CN111518918B - Method for screening cows with different lactoferrin contents and kit used by method - Google Patents

Abstract

The invention discloses a method for screening cows with different lactoferrin contents and a kit used by the method. The method comprises the following steps: detecting whether the genotype of the cow to be detected is genotype I, genotype II or genotype III, wherein the lactoferrin content in the cow milk of the genotype I is less than that of the cow of the genotype II, the lactoferrin content in the cow milk of the cow of the genotype II is less than that of the cow of the genotype III, the genotypes of the cow of the genotype I, the cow of the genotype II and the cow of the genotype III are TT homozygote, TA heterozygote and AA homozygote respectively based on the T53538908A SNP genotype, and T53538908ASNP is 53538908 th nucleotide of the 22 th chromosome of the cow genome. The method provided by the invention can be used for screening dairy cow individuals with higher lactoferrin content in cow milk and has higher accuracy. The invention has great application value for establishing high-quality dairy cow groups.

Description

Method for screening cows with different lactoferrin contents and kit used by method

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for screening dairy cows with different lactoferrin contents and a kit used by the method.

Background

Lactoferrin encoded by the LTF gene (Genebank number 280846) is a non-heme iron-binding glycoprotein with natural activity in milk, is mainly expressed and secreted by mammary epithelial cells, and is one of the important nutrients in milk. Lactoferrin has a wide range of biological activities, is involved in iron transport, plays an important role in the innate immune system, and has been shown to have inhibitory effects on certain cancer cells. Therefore, lactoferrin is a food additive with great development potential. Lactoferrin is an important nutritional ingredient required by infants and has been widely used in infant formulas. Researches show that the lactoferrin can reduce the diarrhea incidence and respiratory tract infection incidence of infants and children, promote nerve development, and play an important role in the processes of brain development and the like of infants. However, lactoferrin is present in milk in relatively low amounts, only 0.02-0.35mg per ml of milk. Because the natural lactoferrin content in cow milk is very low, the natural lactoferrin is still expensive as a food additive and a nutrition enhancer.

The LTF gene has been shown in many studies to be involved in the production performance of cows. In 2010, O 'Halloran F et al found that SNP mutation (A/C) of-28 bp upstream of transcription initiation site on LTF gene had a certain effect on milk protein amount in cow's milk. In 2015, Mao Y et al showed that the SNP mutation of-270 bp from T to C upstream of the transcription start site in the LTF gene had a positive effect on milk yield, milk fat and milk protein rate, while the mutation of-190 bp from G to A had an adverse effect on milk fat and milk protein rate. In 2017, Viale E and the like find that SNP (rs43765462) on LTF gene is positively correlated with the creaminess rate character of Holstein cows. In 2018, Raschiam et al found that SNP (rs43706485) on LTF gene has potential correlation with milk yield of cow in 305 days. Studies have also shown that the lactoferrin content of bovine milk has a positive correlation with the milk protein content. Therefore, by discovering important genetic variation on the LTF gene which influences the milk production traits, the method can be applied to the breeding of the dairy cows and can simultaneously achieve the effects of improving the production performance of the dairy cows and the lactoferrin content in the cow milk.

A Single Nucleotide Polymorphism (SNP) refers to a genetic marker of a DNA sequence polymorphism caused by mutation of a single nucleotide at the genome level, and involves only a single base variation. To date, many SNPs have been shown to affect the development of human diseases and the phenotype of important economic traits in animals and plants by changing amino acid sequences, creating new variable splice bodies, and other regulatory pathways. At present, the detection method of SNP mainly comprises DNA sequencing, transcriptome sequencing, SNP chip and the like.

RNA-seq technology, i.e. transcriptome sequencing technology, is a technology for sequencing total RNA in cells or tissues by means of a second generation high throughput sequencing method. It can detect the expression of genes in cells or tissues, and can detect important genetic variation existing in the population by aligning with a reference genome sequence. At present, the RNA-seq technology has been widely applied to the research of important economic traits of animals and plants, plays an important role in the research of milk production traits of dairy cows, and a lot of research reports at present discover a batch of important milk production trait candidate functional genes.

Disclosure of Invention

The invention aims to screen the dairy cows with different lactoferrin contents.

The invention firstly protects and screens the dairy cows with different lactoferrin contents.

The method for screening the dairy cows with different lactoferrin contents, which is provided by the invention, can be specifically a method one, and can comprise the following steps: detecting whether the genotype of the cow to be detected is genotype I, genotype II or genotype III, wherein the lactoferrin content in the cow milk of the genotype I is less than that of the cow milk of the genotype II;

the genotype I dairy cow is TT homozygous based on the T53538908A SNP genotype;

the genotype II cow is a TA heterozygous cow based on the T53538908A SNP genotype;

the cow with genotype III is a cow with genotype based on T53538908A SNP and AA homozygote;

the T53538908A SNP is the 53538908 th nucleotide of the 22 nd chromosome of the cow genome.

