A molecular marker related to early body weight traits of Sansui ducks and its breeding method

Abstract

The present invention provides a molecular marker and breeding method associated with the early-stage weight trait of Sansui ducks, belonging to the field of selective breeding. Deep whole-genome sequencing of a Sansui duck population was performed. Comparison with the whole-genome sequence of Peking ducks in a gene library revealed a 17-bp insertion mutation at the 3' end of the ALMS1 gene and a 51-bp deletion mutation in the seventh intron region of the PBX1 gene. Trait association analysis results showed that these mutations significantly affected the early-stage weight of Sansui ducks.

Description

Technical Field

[0001] The present invention relates to the technical field of livestock and poultry breeding, and in particular to a molecular marker related to the early body weight trait of Sansui ducks and a breeding method. Background Art

[0002] Sansui duck is an important local duck genetic resource in Guizhou Province. It is a small egg-laying duck breed with the characteristics of early maturity, high egg production, strong vitality, tender meat and unique flavor. At the same time, it has disadvantages such as slow growth. Sansui duck and other low-fat local duck breeds. The rapid growth period is in the first 9-12 weeks, while the rapid growth period of meat duck breeds such as Beijing duck and Cherry Valley duck is in the first 5 weeks. Early growth performance has a great influence on duck market weight, daily weight gain, feed utilization rate and meat performance, and is an important economic trait in Sansui duck farming. In order to carry out breeding to improve the early growth performance of Sansui duck, explore the genetic mechanism of the early weight trait of Sansui duck, explore the major effect genes or genetic markers affecting this trait, and construct genetic marker-assisted breeding technology, which is of great significance to improving the early growth performance and weight traits of Sansui duck, and ultimately improving its market weight and slaughter meat performance, reducing costs and increasing efficiency. The present invention is based on this related research. Summary of the Invention

[0003] The present invention conducted deep whole-genome sequencing on the Sansui duck population and compared it with the whole-genome sequence of Beijing duck in the gene library. It was found that there was a 17bp insertion mutation at the 3' end of the ALMS1 gene and a 51bp deletion mutation in the 7th intron region of the PBX1 gene. The results of trait association analysis showed that the above mutations significantly affected the early weight of Sansui ducks.

[0004] ALMS1 (centrosome and basal body associated protein) is a protein found in the cytoplasm, cytoskeleton, microtubule organization, centrosomes, and the base of cilia. Research suggests that it is ubiquitously distributed. Some studies suggest that it is involved in energy metabolism and homeostasis, cell differentiation, and cell cycle control, but its functions remain incompletely understood. Mutations in the ALMS1 gene can cause a variety of symptoms in humans and mice, including appetite disorders, type 2 diabetes, cardiomyopathy, renal impairment, male infertility, pubertal growth arrest, and short stature.

[0005] PBX1 (PBX homeobox 1, pre-B-cell leukemia homeobox 1) is a three-amino acid loop extension (TALE) transcription factor. Its target genes include ubiquitous developmental genes such as PAX1 / PAX9, WNT9B / WNT3, and PITX3. Therefore, it is a key developmental regulator, driving cell proliferation, pluripotency, and differentiation, and regulating embryonic limb axial development, organogenesis, and hematopoiesis. Mutations in the PBX1 gene are associated with a variety of congenital diseases, including congenital kidney and urinary tract anomalies, skeletal malformations, congenital heart disease, embryonic asplenia, and sexual differentiation disorders. After birth, PBX1 plays a crucial role in maintaining self-renewal and pluripotency.

[0006] The present invention utilizes insertion-deletion mutations in the ALMS1 and PBX1 genes found in a Sansui duck population to conduct molecular marker-assisted breeding. This allows for selection of Sansui ducks for their early weight traits, eliminating inferior individuals, reducing costs and increasing efficiency. From a genetic perspective, it aggregates advantageous genotypes, improving breeding efficiency, and ultimately improving early growth performance or weight traits in the Sansui duck population, indirectly improving adult weight and slaughter traits. Therefore, a molecular marker and breeding method related to the early weight trait of Sansui ducks are provided.

