgac 13560 tgggagaacg gggcctcaaa gccagctcaa actctgccat gctcttccca ggagcacctg 13620 gggtgcaagt gctttgccgc gcagcgggct gatttggagc cgtgagtata aatagtgaat 13680 gggcagtgac cctctctgtt tctttcattc cagatcatga gaattaaacc ccatcagagt 13740 cagcacatag cgcacatgag cttcttacag cacagtaaat gtgactgcag gtgagaatgg 13800 agcagcgctg gttcctcttg caataaactt actctgagcc tttccttctt cctgccgttg 13860 ggttgggact gttgtccaga ggcttctgcg gtctcctcag gtggtttgca gtgggatggc 13920 tgagcctggg aaaagaactc tgctggcgag gctcctccat ctctagatat ggcaggtgga 13980 caatgctagc ctgcagcctg ggggctggtg gcactggtga ggtgcagcag gagagcaaat 14040 gctttgagaa ggactgctta acctgtgcca tgggcgtcct gcagcagcag ctgtacatgt 14100 cctcttccct gtgcttgcgg gaactgggaa taatactgga tgtgtatgtg gtaagcgccc 14160 ttctaatgag gccttttgttc tttcttcctc cacagaccaa agaaagatgt caaaaataaa 14220 caagaaaagt aagtgacaaa cctttcctcc tttctcttttt ggtggttggt tggcatttcc 14280 tctccccact gccccttgcc cttttccttg attggttgga gatcctcact tggagaaggt 14340 gttttggaa atggcatatg gtagtgcatg gggcgacact ctgaatccgt tccaggtggg 14400 cacagaaacc cagatctgac ggtccttcct tcatacccca gacctggagc ttgcatgacc 14460 cacgaccatg gcagtcattc ctaatgcttt acccagagtc agggaagtcc cgtcgtgtgt 14520 gtcctaccag aatggattca aggtgcagcc cctgttctcc cttccctctt cctggggtgt 14580 ttctggttac tgcaaatcgc tgcctttctt tttattat ttttttctt gctgcttgag 14640 tgagaacttg ctggcttct tcacagcagc ttgggtagga actctgctgg ctacagggag 14700 aggtggcagt ggaggcctgg tggaggcagg actgatgcca tgcacgatgt gatgctggtt 14760 ttggtggggt gggggcctgg gagggtataa aaggagatgg atctcctagc tgggtctcgg 14820 tggaggcttt caggagaggt ttttttcaag ctgatggatt tactctggtg acctgaagaa 14880 ggttaaggca caaccttccc tttccttcct ccccgaagta aaaaggttat ggaaagtaga 14940 taaatcggct ctcgccctgc ggtagttgca gggacgatca caaaacgtgg aagaacatgg 15000 aaaaatagag cgacctctct ggctgagagg ctcagccgga gcgcgcatgg ctgggatagc 15060 ggctctgcca cttggaggtg cggagttgga gaggaaaggg gcggctgctg ggcgatgagg 15120 ctgaaacaga gaaagagaga gagcaagcgg gagagtgaga gagaacttct ttcacccctc 15180 tcccggcggc cgtgtatttc tcgccgcctt ccagcctctc ctcctccccc tggcaatccg 15240 ctcgtggctg catcgggcag ccggtgctcc tctctgtgca gggccgtgtc ggggacggtc 15300 ggtctgcgtg gctctgtggt ctgaaggtgt gtgagagttg ttgggtctgt gtgagctctg 15360 gtgaggatgg ggtgtgggtg aagcagccag cccggcctgt gttgcaattc cctttcctct 15420 tctaacagca ccttgcctgc tctttgctgg ttctgcactc ccccggggaa ggaagggaag 15480 ggatgggaga gattgctgtg ctttgacttg ctctcgattg aaagtcctgt tatattggtg 15540 cccctctcct cccttcaggt cgattttcct cccctcaccg cctccctgtg actcctgaca 15600 cgtagggcag aggctgggag tgagctgatg ctcgggcact cgttccagcg agaagacttg 15660 aacggggcct aaagaacacc cgtggtgctt tcccttctgc ccccccaggt cccctccctc 15720 cacagctccc tcccgaggtg ggaaggggg agtccctgct gagctgccat ggtgcccacg 15780 tgtctgacga gccccggtgc tttgcccagg gccttctcct cgcatgcctc tcccttggtg 15840 ctgctctgat gtggctcgag gagcagcgca ggctccagcc tccctccctc tctgtgctct 15900 cgctgggctg tagaggtcca tgggtagcgt cctagcccca tcccgtccca gctcctgccc 15960 ccaaatcagg agcaaggggt gaattttaaa gagagtcagt gccttggctc cgtctgctca 16020 tcaggcccat gtggaaagcg aagggaggga aggggcgggt gaggggaaa aggagcaaag 16080 gcctgaagcc tcttgctggt