The method for screening the dairy cows with different lactoferrin contents, which is provided by the invention, can be specifically a method II, and can comprise the following steps of: detecting whether the genotype of the cow to be detected is genotype I or genotype III, wherein the lactoferrin content in the cow milk of the genotype I is lower than that of the cow milk of the genotype III;

the genotype I dairy cow is TT homozygous based on the T53538908A SNP genotype;

the cow with genotype III is a cow with genotype based on T53538908A SNP and AA homozygote;

the T53538908A SNP is the 53538908 th nucleotide of the 22 nd chromosome of the cow genome.

The method for screening the dairy cows with different lactoferrin contents, which is provided by the invention, can be specifically a third method, and can comprise the following steps: detecting whether the genome of the cow to be detected contains SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and the DNA fragment shown in SEQ ID NO: 4, and the DNA fragment shown in SEQ ID NO: 5 and the DNA fragment shown in SEQ ID NO: 6, and then judging as follows:

if the genome of the cow to be detected contains the DNA segment 1, the DNA segment 3 and/or the DNA segment 5 and does not contain the DNA segment 2, the DNA segment 4 and/or the DNA segment 6, the genotype of the cow to be detected is genotype I;

if the genome of the cow to be detected contains the DNA segment 1, the DNA segment 3 and/or the DNA segment 5, and contains the DNA segment 2, the DNA segment 4 and/or the DNA segment 6, the genotype of the cow to be detected is genotype II;

if the genome of the cow to be detected contains the DNA segment 2, the DNA segment 4 and/or the DNA segment 6 and does not contain the DNA segment 1, the DNA segment 3 and/or the DNA segment 5, the genotype of the cow to be detected is genotype III;

the lactoferrin content in the cow's milk of the ' genotype I cow ' is less than that in the cow's milk of the ' genotype II cow ' is less than that in the cow's milk of the ' genotype III cow '.

The method for screening the dairy cows with different lactoferrin contents, which is provided by the invention, can be specifically the fourth method, and can comprise the following steps: detecting whether the genome of the cow to be detected contains SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and the DNA fragment shown in SEQ ID NO: 4, and the DNA fragment shown in SEQ ID NO: 5 and the DNA fragment shown in SEQ ID NO: 6, and then judging as follows:

if the genome of the cow to be detected contains the DNA segment 1, the DNA segment 3 and/or the DNA segment 5 and does not contain the DNA segment 2, the DNA segment 4 and/or the DNA segment 6, the genotype of the cow to be detected is genotype I;

if the genome of the cow to be detected contains the DNA segment 2, the DNA segment 4 and/or the DNA segment 6 and does not contain the DNA segment 1, the DNA segment 3 and/or the DNA segment 5, the genotype of the cow to be detected is genotype III;

the lactoferrin content in the cow's milk of the' genotype I 'milk is less than that in the cow's milk of the 'genotype III'.

In any of the above methods, the "detecting whether the genome of the cow to be detected contains DNA fragment 1, DNA fragment 2, DNA fragment 3, DNA fragment 4, DNA fragment 5, and DNA fragment 6", the "detecting whether the genotype of the cow to be detected is genotype I, genotype II, or genotype III", or the "detecting whether the genotype of the cow to be detected is genotype I or genotype III" may be a1) or a 2):

a1) direct sequencing;

a2) taking the genome DNA of the cow to be detected as a template, carrying out PCR amplification by adopting a primer pair 1, a primer pair 2 and/or a primer pair 3, and then sequencing;

the primer pair 1 consists of a primer 1 and a primer 2;

the primer pair 2 consists of a primer 3 and a primer 4;

the primer pair 3 consists of a primer 5 and a primer 6;

the nucleotide sequences of the primers 1 to 6 are shown as SEQ ID NO: 7-SEQ ID NO: shown at 12.

The invention also protects a kit which can comprise substances for detecting whether the genome of the cow to be detected contains the DNA fragments 1, 2, 3, 4, 5 and 6, substances for detecting whether the genotype of the cow to be detected is genotype I, II or III, or substances for detecting whether the genotype of the cow to be detected is genotype I or III;

the genotype I dairy cow is TT homozygous based on the T53538908A SNP genotype;

the genotype II cow is a TA heterozygous cow based on the T53538908A SNP genotype;

the cow with genotype III is a cow with genotype based on T53538908A SNP and AA homozygote;

the T53538908A SNP is the 53538908 th nucleotide of the 22 nd chromosome of the cow genome.

The use of the kit may be b1) or b2) or b 3):

b1) screening cows with different lactoferrin contents;

b2) identifying the lactoferrin content of the cow milk;

b3) identifying genotypes based on the T53538908A SNP in the genome of the cow.

In the above kit, the "detecting whether the genome of the cow to be detected contains the substances of the DNA fragment 1, the DNA fragment 2, the DNA fragment 3, the DNA fragment 4, the DNA fragment 5 and the DNA fragment 6", the "detecting whether the genotype of the cow to be detected is genotype I, genotype II or genotype III", or the "detecting whether the genotype of the cow to be detected is genotype I or genotype III" may be the primer pair 1, the primer pair 2 and/or the primer pair 3.