[0007] The specific technical solutions of the present invention are:

[0008] In the first aspect, the present invention provides an application of a molecular marker related to the early weight trait of Sansui duck in the breeding of the early weight trait of Sansui duck, wherein the molecular marker is site A and / or site B, wherein the site A is located at position 3426 of the 3' end of the ALMS1 gene and corresponds to position 348 of the nucleotide sequence shown in SEQ ID NO.1; the site B is located at position 68799 of the 7th intron region of the PBX1 gene and corresponds to position 344 of the nucleotide sequence shown in SEQ ID NO.2.

[0009] Preferably, there is an insertion-deletion mutation at position 348, and the insertion-deletion mutation includes three alleles A1, I1, and D1, wherein A1 corresponds to base A, I1 corresponds to base A and the sequence shown in SEQ ID NO.3, and D1 corresponds to the deletion of base A and the sequence shown in SEQ ID NO.3;

[0010] There is an insertion-deletion mutation at position 344, and the insertion-deletion mutation includes three alleles A2, I2 and D2, wherein A2 corresponds to base A, I2 corresponds to base A and the sequence shown in SEQ ID NO.4, and D2 corresponds to the missing base A and the sequence shown in SEQ ID NO.4.

[0011] In a second aspect, the present invention provides a method for breeding Sansui ducks for early body weight traits, comprising:

[0012] Step 1: Extract the genomic DNA of Sansui duck to be tested.

[0013] Step 2, using a primer pair and the genomic DNA of the Sansui duck to be tested as a template, performing PCR amplification and sequencing on the molecular markers described in the first aspect;

[0014] Step 3, determining the genotype of the molecular marker based on the sequencing results;

[0015] Step 4: Select and cultivate individuals based on the genotype results.

[0016] Preferably, the sequences of the primer pairs for detecting the A site are shown as SEQ ID NO: 5 and SEQ ID NO: 6; the sequences of the primer pairs for detecting the B site are shown as SEQ ID NO: 7 and SEQ ID NO: 8.

[0017] Preferably, in step 3, the genotypes of molecular marker site A are A1A1, I1I1, I1A1 and D1D1, and the genotypes of molecular marker site B are I2I2, I2A2 and D2D2.

[0018] Further preferably, in the molecular marker site A, the PCR product of the A1A1 genotype includes sites 130 to 489 of the sequence shown in SEQ ID NO. 1, corresponding to sites 74459660 to 74460019 on the duck chromosome 4 sequence in the GenBank database, and sites 3208 to 3567 at the 3' end of the ALMS1 gene;

[0019] The PCR product of the I1I1 genotype is inserted into the sequence shown in SEQ ID NO. 3 relative to the A1A1 genotype;

[0020] The PCR product sequences of the I1A1 genotype contained two sequences simultaneously, one identical to the A1A1 genotype and the other identical to the I1I1 genotype;

[0021] The PCR product of the D1D1 genotype has a deletion of base A at position 348 of the SEQ ID NO.1 sequence, position 74459877 of the duck chromosome 4 sequence in the GenBank database, and position 3426 of the 3' end of the ALMS1 gene relative to the A1A1 genotype;

[0022] In the molecular marker site B, the PCR product of the I2I2 genotype includes sites 180 to 540 of the sequence shown in SEQ ID NO. 2, corresponding to sites 10269420 to 10269780 on the duck chromosome 8 sequence in the GenBank database and sites 68635 to 68995 of the PBX1 gene;

[0023] The PCR product of the I2A2 genotype contains two sequences: one sequence is missing the sequence shown in SEQ ID NO. 4 relative to the I2I2 genotype; the other sequence is the same as the I2I2 genotype;

[0024] Compared with the I2I2 genotype, the PCR product of the D2D2 genotype lacks the sequence shown in SEQ ID NO.4, as well as base A at position 344 of the corresponding SEQ ID NO.2 sequence, position 10269583 on the duck chromosome 1 sequence in the GenBank database, and position 68798 of the PBX1 gene.