tatcatctcc tgggtgtcat agctcgttca ggcctccctc 16140 ttcctcctct ggcactcggg aaagggctg ctggaggggc tgctagcaga cagcgaggtc 16200 tcctggagcc cagcgagcca caggcaggca gcggggtggg cgagcagagc ccacgggcta 16260 ggtgacccct tccaagcagc attctccccc cttatcccag cgtgctgagc ccgtcccgtg 16320 gcaggattat caccatccct gggctgcccc ctcctcgcca ccaggtcagg gcgctttcct 16380 cttccctgag ggagcccctc catgcaggtt gaccctcctc agccagcgtg cacctgagct 16440 tgtggcccgg gatgactgcc tgcgctcgct gcgtgacgct tccaccaggc tttccattcc 16500 ctttccctct cccctgtccc actgcccctg tccgggggct gcgaggaccc tcagagacag 16560 ccccgcgtgg aggtggcctc tgcgcacgct gtgtcccctg gcgctggctt ggctgcgggc 16620 aggctgggtg cggtgctggc aggagcgcag gaggccagtg agtgtgtgtg cgaggtggag 16680 atctgtgctt ctggagatct gtgcttctgg agatctgtgc ttcggccccg ggacttgccc 16740 tcctccggtc ctgcctcatg tgcttccccc tcccagcaag gcacatggct gtgttttttt 16800 tttctcctct tcttccttgc gttttcttcc gttttctctc tctctctctc tccctttctt 16860 tccttctgtc tctctctctc tttctctgtt tttttttttt tgtttttctt ccagaaaatc 16920 aaagcgagga aaggggaagg gtcaaaagag aaagcgcaag aaaggccggt acaaaccacc 16980 cagcttgtac gtttgtgtcc tctgcttgta gactctcttc cctccctttt ccccaaacca 17040 gtccgcctgc ttgctgctca ctcaaaatct caccctcccc tcccagtcac tggtgggctg 17100 cagatccccg caggggcagc gctggcggtg gcaccgctcc ggcaccgagg ggtgtctcag 17160 ggaaggcggc tctcgcttgc ttggctctct ctaatcaccc ccggcgaggc tcaggagcgc 17220 ggggcggggt gggcgagctc tctgcacctc ttctcccgct ccttgggctg cggctcttgt 17280 cgcctgctgt ccccccaact tgtgcccgtg ctacggcgac tggcggcggg caggctggtg 17340 cctgccgtgc atgggtgtgt gtgcgtgcgt gcgcgcgtgt gcctgcgtgt ctggtggagt 17400 cacaggcgga gaggagaggt gggacggtgg ggagctggaa aaggggtgct ggggaaccgg 17460 ggggctctct ctctctcttt ctttttctct ctctctctct ctctctccct gctggttttg 17520 gtggcttctc tctctccctg tctctaaacc tgtctctttc gctctgcctc tcacggtggt 17580 tcacacaatt ctgcattgaa agctcacggc ccttccctca cgggagaatc ctaggtagca 17640 aggctgggat gggacttaga gaaaggaggg ccatgccgac tgtggggaca agcacatgat gctggagccc agctgctttc caggggggag agaccctgtg tatttaactc atgtagccgc tggcctcttt ctactcgaat agcccacttc agaaaacgcc tttcattgat acctcaatag ctggttgggga cctctcacct tcccatcctt gcttcccccc ccttcctcct ccctgtggcc 17880 tgtgccgatg gatccaatgc agcatttgtg aatactgtgc agagcctgtt ctcttttctc 17940. tttacgctaa tgccaagggg tgctgtcctg accaggttct ggggctgccg gggtgctgct ggaaaaccat cagcccgcga cacttcagtg ggcagcatgg gggctgtggg agggggcaag gggattgctt tgatgtttag ggtgttgcat gtgtattttc tttctgctga agcgctgtag 18120 cttagacatc tttccttttg cctttttgca gtcactgtga gccttgctca gagaggaga agcacttgtt tgtacaagat ccccagacct gtaaatgttc ctgcaaattc acagactcac gttgcaagtc gaggcagctt gagttaaacg agcgcacttg caggtttgtg tcctgaagga tgaaagagaa acacacaaaa agaaagagag agaaagagaa agagggggga gcttgcttcc 18360 tgctgacttc tggcaccagt gctatatgat tactccgttt gtctatccta cgtgggccag 18420 agggtctaac tgagatgagg acagagattc cctgtgtcag agggcagctc tgcgtttcgg 18480 atgtgctgtc aggacttgga tggggatggc tcccagagac agggcatttg tgtctaaggt 18540 gtccattccc ctagaacgag gtaacggagt tcatgctgct gcagtagcca gggggagggt 18600 ctgcagccct ctgtgcatta gctgtgtgca actctgagag ggtactgttg tacaaagcca 18660 aatccttcct agctgactag gatttcacca