The invention also protects the amino acid sequence shown in SEQ ID NO: 13, and the molecular markers A and SEQ ID NO: 14 or SEQ ID NO: 15 is labeled with gamma.

The invention also protects (z1) or (z2) or (z3) or (z4) or (z5) or (z6) or (z 7).

(z1) the application of any one of the kit, the molecular marker A, the molecular marker B or the molecular marker C in screening cows with different lactoferrin contents also belongs to the protection scope of the invention.

(z2) the application of any one of the kit, the molecular marker A, the molecular marker B or the molecular marker C in the preparation of products for screening dairy cows with different lactoferrin contents also belongs to the protection scope of the invention.

(z3) the application of any one of the kit, the molecular marker A, the molecular marker B or the molecular marker C in the identification of the content of the cow lactoferrin also belongs to the protection scope of the invention.

(z4) the application of any one of the kit, the molecular marker A, the molecular marker B or the molecular marker C in the preparation of products for identifying the content of cow lactoferrin also belongs to the protection scope of the invention.

(z5) the application of any one of the kit, the molecular marker A, the molecular marker B or the molecular marker C in the identification of the genotype of the cow genome based on the T53538908A SNP also belongs to the protection scope of the invention.

(z6) the application of any one of the kit, the molecular marker A, the molecular marker B or the molecular marker C in the preparation of products for identifying the genotype of the dairy cow based on the T53538908A SNP also belongs to the protection scope of the invention.

(z7) the application of any one of the kit, the molecular marker A, the molecular marker B or the molecular marker C in the breeding of dairy cows also belongs to the protection scope of the invention.

Any of the above cows may specifically be a Chinese Holstein cow.

The LTF gene encodes lactoferrin. The lactoferrin content of any of the above cows 'milk is evaluated by detecting the standardized expression level of the LTF gene in cow's milk or blood. The higher the normalized expression level of the LTF gene in cow's milk or blood, the higher the lactoferrin content in cow's milk. The blood may be day 15 lactating blood. The cow's milk may be cow's milk at the early lactation stage. The lactation period generally means 10-15 days after the dairy cow is delivered. In the embodiment of the invention, cows 15 days after delivery are selected to collect cow milk.

Lactoferrin is a natural nutrient component with important immune function in milk and has become an important auxiliary ingredient added in some infant formula milk powder. Researches show that the lactoferrin plays an important role in neural development, immunity improvement and cancer resistance. However, the content of the lactoferrin in milk is very low, and the technical process for extracting the high-purity lactoferrin is complex and difficult, so that the value of the lactoferrin is very high, and the cost can be greatly increased by adding the nutritional ingredients into food. The method provided by the invention can be used for screening dairy cow individuals with higher lactoferrin content in the milk, has higher accuracy, can be used for early breeding of dairy cows, can accurately screen dairy cows even when the dairy cows are born, and greatly accelerates the breeding process of Chinese Holstein dairy cows. The invention has great application value for establishing high-quality dairy cow groups.

Drawings

FIG. 1 shows the results of 1% (m/v) agarose gel electrophoresis of the product of the second PCR amplification in example 2.

FIG. 2 is a statistical result of the standardized expression level of LTF genes of 6 cows in the Shimazu cattle farm in the suburb of Beijing.

FIG. 3 shows the partial sequencing results of cattle No. 3.

FIG. 4 is a statistical result of the standardized expression level of LTF genes of 6 cows in Beijing Sanyinjiu island cattle farm.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

The cows in the following examples are all chinese holstein cows.

Example 1, discovery of T53538908ASNP and Classification of cow genotypes based on this SNP site

Considering the influence of the feeding environment factors on individuals, the cows in the same cow farm (particularly the cow farm in the golden island of the suburb of Beijing) are selected for testing. Meanwhile, in order to control errors caused by differences among individuals, individuals with the same lactation days (specifically the 15 th day of lactation), 2 or 3 fetuses and similar sampling time are selected, and the sampling time and the differences among the individuals are reduced to the minimum as far as possible. Finally, according to the milk production record of the previous fetus of the individual, the milk yield, the milk fat content and the milk protein content of the individual in 305 days are counted; selecting 305 dairy cows with milk yield of more than 12000kg, milk fat of more than 450kg and milk protein of more than 380kg as a high-yield group, and selecting 3 dairy cows from 1500 (respectively named as H1, H2 and H3); the individual cows with milk yield less than 9000kg, milk fat less than 350kg and milk protein less than 280kg in the day 305 were selected as a low-yield group, and 3 cows (designated as L1, L2 and L3, respectively) were selected from 1500 cows. All individuals were healthy individuals.

The milk yield, the milk fat content and the milk protein content of the 6 cows in 305 days are shown in the table 1, and 3 milk production trait phenotypes all reach extremely different levels (P < 0.01).