[0025] Preferably, in step 5, the individual selection criteria are: for site A, select individuals with the A1A1 genotype and eliminate individuals with other genotypes; for site B, select individuals with the I2I2 genotype and eliminate individuals with other genotypes.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0027] By studying molecular marker A, located at position 3426 of the 3' end of the ALMS1 gene and corresponding to position 348 of the nucleotide sequence shown in SEQ ID NO. 1, and molecular marker B, located at position 68799 of the 7th intron region of the PBX1 gene and corresponding to position 344 of the nucleotide sequence shown in SEQ ID NO. 2, the present invention discovered insertion / deletion mutations in both markers. By detecting and genotyping these two loci, a breeding method for early-stage weight traits in Sansui ducks was developed. This method is unaffected by age, sex, and other factors, and DNA can be extracted at any stage of breeding for testing and breeding. This method improves the weight traits of Sansui duck offspring, possessing significant economic value and practical significance. Furthermore, this method is unaffected by the breeding environment and feed nutrition, can genetically improve weight traits, aggregate advantageous genotypes, and rapidly enhance breeding progress. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0029] Figure 1This figure shows the information of primer sequences, insertion mutations, deletion sequences, etc. in SEQ ID NO.1 and SEQ ID NO.2 of the present invention, wherein Figure A corresponds to SEQ ID NO.1 and Figure B corresponds to SEQ ID NO.2.

[0030] Figure 2 Box plot comparing the performance of different genotypes of ALMS1 molecular marker A.

[0031] Figure 3 Box plot comparing the performance of different genotypes of PBX1 molecular marker B.

[0032] Figures 2-3 In the figure, the horizontal axis represents the genotypes of ALMS1 and PBX1, and the vertical axis represents the body weight at 42 days of age. DETAILED DESCRIPTION

[0033] In the description of the present invention, it should be noted that if specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0034] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0035] Example 1

[0036] This example provides the discovery process of the Sansui duck ALMS1 gene and PBX1 gene molecular markers, as follows:

[0037] 1. Test materials

[0038] The experiment used 665 Sansui ducks (483 females and 172 males) from the breeding duck farm of Sansui Qianlishan Food Technology Co., Ltd. Male and female ducks were hatched from the same batch and raised under the same conditions and environment.

[0039] 2. Molecular marker detection

[0040] When the ducks were 160 days old, 1.5 mL of blood was collected from the wing vein, treated with EDTA anticoagulation, and subjected to 10× deep whole-genome resequencing by Beijing Compson Agricultural Technology Co., Ltd.

[0041] After deep sequencing and comparative analysis of the database genome, it was found that a 17bp insertion mutation was found at position 3426 of the 3' end of the Sansui duck ALMS1 (centrosome and basal body associated protein) gene, relative to the Peking duck genome (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCA_015476345.1 / ). This position also corresponds to position 348 of the nucleotide sequence shown in SEQ ID NO.1. Detection found that the site includes three alleles A1, I1, and D1, among which A1 corresponds to base A, I1 corresponds to base A and the 17bp insertion sequence shown in SEQ ID NO.3, and D1 corresponds to the missing base A and the 17bp sequence shown in SEQ ID NO.3 (tccaaacagctccaccg).

[0042] At the same time, it was discovered that, compared to the Peking duck genome, the Sansui duck had a 51bp deletion mutation at position 68799 in the 7th intron region of the PBX1 (PBX homeobox 1, pre-B cell leukemia homeobox 1) gene. This position also corresponds to position 344 of the nucleotide sequence shown in SEQ ID NO.2. Testing revealed that this site includes three alleles, A2, I2, and D2. Among them, A2 corresponds to base A, I2 corresponds to base A and the 51bp deletion sequence shown in SEQ ID NO.4, and D2 corresponds to the deletion of base A and the 51bp deletion sequence shown in SEQ ID NO.4 (gcacggcagc agccaggtta tttttaagcc agtgttttta cacctaccca t).