cccctgtctt ccaatccctt tctggatagc 18720 ttgtggacac tctgcagtgt gcagagaagg gaaggccagg ctgggaacct ccctcaccct 18780 cagggatgtc ctcatactct catttgcctt tgtgttcttg aaagccaggg agagggccct 18840 gtcctcttgc actgtgctca cagtgtactg tccacggctt ggccagagtg gtggtttgag 18900 ggactcaggg ctgtgatgtg ggattcagcc tgtctctacg tgaaggcagg ccggggtctg 18960 tatttcagat gccaggtgag aggtgcaaag acaccggtta gtgtctgctc agaaatccac 19020 actgtgcatg cgtgtctga atgctactta ccatgcacca cccgtggcat gggcacgtgc 19080 agaaaccgat tcctggttct cacctgagca ctgtggtgaa ctcagtgggg tcacttgact 19140 cgcaccagct gtgtgtgcac ctctggtgca tgaacatcct acctatctgt agagagaggc 19200 atagaggtgg gtttgctaat gagcccttgg ttgtgcctcc aaactcagct cacagagatc 19260 taggagcttc ctcctgccag aatattgcag gagcagtggc tggatgagaa agaataaggg 19320 agagagatcc agggtcctca ccaggggcta catcctgaca gaggggacat ctgtttgcct 19380 gtctagaatt ggctaggtga ctagggctgt gcttccctta aatgtgaaag tgctgtgtgc 19440 caaaatcagg gaggcatcta ggcactgcca gagtgctgga aactgctctg caaacagagg 19500 aatttctgg ggttctgtaa tccgagtggg aaagtgattg atggaggggc cagagtacag 19560 cagcttccag agttacactt ctcccagtga gcatagcccc cgaagaacga ggccagaccc 19620 aaccttgttt ttttttccct ctggtcctgc tccctgtcca cagagtaact ggaaattacc 19680 ctacagagtt tgaagagtga gaaacagtac aaagtacaaa gaaaaacaaa caatacccca 19740 aagaagtcat gatggcagga gggggttact tgcttctttt actagtcgct ccctgtggtg 19800 ggatagttgg cactcaggga gatttccaga gctggtgtgt tggccagaag gatagggagg 19860 agtcgtaaga gtgggtagct tatctgtttc cagatacaaa gctggttaag ctgtagttct 19920 tcctgttcca tctctttgta gagttagaat tgggccctgg aggggagggc aaatttttgt 19980 ttctcctgcc gcttcttcg gctgctgctg ctgctcttct gtggcgctaa cccctgctcg 20040 tggcagccag tgaaatgaga tgcattttct tttcaccttc tcgcctgcat gaggctttgt 20100 tgtctgcatt gcttccaacc caaacaacag gctaggtggg gagctgaggg aggtgtcct 20160 tctgcatgcg ttgcttagcc ttgtttttgt gtgtgtgcgt gtatctatgt gtgcatggtg 20220 tgagagggtg caaacaagac actcagcttg tgagcgagga gacctctggt tctgctccta 20280 aatttacccc tgtaccagtg tccccctgca tctgggtgag cctgggggaa taaattcat 20340 cgacccattc acgggtcgag gttctctgct gttttgccct gactgcacag cacttcaccc 20400 caccagtgac gtcatgccat gtcttaacgc cattagctt taaacaccc 20460 cttcctcccc ttccctgtcc tacctgcacc cccgtcacaa acgttaatct cttctctctt 20520 tctttccttc cctctcctct ctgacagatg tgaaaaaccg agacggtgag cagcggaaaa 20580 gaagggggaa cagccctgtt tctggagcct gatttctcgc ctggacagaa aaatacaaa 20640 aaaaacact aatccaatg aaccctaaaa atgaaggatc ggtagagcac agcactttca 20700 gtacggacga tggactccgg aaggacatt cccaaggca cccgccgcac agaattcacc 20760 ttgggtttt gaactgtttt atttttt tctttcatg ctgctaaata acgagccag 20820 gaagactata ggggtgtatt tgatgggttt tcccatgcat acatatatat gtgtgtatac 20880 atgcatatac atatatatat atgcatatat attttaacca catctatata tacatatatg 20940 fatherfather dragonfly fatherfather fatherfather fatherfather tatactgatt 21000 attttttta acatgctaa tgttattggt gtctcactg gataacc gactgctgtg 21060 gacacaagcg ggctacttgg ggcataagaa acaagctaag actggacttg agtagaagcg 21120 ctgggtgtaa acttcttaac tctaactgac ttgtgcggcc tccgctgttt ctctgtagag 21180 tgtgctgtac cataaaccac cttctccttg cctgggacag gaaggagagg tgatggagag 21240 gatgggcagg gagttccaaa