TABLE 1

	Milk yield in 305 days (kg)	Amount of milk fat (kg)	Milk protein amount (kg)
				H1	12514	472.28	394.19
H2	12966	466.78	414.91
				H3	13188	474.77	422.02
L1	6223	227.14	197.89
				L2	7494	348.47	251.80
L3	8623	313.01	251.80

1. Blood was collected from 6 cows on day 15 of lactation.

2. After step 1, total RNA of blood of 6 cows was extracted, respectively, and then transcriptome sequencing was performed. The method comprises the following specific steps:

(1) total RNA of blood of 6 cows was extracted by Trizol method, and then purity and concentration of total RNA of cow blood were measured by Nanodrop nucleic acid analyzer (Thermo Scientific, USA), and integrity of total RNA of cow blood was measured by Agilent2100(Agilent, USA).

The results show that the total RNA concentration of the cow blood is more than 100 ng/muL, and the RIN value is more than 7.5.

(2) After the step (1) is finished, respectively taking total RNA of blood of 6 cows, and referring to IlluminaTruSeq^TMInstructions for RNA sample preparation kit (Illumia, USA) construction of the corresponding cDNA library; and (6) quality inspection.

The result shows that 6 cDNA libraries constructed by total RNA of the blood of 6 cows are qualified in quality inspection.

(3) After the step (2) is completed, taking 6 cDNA libraries, and performing double-end sequencing by using an IlluminaHiseq 2500 sequencing platform, wherein the sequencing read length is 100 bp; removing band connectors, poly-N and low-quality reads from the sequenced original sequences (Raw reads) to form Clean reads; clean reads were then aligned to the bovine reference genomic sequence (UMD3.1) and the corresponding gene annotation file (UMD3.1) with the help of BWA software.

The sequencing data volume (i.e., Clean reads), quality control result (Q30) and alignment rate with reference genome of 6 cows are shown in table 2.

TABLE 2

Individual numbering	Clean bases	Q30(％)	Comparison with reference genome (%)
				H1	5.80Gb	91.10	95.48
H2	6.24Gb	92.08	96.33
				H3	6.06Gb	91.96	96.95
L1	5.84Gb	91.74	96.31
				L2	8.14Gb	89.67	90.22
L3	6.06Gb	90.98	96.64

3. After completion of step 2, genetic variations including SNP and InDel on the LTF gene of 6 cows were identified by SAMtools and BCFtools software based on sequencing data, and the detected genetic variations (provided DP >5, QUAL >30 and MQ >40) were filtered using vccffilter software.

The result shows that the intron region of the LTF gene of the cow contains 1 SNP locus, and the SNP locus is named as T53538908A SNP. The T53538908A SNP is located at the 53538908 th chromosome of the 22 nd chromosome of a cow genome, and the genotype is TT homozygous, AA homozygous or TA heterozygous. Since genomic DNA is a double-stranded DNA molecule composed of two single-stranded DNA molecules that are complementary to each other in the opposite direction, a DNA molecule encoding a protein, that is, a DNA molecule having an initiation codon to a termination codon, is generally named as a sense DNA molecule; a DNA molecule complementary to the reverse direction of the sense DNA molecule is designated as an antisense DNA molecule. The genotype at the T53538908A SNP is that of sense DNA.

Cows were classified into 3 genotypes based on the difference in T53538908A SNPs: genotype I, genotype II, and genotype III.

The cow with genotype I is TT homozygous based on the genotype of T53538908A SNP.

The genotype of the cow with genotype II is TA heterozygous based on the genotype of T53538908A SNP.

The genotype III cows are AA homozygous based on the genotype of the T53538908A SNP.

The genotypes of 6 cows based on the T53538908A SNP are specifically shown in table 3.

TABLE 3

Example 2 establishment of T53538908A SNP-based genotyping method for dairy cows to be tested

First, preparation of primer pairs

Primer pair 1, primer pair 2, primer pair 3, primer pair 4 and primer pair 5 were designed for amplification of a target sequence including the T53538908A SNP.

The nucleotide sequences and amplified fragment sizes of the primers upstream and downstream of each primer pair are shown in Table 4.

TABLE 4

Secondly, establishing a T53538908A SNP-based genotyping method for dairy cows to be detected

1. And (3) performing PCR amplification by using the genome DNA of the cow to be detected as a template and adopting a primer pair (a primer pair 1, a primer pair 2, a primer pair 3, a primer pair 4 or a primer pair 5) to obtain a PCR amplification product.

2. After completing step 1, the PCR amplification product was detected by 1% (m/v) agarose gel electrophoresis.

Part of the detection results are shown in FIG. 1(M is DNA Marker, 1 is primer pair 1, 2 is primer pair 2, and 3 is primer pair 3). The result shows that the PCR amplification is carried out by adopting the primer pair 1, the primer pair 2 or the primer pair 3, and the banding pattern of a PCR amplification product is single and clear; and the corresponding PCR amplification product is not obtained by adopting the primer pair 4 or the primer pair 5 for PCR amplification. Therefore, the primer pair 1, the primer pair 2 and the primer pair 3 can be used for detecting the genotype of the dairy cow to be detected based on the T53538908A SNP. Neither primer pair 4 nor primer pair 5 can be used for detecting the genotype of the dairy cow to be detected based on the T53538908A SNP.