[0043] Example 2

[0044] This example provides a method for detecting ALMS1 and PBX1 molecular markers and trait association analysis, as follows:

[0045] 1. Test materials

[0046] The experiment used 65 Sansui ducks from the breeding farm of Sansui County Qianlishan Food Technology Co., Ltd. Male and female ducks were mixed and raised under the same breeding conditions and environment.

[0047] 2. ALMS1 gene molecular marker detection

[0048] Blood was collected from the wing vein at around 50 days of age, and DNA was extracted using the TIANamp Blood DNA Kit. Primers were designed using the GenBank database online tool, and primer sequences were synthesized (commissioned to a biochemical technology company).

[0049] The PCR amplification primer sequence information is as follows:

[0050] Upstream primer F: 5'-TCAGGCATCTCTGGCATGTG-3'; (as shown in SEQ ID NO. 5)

[0051] Downstream primer R: 5'-CAGGTGCTGAAAGGCAACAC-3'; (as shown in SEQ ID NO. 6).

[0052] PCR reaction system: 1 μL DNA template, 1 μL each of upstream and downstream primers, 10 μL of 2× Taq PCR MasterMix reagent, and ddH₂O to 20 μL. PCR amplification protocol: ① Initial denaturation at 95°C for 5 min. ② Amplification reaction: 35 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s; final extension at 72°C for 5 min. PCR products were sequenced by Nosai Genomics Research Center Co., Ltd.

[0053] After sequencing, DNAMAN software was used for sequence alignment and genotyping. The results of the sequence alignment for each genotype are as follows:

[0054] (1) The nucleotide sequence of the PCR amplification product of the A1A1 genotype is shown in SEQ ID NO.1, including the region 130-489, corresponding to sites 74459660 to 74460019 on the duck chromosome 4 sequence in the GenBank database and sites 3208 to 3567 at the 3' end of the ALMS1 gene. The primer sequences, insertion mutations, deletion sequences, etc. in SEQ ID NO.1 are shown in Figure 1 shown.

[0055] (2) The PCR product of the I1I1 genotype is 377 bp in length. Compared with the A1A1 genotype, the 17 bp sequence shown in SEQ ID NO. 3 is inserted at position 348 shown in SEQ ID NO. 1.

[0056] (3) There are two lengths of PCR products of the I1A1 genotype: one is 360 bp in length, with the same sequence as the A1A1 genotype, and the other is 377 bp in length, with the same sequence as the I1I1 genotype.

[0057] (4) The PCR product of the D1D1 genotype is 359 bp in length. Compared with the A1A1 genotype, it lacks base A at position 348 of SEQ ID NO.1, position 74459877 of duck chromosome 4 sequence in the GenBank database, and position 3426 of the 3' end of the ALMS1 gene.

[0058] The genotyping results of ALMS1 molecular marker detection are shown in Table 1. Among the 65 Sansui duck samples, 48 ​​individuals with A1A1 genotype, 3 individuals with I1I1 genotype, 12 individuals with I1A1 genotype, and 2 individuals with D1D1 genotype were finally detected.

[0059] 3. PBX1 gene molecular marker detection

[0060] Blood was collected from the wing vein at around 50 days of age, and DNA was extracted using the TIANamp Blood DNA Kit. Primers were designed using the GenBank database online tool, and primer sequences were synthesized (commissioned to a biochemical technology company).

[0061] The PCR amplification primer sequence information is as follows:

[0062] Upstream primer F: 5'-GCCCCAAAACGGCATCTTTT-3'; (as shown in SEQ ID NO. 7)

[0063] Downstream primer R: 5'-TCTGTTTAAGCCGCTCCCTG-3'; (as shown in SEQ ID NO. 8).

[0064] PCR reaction system: 1 μL DNA template, 1 μL each of upstream and downstream primers, 10 μL of 2× Taq PCR MasterMix reagent, and ddH₂O to 20 μL. PCR amplification protocol: ① Initial denaturation at 95°C for 5 min. ② Amplification reaction: 35 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s; final extension at 72°C for 5 min. PCR products were sequenced by Nosai Genomics Research Center Co., Ltd.

