Primer pair and molecular marker for simultaneously predicting and identifying natural length and crimpness of sheep wool and application

Abstract

The invention relates to the technical field of animal molecular genetics, in particular to a primer pair for simultaneously predicting and identifying the natural length and crimpness of sheep wool, a molecular marker and application. According to the application of a primer pair of the molecular marker for simultaneously indicating and identifying the natural length and the crimpness of the sheep wool in the detection of the natural length and the crimpness of the sheep wool, the molecular marker is located at a T76025431C site of a second intron region of a YWHAZ gene, and the site is obtained by mutating a C base from a T base; when the natural length and the crimpness of the sheep wool are selected through the marker genotype, the natural length and the crimpness of the sheep wool have great genetic progress, and the method has the advantages of being easy to operate, low in cost and high in accuracy.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of animal molecular genetics, and particularly relates to a primer pair and a molecular marker for simultaneously predicting and identifying natural length and curliness of sheep wool and application thereof. BACKGROUND

[0002] The natural length and curliness of wool are important indexes of wool quality traits of sheep. The natural length of wool is directly measured by a ruler in a natural state. The curliness of wool refers to the number of curls of wool in a natural length of 2.5 cm. Different fine wool breeds have different natural lengths and curliness of wool. For example, the natural length and curliness of wool of Australian Merino sheep are superior to those of many fine wool breeds. The natural length and curliness of wool are second only to the average fiber diameter of wool in terms of significance in textile technology. They not only affect the parameter setting of textile technology, but also affect the comfort of wool textiles. In addition, with the change of market demand, the influence of the natural length of wool on the market price of wool has exceeded that of the average fiber diameter and curliness of wool, and has become the largest factor affecting the market price of wool. In order to meet market demand and increase the economic income of herdsmen, how to further improve the natural length and curliness of wool, cultivate high-quality fine wool sheep with long natural length and large curliness of wool, and rapidly expand the population scale is still an important problem.

[0003] YWHAZ belongs to the 14-3-3 gene family and plays an important role in the regulation of signal transduction, cell cycle and apoptosis. Studies have shown that YWHAZ is highly expressed in cancer cells such as lung cancer and breast cancer. Overexpression of YWHAZ can down-regulate the expression of the tumor suppressor gene p53 and promote cancer cell proliferation. In addition, the scratch experiment shows that overexpression of YWHAZ can promote tumor cell metastasis. The relationship between YWHAZ gene and wool quality traits in livestock and poultry, especially sheep, has not been reported. SUMMARY

[0004] In view of the deficiencies in the prior art, the application provides a primer pair and a molecular marker for simultaneously predicting and identifying the natural length and curliness of sheep wool and application thereof. The molecular marker and the primer pair of the application can be used to simultaneously select the natural length and curliness of wool, thereby providing an effective molecular marker breeding method for improving the quality traits of sheep wool and a more effective, simple and easy molecular marker method for marker-assisted selection in sheep breeding.

[0005] The application provides a primer pair of a molecular marker for simultaneously predicting and identifying the natural length and curliness of sheep wool. The molecular marker is located at a T76025431C site in a second intron region of a YWHAZ gene. The site is mutated from a T base to a C base. The nucleotide sequence of the second intron region of the YWHAZ gene is shown in SEQ ID NO. 1. The primer pair comprises an upper primer and a lower primer, wherein the nucleotide sequence of the upper primer is shown in SEQ ID NO. 2, and the nucleotide sequence of the lower primer is shown in SEQ ID NO. 3.

[0006] The application further provides application of the primer pair of the molecular marker for simultaneously predicting and identifying natural length and curl degree of sheep wool in detection of natural length and curl degree of sheep wool.