3. Sequencing a PCR amplification product obtained by performing PCR amplification on the primer pair 1, the primer pair 2 or the primer pair 3, and judging according to the following steps:

if the nucleotide sequence of the PCR amplification product obtained by using the primer pair 1 is only as shown in SEQ ID NO: 1, the nucleotide sequence of the PCR amplification product obtained by adopting the primer pair 2 is only shown as SEQ ID NO: 3 and/or the nucleotide sequence of the PCR amplification product obtained with primer pair 3 is only as shown in SEQ ID NO: 5, the genotype of the to-be-detected cow based on the T53538908A SNP is TT homozygosis, namely the genotype of the to-be-detected cow is genotype I;

if "there are two kinds of PCR amplification products obtained by using the primer pair 1, one nucleotide sequence is shown as SEQ ID NO: 1, and the other nucleotide sequence is shown as SEQ ID NO: 2 and two PCR amplification products obtained by adopting the primer pair 2, wherein one nucleotide sequence is shown as SEQ ID NO: 3, and the other nucleotide sequence is shown as SEQ ID NO: 4 or the PCR amplification products obtained by adopting the primer pair 3 have two types, one nucleotide sequence is shown as SEQ ID NO: 5, and the other nucleotide sequence is shown as SEQ ID NO: 6, the genotype of the to-be-detected cow based on the T53538908A SNP is a TA heterozygote, namely the genotype of the to-be-detected cow is genotype II;

if the nucleotide sequence of the PCR amplification product obtained by using the primer pair 1 is only as shown in SEQ ID NO: 2, the nucleotide sequence of the PCR amplification product obtained by adopting the primer pair 2 is only shown as SEQ ID NO: 4 and/or the nucleotide sequence of the PCR amplification product obtained by using the primer pair 3 is only shown as SEQ ID NO: 6, the genotype of the to-be-detected cow based on the T53538908A SNP is AA homozygous, namely the genotype of the to-be-detected cow is genotype III.

Example 3 evaluation of lactoferrin content in cow milk by detecting T53538908A SNP-based genotype of cow to be tested

In the kit for detecting the content of lactoferrin in cow milk, the repeatability of the detection result based on the elisa enzyme-linked immunosorbent assay principle is low, and only 1-2 species are available in China; foreign kits are not generally in stock, the waiting period is 3-4 months, and the storage time of test samples can influence the detection result. The LTF gene codes lactoferrin, and the detection of the expression quantity of the LTF gene is simple, convenient, easy, accurate and stable. Therefore, the lactoferrin content can be evaluated by detecting the normalized expression level of LTF gene. The higher the normalized expression level of LTF gene, the higher the lactoferrin content.

Method for evaluating lactoferrin content in cow milk of 6 cows in Beijing suburb gold and silver island cattle farm by detecting genotype of to-be-detected cows based on T53538908A SNP

6 cows are 6 cows in the cattle farm in the suburb of Beijing south China in example 1.

1. The genotypes of 6 cows based on the T53538908A SNP are shown in table 3.

2. Obtaining the normalized expression level of LTF gene in blood

After completion of step 2 of example 1, standardized expression levels of LTF genes of 6 cows were obtained based on the sequencing data.

The method comprises the following specific steps:

(3-1) counting counts by means of featurecounts software;

(3-2) normalizing the counts by means of the DESeq2 package and completing the differential expression analysis of the genes, and outputting the differential expression genes and the normalized counts thereof with P <0.05 as a threshold value, namely obtaining the normalized expression level of the LTF genes.

And respectively counting the standardized expression quantity of the individual LTF genes of genotype I, genotype II and genotype III, and averaging the results.

The statistical results are shown in FIG. 2(TT is genotype I, AT is genotype II, and AA is genotype III). The results showed that the normalized expression level of LTF gene of "genotype I cow" was < "the normalized expression level of LTF gene of" genotype II cow "was <" the normalized expression level of LTF gene of "genotype III cow". Therefore, the lactoferrin content in cow's milk of "genotype I cow" is less than that of "genotype II cow" is less than that of "genotype III cow". The < "is a statistical <".

Secondly, evaluating the content of lactoferrin in cow milk of 6 cows in Beijing three-way gold and silver island cattle farm by detecting the genotype of the to-be-detected cow based on T53538908A SNP

6 cows (numbered 1 cow, 2 cow, 3 cow, 4 cow, 5 cow and 6 cow) with similar gestation, same lactation period, same lactation time and similar sampling time in a Beijing three-element gold and silver island cow farm are selected for testing.

1. Obtaining the genotype of 6 cows based on T53538908A SNP

A. Genotype of 6 cows based on T53538908A SNP obtained by sequencing

(1) Respectively collecting cow milk of 6 cows at the early lactation stage.

The lactation period generally means 10 to 15 days after the cow is delivered. Cows 15 days after delivery were selected to collect cow milk in this study.

(2) And (3) after the step (1) is finished, extracting the total RNA of the cow milk of 6 cows respectively, and then carrying out transcriptome sequencing. The method comprises the following specific steps:

(a1) the total RNA of cow milk of 6 cows is respectively extracted by adopting a Trizol method, then the purity and the concentration of the total RNA of cow milk are detected by utilizing a Nanodrop nucleic acid analyzer, and the integrity of the total RNA of cow milk is detected by utilizing Agilent 2100.