[0065] After sequencing, DNAMAN software was used for sequence alignment and genotyping. The results of the sequence alignment for each genotype are as follows:

[0066] (1) The nucleotide sequence of the PCR amplification product of the I2I2 genotype is shown in SEQ ID NO. 2, which is 361 bp in total and includes sites 180 to 540 of the sequence shown in SEQ ID NO. 2, corresponding to sites 10269420 to 10269780 on the duck chromosome 8 sequence in the GenBank database and sites 68635 to 68995 of the PBX1 gene; the primer sequences, insertion mutations, deletion sequences, etc. in SEQ ID NO. 2 are shown in Figure 1 shown.

[0067] (2) There are two lengths of PCR products for the I2A2 genotype: one is 310 bp, which lacks the 345-395 position shown in SEQ ID NO. 2, i.e., the 51 bp sequence shown in SEQ ID NO. 4, compared to the I2I2 genotype; the other is 361 bp, with the same sequence as the I2I2 genotype;

[0068] (3) The PCR product of the D2D2 genotype was 309 bp in length. Compared with the I2I2 genotype, it lacked the sequence shown in SEQ ID NO. 2, as well as base A at position 344 of the corresponding SEQ ID NO. 2 sequence, position 10269583 on the duck chromosome 1 sequence in the GenBank database, and position 68798 of the PBX1 gene.

[0069] (4) The genotyping results of PBX1 gene molecular marker detection are shown in Table 1. As shown in Table 1, 52 individuals with I2I2 genotype, 3 individuals with D2D2 genotype, and 10 individuals with I2A2 genotype were detected in 65 Sansui duck samples.

[0070] 4. Growth Data Determination

[0071] The birth weight was measured, and then the weight of Sansui ducks was measured every 2 weeks. The weight at 42 days of age (D42) was used as an indicator of early weight traits for association analysis. The data results of the weight at 42 days of age (D42) are shown in Table 1.

[0072] Table 1 Molecular marker genotyping and 42-day body weight (D42, g) data of each sample

[0073]

[0074]

[0075] In this embodiment, the weight at 42 days of age (D42) is used as an early body weight trait for only an example. The weight below 70 days of age can be used as an early body weight trait, preferably at 40-50 days of age.

[0076] 5. Association analysis between molecular marker genotypes and D42 traits

[0077] SPSS 22.0 software was used to draw the box plots of different genotypes of ALMS1. Figure 2 .from Figure 2 It can be seen that among the four genotypes, the body weight at 42 days of age (D42) of individuals with the ALMS1 gene molecular marker A1A1 genotype was significantly better than that of individuals with other genotypes.

[0078] SPSS 22.0 software was used to draw the box plots of different genotypes of PBX1. Figure 3 .from Figure 3It can be seen that among the three genotypes, the body weight at 42 days of age (D42) of individuals with the PBX1 gene molecular marker I2I2 was significantly better than that of individuals with other genotypes.

[0079] The results of multiple comparisons and significance tests of various traits of different genotypes are shown in Table 2. The letters above the mean values ​​in the table indicate significant test results. Different lowercase letters indicate significant differences (P < 0.05), different uppercase letters indicate extremely significant differences (P < 0.01), and the same letters or no mark indicate no significant difference.

[0080] As shown in Table 2, the D42 trait of the ALMS1 gene A1A1 genotype was significantly higher than that of individuals with other genotypes. There was a significant difference in the D42 trait between I1A1 and I1I1, but there was no significant difference in the D42 trait between individuals with the D1D1 genotype and those with the I1A1 or I1I1 genotypes.

[0081] As shown in Table 2, the D42 trait of the PBX gene I2I2 genotype was significantly higher than that of individuals with other genotypes, and there was no significant difference in the D42 trait between I2A2 and D2D2 individuals.

[0082] Table 2 Comparison results of D42 traits among genotypes

[0083]

[0084] Example 3

[0085] This example uses ALMS1 and PBX1 molecular markers for breeding of D42 traits, as follows:

[0086] 1. Test materials and growth data determination:

[0087] The sources of the experimental materials were the same as those in Example 2. The results of the D42 trait and molecular marker genotype detection of 65 samples are shown in Table 1.