gagcggcatc ctagggtgcg atgggtcaaa ggccaaggaa 21300 atggccatct gcgtgatcaa ggattcctcc tcccctctct tcccccaacc ttcgccctta 21360 ctcgtctcat aacctgcctg ccttgctggg acatgggatg tcagtgtaca ttttgcgttg 21420 actttattcg ctatagaaac ttcgccagag aggcaatgct ggcttctctt aaggatggtg 21480 ggcagctgat gcgcactaac tttccattca aggaagtatc acaggagtct agattccccg 21540 cccttggtgg ctgcaaaagg acataaggcc tacagtgtgc tgaagcaaga atggggaccg 21600 gtgcatagcc acacaattgt tcagcactgt ctgctttctt catgcacaga gctgccactc 21660 aagagcatcc aagatgctta tggaacattt ctggaaaggg atattaatca aaagtatata 21720 cacagtaggg gtgtgtgtgt gtgtatgtga gcgtcaagat ttttttttaa tggcgtactg 21780 taaatatcag gcccttttc cagctctgg gctgggaat caatgtgaa ggcagccgga 21840 atccccaagc cgtgtctgaa aatcttggct atgtgtgtat ctacagtata cgctgtggtg 21900 tatgtgaaac cccaccttaca cacgcgtgca cacacacc cctgaaagcg tgtttgtaca 21960 tctatgtgga tatatataat ctagttctgt gattaaaatt attaata attaata 22020 tcaaaaggt actcggtctg tgtcagaat ctgccaaaca tcatctgcaa ttgaattttt 22080 caacgcctga taatgtaca cacggaagc ttccaaaaa caacagacag tctaaaaaca 22140 gactcacaac aagtgactac taaccactag gaaccccca tccgaccgat ggctttcaga 22200 aggaaaaa aaacacacgg gtgggtgctt gcatctgcaa tgtatatac ttatgctgca 22260 tcgtgcgcaa gagaggct tccgatgggg ggggaggga gagaaagtgt tttatatact 22320 tattatta cccttttaa attagaatt aaacagatta tattattaa gagttaggt 22380 ttttcagtat tctttgttaa tattattt caactattta taagccgtac ctcttttttt 22440 tttttttttt tttttttttt tttttatttg tactggttgt gtataaattt aaccttcctg 22500 ttgtcccatc ccatctctcc ccaattgttg gcagagtcag tagcatgtta tgggcagtta 22560 tttaattaat ttctctaaca cctaccccta cacattccta ttgagacaag ggttagatat 22620 acatctacat actatatata tatttggcta tgtgtttgta tatatatata tatgtttatg 22680 tatatgtgtg atttggatta aatagacgct acgatttttt tatatatgta aaaagaagag 22740 acaggaaaa tagaaaattc tacatcttga atctctctcc tttttaattt taatatttgt 22800 tatcatttta tttattggtg ctactgttta tctgtaataa ttgcggggaa aaaagatatt 22860 aacactacat attgtgtcta gtgaattttt tcaagatatt ccttagtaca tatttatttt 22920 taaacaaaga aattacagat atatcttaaa aaaacgacga cgacgacgac aaacaaactt 22980 tttttgtatt aaagaattta attctgacct tga 23013

Claims

1. A molecular marker-assisted breeding method for high egg production and weight trait of Leizhou black duck, characterized in that: The following steps are involved: a. Blood was collected from the wing vein of Leizhou black ducks to extract genomic DNA; b. Using the primer pair: upstream primer F: 5′-ATCATGAGAATTAAACCCCATCAGAGTCAG-3′ and downstream primer R: 5′-CTTGAATCCATTCTGGTAGGACACACAC-3′, PCR amplification was performed using the above genomic DNA as a template; c. The PCR amplification product was purified and sequenced, and the sequencing results were compared with the VEGFA gene sequence shown in SEQ ID NO.1 to determine the genotype of the Leizhou black duck individual SNP site I4-14004 G>T. Individuals with the GT genotype at this site were selected, i.e., Leizhou black duck individuals with high egg production weight traits; the SNP site I4-14004G>T indicated that the SNP site mutated from G to T at the 14004th base on the VEGFA gene sequence.