The results show that the total RNA concentration of cow milk of 6 cows is more than 100 ng/muL, and the RIN value is more than 7.5.

(a2) After the step (a1) is completed, respectively taking total RNA of cow milk of 6 cows, referring to IlluminaTruSeq^TMInstructions for RNA sample preparation kit (Illumia, USA) construction of the corresponding cDNA library; and (6) quality inspection.

The result shows that 6 cDNA libraries constructed by the total RNA of 6 cows' milk are qualified in quality inspection.

(a3) After the step (a2) is completed, taking 6 cDNA libraries, and performing double-end sequencing by using an illumina hiseq 2500 sequencing platform, wherein the sequencing read length is 100 bp; removing band connectors, poly-N and low-quality reads from the sequenced original sequences (Raw reads) to form Clean reads; clean reads were then aligned to the bovine reference genomic sequence (UMD3.1) and the corresponding gene annotation file (UMD3.1) with the help of BWA software.

The sequencing data volume (i.e., Clean reads), quality control result (Q30) and alignment rate with reference genome of 6 cows are shown in table 5.

TABLE 5

Individual numbering	Clean bases	Q30(％)	Comparison with reference genome (%)
				No. 1 cattle	4.88Gb	95.45	90.40
No. 2 cattle	3.23Gb	94.57	93.03
				No. 3 cattle	4.11Gb	96.01	89.99
No. 4 cattle	6.58Gb	96.37	92.96
				No. 5 cattle	4.97Gb	95.40	94.03
No. 6 cattle	4.14Gb	95.66	92.90

(3) And (3) after the step (2) is completed, obtaining the genotype of 6 cows based on the T53538908A SNP according to sequencing data.

B. Genotype of 6 cows based on T53538908A SNP was obtained by the method of example 2

The genotype of 6 cows based on the T53538908A SNP is detected by the method of step two in example 2 (specifically, by using primer pair 1).

The partial sequencing results for bovine # 3 are shown in FIG. 3 (T marked in gray is the T53538908A SNP).

The results show that the genotypes of the 6 cows obtained by the two methods based on the T53538908A SNP are completely consistent.

The genotypes of 6 cows based on the T53538908A SNP are shown in table 6.

TABLE 6

	Genotype based on T53538908A SNP
		No. 1 cattle	Genotype III
No. 2 cattle	Genotype I
		No. 3 cattle	Genotype I
No. 4 cattle	Genotype III
		No. 5 cattle	Genotype III
No. 6 cattle	Genotype III

2. Obtaining the standardized expression level of the LTF gene in cow milk

And (3) after the step (2) in the step (1) is completed, obtaining the standardized expression quantity of the LTF gene of 6 cows according to a sequencing result. The method comprises the following specific steps:

(1) counting the counts by means of featurecounts software;

(2) normalizing the counts by the aid of the DESeq2 package, performing differential expression analysis of the genes, and outputting the differentially expressed genes and the normalized counts thereof by taking P <0.05 as a threshold value, namely obtaining the normalized expression quantity of the LTF genes.

And respectively counting the standardized expression quantity of the individual LTF genes of genotype I, genotype II and genotype III, and averaging the results.

The statistics are shown in FIG. 4 (genotype I for TT and genotype III for AA). The results showed that the normalized expression level of LTF gene of "genotype I cow" and the normalized expression level of LTF gene of "genotype III cow". Therefore, the lactoferrin content in cow's milk of "genotype I cow" is less than that of "genotype III cow". The < "is a statistical <".

The results show that the lactoferrin character of the milk can be screened by detecting the genotype of the to-be-detected milk cow based on the T53538908A SNP, and the method has important application value in the process of breeding the milk cow.

<110> university of agriculture in China

<120> method for screening cows with different lactoferrin contents and kit used by same

<160> 15

<170> PatentIn version 3.5

<210> 1

<211> 474

<212> DNA

<213> Artificial sequence

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gatcttaaga acaactcaca atgaatgcag gtaatctgga aaatttagaa agatgtgatg 360