[0088] 2. Individual selection and elimination

[0089] Individuals are selected and eliminated based on the genotype of the loci. Individuals with the A1A1 genotype at locus A and the I2I2 genotype at locus B are selected, and individuals with other genotypes are eliminated.

[0090] Among the 65 Sansui duck samples, 48 ​​individuals had the A1A1 genotype at the ALMS1 gene molecular marker A site, but two individuals (A066 and S3944) had the D2D2 genotype at the PBX1 gene molecular marker B site, and four individuals (A090, S3910, S3958 and S9567) had the I2A2 genotype at the PBX1 gene molecular marker B site. Therefore, 42 individuals were finally retained and 23 individuals were eliminated.

[0091] 3. Evaluation of the selection and retention effect

[0092] To evaluate the effectiveness of this breeding method, the ducks were not culled. Instead, they were reared under the same conditions until market release, and their average daily weight gain (AGR) was calculated. Growth and meat performance were then measured at slaughter. These performance indicators included AGR, live weight before slaughter, carcass weight, eviscerated weight, eviscerated weight, breast muscle weight, and leg muscle weight. These performance indicators were measured in accordance with the "Terminology and Statistical Methods for Measurement of Poultry Production Performance: Agricultural Industry Standard of the People's Republic of China, NY / T823-2004."

[0093] Performance comparisons were performed between the retained and eliminated individuals. Data for each indicator are presented as mean ± standard deviation. Letters superimposed above the mean values ​​indicate significant differences (P < 0.05). Different lowercase letters indicate significant differences (P < 0.01). Identical letters or no letter indicate nonsignificant differences. The comparison results are shown in Table 3.

[0094] Table 3 Comparison of traits of each population Unit: g

[0095] D42 Daily average weight gain Pre-slaughter weight Carcass weight all 808.15±123.23 3.22±0.97 1285.25±169.01 1182.52±161.09 Selection <![CDATA[868.82±79.95 A ]]> 3.12±1.07 <![CDATA[1330.48±167.20 A ]]> <![CDATA[1221.88±158.52 A ]]> disuse <![CDATA[697.38±111.42 B ]]> 3.41±0.76 <![CDATA[1202.66±141.28 B ]]> <![CDATA[1110.66±142.51 B ]]> Semi-eviscerated weight Eviscerated weight Chest muscle weight Leg muscle weight all 1070.98±151.06 792.61±100.45 125.62±22.71 121.21±19.96 Selection <![CDATA[1108.61±150.68 A ]]> <![CDATA[813.37±105.48 a ]]> 129.31±23.87 <![CDATA[125.61±21.29 a ]]> disuse <![CDATA[1002.25±128.17 B ]]> <![CDATA[754.69±79.36 b ]]> 118.86±19.09 <![CDATA[113.16±14.48 b ]]>

[0096] Table 3 shows that the early body weight (D42) of the retained group was significantly higher than that of the culled group. After market release (160 days of age), the live weight, carcass weight, and eviscerated weight of the retained group were significantly higher than those of the culled group. The eviscerated weight and leg muscle weight of the retained group were significantly higher than those of the culled group. There was no significant difference in average daily weight gain and breast muscle weight.

[0097] In summary, the present invention, through deep whole-genome sequencing and association analysis with traits, found that insertion-deletion mutations in the 3' end of the ALMS1 gene and the 7th intron region of the PBX1 gene were significantly associated with the early growth traits (weight at 42 days of age) of Sansui ducks. The present invention constructed a molecular marker-assisted breeding method based on the ALMS1 and PBX1 genes to improve the early growth traits of Sansui ducks, providing a new breeding technology for the selection of Sansui duck meat product lines. Since early growth traits can affect the final meat traits, the application of this method indirectly improves the adult weight and slaughter traits of Sansui ducks, which has important economic value and scientific significance for accelerating the genetic improvement of Sansui ducks, tapping their meat performance potential, and cultivating new low-fat, high-quality local meat duck lines, and improving the germplasm resources of local ducks and reducing costs and increasing efficiency.