[0007] A molecular marker method for simultaneously predicting and identifying natural length and curl degree of sheep wool comprises the following steps. Step 1: extracting sheep genomic DNA; Step 2: performing PCR amplification on the sheep genomic DNA according to the primer pair of the T76025431C site in the 2nd intron region of the YWHAZ gene, and then performing enzyme cutting by using endonuclease XspI to obtain an enzyme cutting product; Step 3: performing electrophoretic separation on the enzyme cutting product, when two bands are presented in the electrophoresis, the molecular marker site is in the CC genotype; when one band is presented in the electrophoresis, the molecular marker site is in the TT genotype; and when three bands are presented in the electrophoresis, the molecular marker site is in the TC genotype; wherein the TT genotype is a sheep individual with high natural length and curl degree of sheep wool, the CC genotype is a sheep individual with low natural length and medium curl degree of sheep wool, and the TC genotype is a sheep individual with low natural length and curl degree of sheep wool.

[0008] Compared with the prior art, the application has the following beneficial technical effects: The application has the advantages of simple operation, low cost, high accuracy and automatic detection. When the marker genotype is used for selecting sheep wool natural length and curl degree, great genetic progress can be achieved in the natural length and curl degree of sheep wool. The application can be effectively applied to the field of molecular assisted breeding of sheep with long natural length and large curl degree of wool, early selection of sheep is realized, and selection and reservation can be performed after birth, thereby accelerating the breeding process of sheep. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 The figure is a SNP-RELP analysis result diagram of the T76025431C site in the 2nd intron region of the YWHAZ gene provided for the embodiment 2 of the application.

[0010] Figure 2 The figure is a sequence alignment result diagram of the template sequence of the YWHAZ gene and the upper primer sequence in the primer pair provided for the embodiment 2 of the application.

[0011] Figure 3 Figure of electrophoretic separation result provided for embodiment 3 of the present application. DETAILED DESCRIPTION

[0012] The technical solutions in the embodiments of the present application will be clearly and completely described in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the protection scope of the present application.

[0013] The experimental methods in the following embodiments are all conventional experimental methods unless otherwise specified. The reagents used in the following embodiments are all purchased from conventional biochemical reagent companies unless otherwise specified; 524 Chinese Merino sheep (Xinjiang Junken type) are used as the test population in the embodiments of the present application to verify the application of the molecular markers and primer pairs for predicting and identifying the natural length and curliness of sheep wool.

[0014] The following will be described in combination with specific embodiments: EMBODIMENT

[0015] Obtaining of Chinese Merino sheep genomic DNA Sheep ear tissues are collected and stored at -20℃ for standby use. The sheep genomic DNA is extracted by using the conventional phenol / chloroform method. The specific method is as follows: (1) 5 g of Chinese Merino sheep (Xinjiang Junken type) ear tissues are taken, the connective tissues are removed, the tissue blocks are washed and sterilized with 70% alcohol, and are placed in an Eppendorf tube, and are cut or ground into pieces by using scissors; (2) After the alcohol in the Eppendorf tube is completely volatilized, 700 μL of separation buffer is added, the cut tissues are suspended, and 5.0 μL of proteinase K (20 mg / mL) is added, and is acted at 55℃ for 8-12 h until no tissue blocks are left; (3) The digested tissue liquid is taken out, an equal amount of saturated phenol of the tissue liquid is added, and is mixed for 10 min, centrifuged at 4℃ and 12000 r / m for 10 min; (4) The supernatant is taken, an equal amount of phenol / chloroform is added, and is mixed gently for 10 min, centrifuged at 4℃ and 12000 r / m for 10 min; (5) The supernatant is taken, an equal amount of chloroform is added, and is mixed for 10 min, centrifuged at 4℃ and 12000 r / m for 10 min; (6) The supernatant is taken, 2 times of anhydrous ethanol is added for precipitation, and is mixed by inverting after being placed at room temperature for 10-20 min, and the DNA precipitate forms white flocculent substances; (7) Discard the supernatant, add 70% ethanol for cleaning, discard the supernatant, absorb the excess liquid on the water absorption paper, dry naturally, add an appropriate amount of TE for dissolving, and store at -20℃; (8) If there are insoluble particles in the DNA solution, centrifuge at 5000 r / m for a short time, and take the supernatant; if the RNA is to be removed, 5 μL of RNase A (10 μg / μL) can be added, and incubated at 37℃ for 30 min, and then extracted with phenol, and the DNA is reprecipitated according to steps 4-7.