taaaaccaaa cagcaatgga gcctcacacg gggctaccac agttgagtgc gtgttagtca 420

ctcagtcatg tctgacccct tgtggcccca tggactgtag ccctcctctt ccca 474

<210> 2

<211> 474

<212> DNA

<213> Artificial sequence

<400> 2

gggacctgag gagcagaaga agtgccagca gtggagccag cagagcggcc agaacgtgac 60

ctgtgccacg gcgtccacca ctgacgactg catcgtcctg gtgctggaag ggggcctgct 120

gccggcctgc aagccgtgcg ggtggggggc acaggtcacc gcatctggtt ggaggggagc 180

aggggctcag tggtgctccg cccccagggg ggcaggggct gctcgtgctg cctgcccaga 240

gtggggacag acagagcttt ctgtcctcca gagtgtctac tgtggacttc ccactcttct 300

gatcttaaga acaactcaca atgaatgcag gtaatctgga aaatttagaa agatgtgatg 360

taaaaccaaa cagcaatgga gcctcacacg gggctaccac agttgagtgc gtgttagtca 420

ctcagtcatg tctgacccct tgtggcccca tggactgtag ccctcctctt ccca 474

<210> 3

<211> 382

<212> DNA

<213> Artificial sequence

<400> 3

gggacctgag gagcagaaga agtgccagca gtggagccag cagagcggcc agaacgtgac 60

ctgtgccacg gcgtccacca ctgacgactg catcgtcctg gtgctggtag ggggcctgct 120

gccggcctgc aagccgtgcg ggtggggggc acaggtcacc gcatctggtt ggaggggagc 180

aggggctcag tggtgctccg cccccagggg ggcaggggct gctcgtgctg cctgcccaga 240

gtggggacag acagagcttt ctgtcctcca gagtgtctac tgtggacttc ccactcttct 300

gatcttaaga acaactcaca atgaatgcag gtaatctgga aaatttagaa agatgtgatg 360

taaaaccaaa cagcaatgga gc 382

<210> 4

<211> 382

<212> DNA

<213> Artificial sequence

<400> 4

gggacctgag gagcagaaga agtgccagca gtggagccag cagagcggcc agaacgtgac 60

ctgtgccacg gcgtccacca ctgacgactg catcgtcctg gtgctggaag ggggcctgct 120

gccggcctgc aagccgtgcg ggtggggggc acaggtcacc gcatctggtt ggaggggagc 180

aggggctcag tggtgctccg cccccagggg ggcaggggct gctcgtgctg cctgcccaga 240

gtggggacag acagagcttt ctgtcctcca gagtgtctac tgtggacttc ccactcttct 300

gatcttaaga acaactcaca atgaatgcag gtaatctgga aaatttagaa agatgtgatg 360

taaaaccaaa cagcaatgga gc 382

<210> 5

<211> 265

<212> DNA

<213> Artificial sequence

<400> 5

tgttacagcc tgttgaccgc tgtgtccccg ggcagctgcg gaggaggtga aggcgcggta 60

caccagggtc gtgtggtgtg ccgtgggacc tgaggagcag aagaagtgcc agcagtggag 120

ccagcagagc ggccagaacg tgacctgtgc cacggcgtcc accactgacg actgcatcgt 180

cctggtgctg gtagggggcc tgctgccggc ctgcaagccg tgcgggtggg gggcacaggt 240

caccgcatct ggttggaggg gagca 265

<210> 6

<211> 265

<212> DNA

<213> Artificial sequence

<400> 6

tgttacagcc tgttgaccgc tgtgtccccg ggcagctgcg gaggaggtga aggcgcggta 60

caccagggtc gtgtggtgtg ccgtgggacc tgaggagcag aagaagtgcc agcagtggag 120

ccagcagagc ggccagaacg tgacctgtgc cacggcgtcc accactgacg actgcatcgt 180

cctggtgctg gaagggggcc tgctgccggc ctgcaagccg tgcgggtggg gggcacaggt 240

caccgcatct ggttggaggg gagca 265

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence

<400> 7

gggacctgag gagcagaaga a 21

<210> 8

<211> 21

<212> DNA

<213> Artificial sequence

<400> 8

tgggaagagg agggctacag t 21

<210> 9

<211> 21

<212> DNA

<213> Artificial sequence

<400> 9

gggacctgag gagcagaaga a 21

<210> 10

<211> 22

<212> DNA

<213> Artificial sequence

<400> 10

gctccattgc tgtttggttt ta 22

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence

<400> 11

tgttacagcc tgttgaccgc 20

<210> 12

<211> 19

<212> DNA

<213> Artificial sequence

<400> 12

tgctcccctc caaccagat 19

<210> 13

<211> 474

<212> DNA

<213> Artificial sequence

<222> （108）…（108）

<223> n is t or a

<400> 13

gggacctgag gagcagaaga agtgccagca gtggagccag cagagcggcc agaacgtgac 60

ctgtgccacg gcgtccacca ctgacgactg catcgtcctg gtgctggnag ggggcctgct 120

gccggcctgc aagccgtgcg ggtggggggc acaggtcacc gcatctggtt ggaggggagc 180

aggggctcag tggtgctccg cccccagggg ggcaggggct gctcgtgctg cctgcccaga 240

gtggggacag acagagcttt ctgtcctcca gagtgtctac tgtggacttc ccactcttct 300

gatcttaaga acaactcaca atgaatgcag gtaatctgga aaatttagaa agatgtgatg 360

taaaaccaaa cagcaatgga gcctcacacg gggctaccac agttgagtgc gtgttagtca 420

ctcagtcatg tctgacccct tgtggcccca tggactgtag ccctcctctt ccca 474

<210> 14

<211> 382

<212> DNA

<213> Artificial sequence

<222> （108）…（108）

<223> n is t or a

<400> 14

gggacctgag gagcagaaga agtgccagca gtggagccag cagagcggcc agaacgtgac 60

ctgtgccacg gcgtccacca ctgacgactg catcgtcctg gtgctggnag ggggcctgct 120

gccggcctgc aagccgtgcg ggtggggggc acaggtcacc gcatctggtt ggaggggagc 180

aggggctcag tggtgctccg cccccagggg ggcaggggct gctcgtgctg cctgcccaga 240

gtggggacag acagagcttt ctgtcctcca gagtgtctac tgtggacttc ccactcttct 300

gatcttaaga acaactcaca atgaatgcag gtaatctgga aaatttagaa agatgtgatg 360

taaaaccaaa cagcaatgga gc 382

<210> 15

<211> 265

<212> DNA

<213> Artificial sequence

<222> （192）…（192）