[0098] Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents. The embodiments described above merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they cannot be construed as limiting the scope of the patent of the present invention. It should be pointed out that, without departing from the concept of the present invention, a number of variations and improvements may be made by those skilled in the art, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the appended claims.

Claims

1. A method for breeding Sansui ducks for early body weight traits, characterized in that: include: Step 1: Extract the genomic DNA of Sansui duck to be tested. Step 2: using a primer pair and the genomic DNA of the Sansui duck to be tested as a template, PCR amplification and sequencing of molecular markers related to the early weight traits of Sansui ducks; the molecular markers are site A and / or site B, wherein site A is located at position 3426 of the 3' end of the ALMS1 gene and corresponds to position 348 of the nucleotide sequence shown in SEQ ID NO.1; and site B is located at position 68799 of the 7th intron region of the PBX1 gene and corresponds to position 344 of the nucleotide sequence shown in SEQ ID NO.2; Step 3, based on the sequencing results, determining the genotype of the molecular marker, wherein the genotype of the molecular marker site A is A1A1, I1I1, I1A1 and D1D1, and the genotype of the molecular marker site B is I2I2, I2A2 and D2D2; Step 4: select and cultivate individuals based on genotype results; There is an insertion-deletion mutation at position 348, and the insertion-deletion mutation includes three alleles A1, I1, and D1, wherein A1 corresponds to base A, I1 corresponds to base A and the sequence shown in SEQ ID NO. 3, and D1 corresponds to the deletion of base A and the sequence shown in SEQ ID NO. 3; There is an insertion-deletion mutation at position 344, and the insertion-deletion mutation includes three alleles A2, I2, and D2, wherein A2 corresponds to base A, I2 corresponds to base A and the sequence shown in SEQ ID NO. 4, and D2 corresponds to the deletion of base A and the sequence shown in SEQ ID NO. 4; In step 4, the individual selection criteria are: for site A, individuals with the A1A1 genotype are selected and individuals with other genotypes are eliminated; for site B, individuals with the I2I2 genotype are selected and individuals with other genotypes are eliminated.

2. The breeding method according to claim 1, characterized in that: The sequences of the primer pairs for detecting the A site are shown in SEQ ID NO: 5 and SEQ ID NO: 6; the sequences of the primer pairs for detecting the B site are shown in SEQ ID NO: 7 and SEQ ID NO:

8.

3. The breeding method according to claim 2, characterized in that: In the molecular marker site A, the PCR product of the A1A1 genotype includes sites 130 to 489 of the sequence shown in SEQ ID NO.1, corresponding to sites 74459660 to 74460019 on the duck chromosome 4 sequence in the GenBank database, and sites 3208 to 3567 at the 3' end of the ALMS1 gene; The PCR product of the I1I1 genotype is inserted into the sequence shown in SEQ ID NO. 3 relative to the A1A1 genotype; The PCR product sequences of the I1A1 genotype contained two sequences simultaneously, one identical to the A1A1 genotype and the other identical to the I1I1 genotype; The PCR product of the D1D1 genotype has a deletion of base A at position 348 of the SEQ ID NO.1 sequence, position 74459877 of the duck chromosome 4 sequence in the GenBank database, and position 3426 of the 3' end of the ALMS1 gene relative to the A1A1 genotype; In the molecular marker site B, the PCR product of the I2I2 genotype includes sites 180 to 540 of the sequence shown in SEQ ID NO. 2, corresponding to sites 10269420 to 10269780 on the duck chromosome 8 sequence in the GenBank database and sites 68635 to 68995 of the PBX1 gene; The PCR product of the I2A2 genotype contains two sequences: one sequence is missing the sequence shown in SEQ ID NO. 4 relative to the I2I2 genotype; and the other sequence is the same as the I2I2 genotype; Compared with the I2I2 genotype, the PCR product of the D2D2 genotype lacks the sequence shown in SEQ ID NO.4, and lacks base A at position 344 of the corresponding SEQ ID NO.2 sequence, position 10269583 on the duck chromosome 1 sequence in the GenBank database, and position 68798 of the PBX1 gene.

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