[0016] Example 2 Obtaining of the cleavage product of the sheep YWHAZ gene As shown in Figure 1 and Figure 2 , a pair of primers was designed according to the T76025431C site in the 2nd intron region of the YWHAZ gene in the sheep genome, including an upper primer YWHAZ-F and a lower primer YWHAZ-R, and then the sheep genomic DNA was subjected to PCR amplification to obtain the PCR amplification product, and then the PCR amplification product was subjected to enzyme cleavage with endonuclease XspI to obtain the cleavage product.

[0017] The primer sequences are as follows: YWHAZ-F: 5'-TAGATTTCCAAGTGTTTCAGAGTGT-3' YWHAZ-R: 5'-TAGAACTACTACAAATATTCTAACCACAAC-3' The PCR amplification system is as follows:

[0018] The PCR amplification conditions are as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 53.8℃ for 30 s, extension at 72℃ for 30 s, a total of 36 cycles, and final extension at 72℃ for 10 min, and termination of the reaction at 4℃.

[0019] The enzyme cleavage reaction system is as follows:

[0020] The enzyme cleavage conditions are as follows: enzyme cleavage at 37℃ for 30 min.

[0021] Example 3 Determination of the genotype of the sheep YWHAZ gene The cleavage product in Example 1 was subjected to electrophoretic separation using an agarose gel with a concentration of 2% ~ 3%, and the genotype was determined according to the electrophoretic separation results, as shown in Figure 3As shown, lane 1 is the control group, specifically the DL2000 marker. The criteria for judgment are: (1) Electrophoresis shows two bands ( Figure 3 Lanes 6 and 7 in the sheep YWHAZ gene have sizes of 136 bp and 54 bp, respectively. When the T76025431C site in the second intron region of the sheep YWHAZ gene is mutated, the PCR amplification product can be completely cleaved by the XspI enzyme and named as CC genotype; (2) Electrophoresis shows a band ( Figure 3 Lanes 2 and 3 in the sheep YWHAZ gene are 190 bp in size. When the T76025431C site in the second intron region of the sheep YWHAZ gene is not mutated, the PCR amplification product cannot be digested by XspI enzyme and is named the TT genotype. (3) Electrophoresis shows three bands. Figure 3 Lanes 4 and 5 of the sheep YWHAZ gene have sizes of 190 bp, 136 bp, and 54 bp, respectively. Therefore, the T76025431C site in the second intron region of the sheep YWHAZ gene is in a heterozygous state, and the PCR amplification product cannot be completely cleaved by the XspI enzyme. This is named the TC genotype.

[0022] The 524 Chinese Merino sheep in this invention were all Xinjiang military reclamation type ewes aged 1 to 12 years. Three genotypes were detected at the T76025431C site of the second intron region of the YWHAZ gene, namely CC genotype (12 sheep), CT genotype (48 sheep) and TT genotype (464 sheep).

[0023] Example 4 Construction of statistical models of genotype effects Based on the characteristics of the Chinese Merino sheep experimental population, a statistical model of genotype effect was constructed: Y = μ + G + A + G×A + e, where Y is the observed value of the trait, μ is the population mean, G is the genotype effect, A is the age effect, G×A is the interaction effect between genotype and age, and e is the residual effect.

[0024] The determination of the natural length and crimp of wool in this invention is based on the fiber inspection standards and with reference to the book "Wool and Wool Quality" written by Academician Liu Shouren. The natural length of wool is directly measured with a ruler in its natural state, and the crimp is the number of crimps observed in the wool at a natural length of 2.5cm.