2. The method according to claim 1, characterized in that The PCR amplification reaction system is 25 μL, which contains 11 μL of ddH2O, 0.5 μL of upstream and downstream primers, 12.5 μL of Green Taq Mix, and 0.5 μL of Leizhou black duck genomic DNA template.

3. The method according to claim 1, characterized in that The PCR amplification reaction procedure is as follows: pre-denaturation at 94°C for 4 min; 35 cycles of 94°C for 30 s, 53.4°C for 30 s, and 72°C for 20 s; and extension at 72°C for 10 min.

4. A molecular marker-assisted breeding method for high egg production performance of Leizhou black ducks, characterized in that: The following steps are involved: a. Blood was collected from the wing vein of Leizhou black ducks to extract genomic DNA; b. Using the primer pair: upstream primer F: 5′-ATCATGAGAATTAAACCCCATCAGAGTCAG-3′ and downstream primer R: 5′-CTTGAATCCATTCTGGTAGGACACACAC-3′, PCR amplification was performed using the above genomic DNA as a template; c. The PCR amplification product was purified and sequenced, and the sequencing results were compared with the VEGFA gene sequence shown in SEQ ID NO.1 to obtain the genotype of the Leizhou black duck individual at the following 8 SNP sites: I4-13876 C>T, I4-13925 C>T, I4-13926 C>G, I4-14004 G>T, I4-14063 C>T, I4-14076 G>A, I4-14173 T>C, and I4-14175 T>C, and the haplotype combination of the Leizhou black duck individual was determined; the SNP sites: I4-13876 C>T, I4-13925 C>T, I4-13926 C>G, I4-14004 G>T, I4-14063 C>T, I4-14076 G>A, I4-14173 T>C, and I4-14175 T>C. T>C and I4-14175 T>C respectively indicate that the SNP sites on the VEGFA gene sequence are mutated from C to T at base 13876, from C to T at base 13925, from C to G at base 13926, from G to T at base 14004, from C to T at base 14063, from G to A at base 14076, from T to C at base 14173, and from T to C at base 14175; d. For the trait of age at first laying, give priority to breeding individuals with the haplotype combination of H1H6 or H2H5, and screen out individuals with the haplotype combination of H1H3; for the trait of egg weight at first laying, give priority to breeding individuals with the haplotype combination of H1H3, H2H3, or H3H5. Eliminate individuals with the haplotype combinations of H4H6, H1H7, or H5H6; prioritize individuals with the haplotype combinations of H1H6 or H5H5 for the egg production trait at 300 days of age, and eliminate individuals with the haplotype combinations of H2H3 or H3H5; The haplotype combination at SNP sites I4-13876 C>T, I4-13925 C>T, I4-13926 C>G, I4-14004 G>T, I4-14063 C>T, I4-14076 G>A, I4-14173 T>C, I4-14175 T>C, the H1H3 haplotype combination is CC, CC, CC, GT, CT, AA, CC, TT genotypes, The H1H6 haplotype combinations are CT, CC, CC, GT, CT, AG, CC, and TT genotypes. The H1H7 haplotype combinations are CC, CC, CC, TT, CT, AA, CC, TT genotypes. The H2H3 haplotype combinations are CC, CC, CC, GT, CT, AA, CT, CT genotypes, The H2H5 haplotype combinations are CC, CT, CG, GT, CT, AG, CT, CT genotypes, The H3H5 haplotype combinations are CC, CT, CG, GG, CC, AG, CC, and TT genotypes. The H4H6 haplotype combinations are CT, CC, CC, GG, CC, AG, CT, CT genotypes, The H5H5 haplotype combinations are CC, TT, GG, GG, CC, GG, CC, TT genotypes. The H5H6 haplotype combinations are CT, CT, CG, GG, CC, GG, CC, and TT genotypes.

5. The method according to claim 4, characterized in that The PCR amplification reaction system is 25 μL, which contains 11 μL of ddH2O, 0.5 μL of upstream and downstream primers, 12.5 μL of Green Taq Mix, and 0.5 μL of Leizhou black duck genomic DNA template.

6. The method according to claim 4, characterized in that The PCR amplification reaction procedure is as follows: pre-denaturation at 94°C for 4 min; 35 cycles of 94°C for 30 s, 53.4°C for 30 s, and 72°C for 20 s; and extension at 72°C for 10 min.

Images