<223> n is t or a

<400> 15

tgttacagcc tgttgaccgc tgtgtccccg ggcagctgcg gaggaggtga aggcgcggta 60

caccagggtc gtgtggtgtg ccgtgggacc tgaggagcag aagaagtgcc agcagtggag 120

ccagcagagc ggccagaacg tgacctgtgc cacggcgtcc accactgacg actgcatcgt 180

cctggtgctg gnagggggcc tgctgccggc ctgcaagccg tgcgggtggg gggcacaggt 240

caccgcatct ggttggaggg gagca 265

Claims (6)

1. A kit comprises a substance for detecting whether the genotype of a cow to be detected is genotype I, genotype II or genotype III; the substance for detecting the genotype of the cow to be detected as genotype I, genotype II or genotype III is a primer pair 1, a primer pair 2 and/or a primer pair 3;

the primer pair 1 consists of a primer 1 and a primer 2;

the primer pair 2 consists of a primer 3 and a primer 4;

the primer pair 3 consists of a primer 5 and a primer 6;

the nucleotide sequences of the primers 1 to 6 are shown as SEQ ID NO: 7-SEQ ID NO: 12 is shown in the specification;

the kit is used for identifying the lactoferrin content in cow milk; the lactoferrin content in the cow's milk of the ' genotype I cow ' is less than that in the cow's milk of the ' genotype II cow ' is less than that in the cow's milk of the ' genotype III cow ';

the genotype I dairy cow is TT homozygous based on the T53538908A SNP genotype;

the genotype II cow is a TA heterozygous cow based on the T53538908A SNP genotype;

the cow with genotype III is a cow with genotype based on T53538908A SNP and AA homozygote;

the T53538908A SNP is SEQ ID NO: 13 from the 5' end at nucleotide position 108, wherein the 108 th base of the sequence is t or a;

the position of the T53538908A SNP was determined based on the bovine reference genomic sequence alignment; the version number of the bovine reference genome sequence is UMD 3.1;

the dairy cow is a Chinese Holstein dairy cow.

2. A SNP molecular marker A has a sequence shown in SEQ ID NO: 13, wherein the 108 th base of the sequence is t or a.

3. A SNP molecular marker B, the sequence of which is shown as SEQ ID NO: 14, wherein the 108 th base of the sequence is t or a.

4. A SNP molecular marker C has a sequence shown in SEQ ID NO: 15 wherein the base at position 192 of the sequence is t or a.

5. Use of the kit of claim 1, the SNP molecular marker A of claim 2, the SNP molecular marker B of claim 3, or the SNP molecular marker C of claim 4 for identifying the genotype of a cow based on the T53538908A SNP as genotype I, genotype II or genotype III; the lactoferrin content in the cow's milk of the ' genotype I cow ' is less than that in the cow's milk of the ' genotype II cow ' is less than that in the cow's milk of the ' genotype III cow ';

the genotype I dairy cow is TT homozygous based on the T53538908A SNP genotype;

the genotype II cow is a TA heterozygous cow based on the T53538908A SNP genotype;

the cow with genotype III is a cow with genotype based on T53538908A SNP and AA homozygote;

the T53538908A SNP is SEQ ID NO: 13 from the 5' end at nucleotide position 108, wherein the 108 th base of the sequence is t or a;

the position of the T53538908A SNP was determined based on the bovine reference genomic sequence alignment; the version number of the bovine reference genome sequence is UMD 3.1;

the dairy cow is a Chinese Holstein dairy cow.

6. Use of the kit of claim 1, the SNP molecular marker A of claim 2, the SNP molecular marker B of claim 3, or the SNP molecular marker C of claim 4 for preparing a product for identifying genotype I, genotype II or genotype III based on the T53538908A SNP in the genome of a cow; the lactoferrin content in the cow's milk of the ' genotype I cow ' is less than that in the cow's milk of the ' genotype II cow ' is less than that in the cow's milk of the ' genotype III cow ';

the genotype I dairy cow is TT homozygous based on the T53538908A SNP genotype;

the genotype II cow is a TA heterozygous cow based on the T53538908A SNP genotype;

the cow with genotype III is a cow with genotype based on T53538908A SNP and AA homozygote;

the T53538908A SNP is SEQ ID NO: 13 from the 5' end at nucleotide position 108, wherein the 108 th base of the sequence is t or a;

the position of the T53538908A SNP was determined based on the bovine reference genomic sequence alignment; the version number of the bovine reference genome sequence is UMD 3.1;

the dairy cow is a Chinese Holstein dairy cow.

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