[0025] The least square analysis is carried out on the average fiber diameter, average fiber diameter standard deviation, wool crimp and natural length of 524 individuals of the Chinese Merino sheep (Xinjiang Jungar type) test population with respect to the three genotypes of the T76025431C single base mutation of the 2nd intron region of YWHAZ gene, and the results show that the different genotype polymorphisms of the T76025431C site of the 2nd intron region of YWHAZ gene of sheep are extremely significantly related to the natural length and crimp of the Chinese Merino sheep (Xinjiang Jungar type) test population, and the P values are 0.0054 and 0.001 respectively; the least square means among the three genotypes are compared, and the results show that the natural length of the individual with the TT genotype is significantly higher than that of the individuals with the CC genotype and the TC genotype (P<0.05); the crimp of the individual with the TT genotype is significantly higher than that of the individual with the TC genotype (P<0.05), and the results are shown in Table 2.

[0026] Table 2 Influence of different genotypes of the T76025431C site of YWHAZ gene of sheep on natural length and crimp of wool

[0027] The above results show that YWHAZ gene can be used as one of the main candidate genes of the natural length and crimp of sheep wool, and the TT genotype can be used as a molecular genetic marker for simultaneously predicting the natural length and crimp of sheep wool. A sheep breeding population mainly composed of individuals with the TT genotype can be established, and a sheep strain with long natural length and large crimp can be effectively bred.

[0028] Example 5 Establishment of a molecular marker method for the natural length and crimp of sheep wool According to the genotypes, the Chinese Merino sheep test population is divided into three types, and according to Example 4, the molecular marker assisted breeding for simultaneously predicting and identifying the natural length and crimp of sheep wool is realized, that is, the molecular marker method for simultaneously predicting and identifying the natural length and crimp of sheep wool is completed.

[0029] It should be noted that in this document, terms such as "comprising", "including" or any other variant thereof are intended to cover non-exclusive inclusion, so that processes, methods, articles or devices including a series of elements not only include those elements, but also include other elements not explicitly listed or inherent to such processes, methods, articles or devices.

[0030] Although the embodiments of the present application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to the embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A primer pair of molecular markers that simultaneously predict and identify the natural length and crimp of sheep wool, characterized in that, The molecular marker is located at the T76025431C site in the second intron region of the YWHAZ gene, where a T base is mutated to a C base. The nucleotide sequence of the second intron region of the YWHAZ gene is shown in SEQ ID NO.

1. The primer pair comprises an upper primer and a lower primer, wherein the nucleotide sequence of the upper primer is shown in SEQ ID NO.2 and the nucleotide sequence of the lower primer is shown in SEQ ID NO.

3.

2. The application of a primer pair containing molecular markers that simultaneously predict and identify the natural length and crimp of sheep wool in the detection of the natural length and crimp of sheep wool, characterized in that, When the molecular marker site described in claim 1 is mutated, it is the CC genotype; when the molecular marker site described in claim 1 is not mutated, it is the TT genotype; when the molecular marker site described in claim 1 is in a heterozygous state, it is the TC genotype.

3. A molecular marker method for simultaneously predicting and identifying the natural length and crimp of sheep wool, characterized in that, Includes the following steps: Step 1: Extract sheep genomic DNA; Step 2: Using the primer pair for the T76025431C site of the second intron region of the YWHAZ gene as described in claim 1, PCR amplification of sheep genomic DNA was performed, followed by digestion with restriction enzyme XspI to obtain the digestion product. Step 3: Separate the enzyme digestion products by electrophoresis. If two bands are observed on the electrophoresis, the molecular marker site described in claim 1 is the CC genotype; if one band is observed, the molecular marker site described in claim 1 is the TT genotype; if three bands are observed, the molecular marker site described in claim 1 is the TC genotype. Among them, the TT genotype is sheep individuals with high natural wool length and crimp, the CC genotype is sheep individuals with low natural wool length and medium crimp, and the TC genotype is sheep individuals with low natural wool length and crimp.

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