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[PCR]#18 한우 경제형질 관련 유전자 SNP 발굴

이건우
2021-11-11
조회수 3370

목차

1.개요 및 목적

2. 실험 재료 및 기기

3. 실험 방법 및 절차

4. 실험 결과

5. 주의 사항

1. 개요 및 목적


1. 개요 및 목적

 - 가축 경제 형질에 영향을 미치는 주요 유전자의 특성에 관현 연구, SNP에 의해 개체간의 형질이 다르게 나타나는 유전적 변이 이용

 - 다양하고 산업적 활용가치가 높은 경제형질 관련 DNA 분자표지의 개발로 한우의 성장률을 높일 수 있음.

 - 유전자 CRH, DECR1, POMC, MC4R 

2. 실험 재료 및 기기


2. 실험 재료 및 기기

 - 서로 가계성이 없는 보증종모우 24두 한우의 모근

 - ND-1000 (NanoDrop)

 - 9700 Thermal Cycler (Applied Biosystems)

 - 3130 Genetic Analyzer (Applied Biosystems)

3. 실험 방법 및 절차


3. 실험 방법 및 절차

 1) 모근샘플에서 Genomic DNA Extraction Kit (Bioneer)를 이용 DNA 추출

 2) ND-1000으로 농도와 순도 측정

 3) 변이가 예상되는 지역을 대상으로 Primer 제작

▶ Primer 정보

Table 1. PCR primers for amplification of DECR1 gene in this study

Marker

Forward primer(5`-3`)

Reverse primer(5`-3`)

DECR1_1

AGGGTCATCCCAGTACACCAG

GAGGTGGATGAAACTGGAACC

DECR1_2

GAAGTTATGGGACCGGATGC

TTCAGGGTTCCCCAAATACC

DECR1_3

ACAGGGATCAAGACCATCCC

TTTCTGTCCTTTATCGAGCCC

DECR1_4

TTGGAAAACTCAGCAGTGGC

TTCAAGAGGCAGGTCAGGTG

DECR1_5

AGCATTTATTACCGGGGGAG

TGCCCGTCTTATAGGAAATGG

DECR1_6

GCCAGCTCTTTTTGCTAGGG

AAGGCTTCTTTCCAATATGCG

DECR1_7

TTTGCTTTTGTTTGGTTCGC

CAAATTTTGGAATTCTAATCCG

DECR1_8

GGCACTTTAATTGAGATTCCTTG

ACAAAGGCTGTGCCATTCAG

DECR1_9

CTGGCCATGACTTTCCTTCC

CATGAGAAACACTGGGCTGG

DECR1_10

TGATTCCAGCTTGTGCTTCC

AGGGGGTGACAGAGGATGAG

DECR1_11

GGGACCCCTGTAATGCAAAT

GGGACAAGATCAAACCCCAG

DECR1_12

TCCTCATTCTCACCGAGCTT

TGTTGGAAGAGGGTGTTTGC

DECR1_13

GCAAAAACAAGACCTGGAGATG

AGCAGCAGCAGTAGGGTGTG

 

Table 2. PCR primers for amplification of MC4R gene in this study

Marker

Forward primer(5`-3`)

Reverse primer(5`-3`)

MC4R_1

TGAGAGCATGCGCACATAGA

AGTAGCCTTTTGCCAGGGAC

MC4R_2

GATGCTGACCAGAGCCACAC

TTGGAAACGCTCACCAACAT

MC4R_3

GCTCTTTGTCTCTCCCGAGG

GCCAGCATGGTGAAGAACAC

MC4R_4

GTCGGGCGTCTTGTTCATC

GCTTGTGTTTAGCATCGCGT

MC4R_5

TGCAATTCCATCATTGACCC

AGCAAGCAAAGTGTCACATCC

MC4R_6

TCCACATCACAGGTTATAGGCAC

TCCCAAATTGCCTGTGAGAA

MC4R_7

TTTCTCATTCATTCTTCCTGTGC

GTAAGTCACTTGCATGTGGAAAA

 

Table 3. PCR primers for amplification of NPY gene in this study

Marker

Forward primer(5`-3`)

Reverse primer(5`-3`)

NPY_1

TGTGCACAGTGCCAAGACTG

CCCAGAGCAGCATCAATGAC

NPY_2

TTAGATTGGGGAAGCATAGGG

GAAAGCCCCAAGTTTCCAAG


Table 4. PCR primers for amplification of CRH gene in this study

Marker

Forward primer(5`-3`)

Reverse primer(5`-3`)

CRH_1

AGAAGGGAAGAGAACCGCTG

GCTCGTTTACACAGCGAGGA

CRH_2

CGTGCCACAAGATTCCAATAG

TCCCCTGTCTAACGATTCCC

CRH_3

GAGCCCTTGAGACCACGAAT

GCGCTAATTGCGGTACAGAG

CRH_4

TTTTCTCTCTCATTCCGCCC

AGAAAGGCGACTGCTGCTTC

 

Table 5. PCR primers for amplification of POMC gene in this study

Marker

Forward primer(5`-3`)

Reverse primer(5`-3`)

POMC

CCTCGAATTCAAGAGGGAGC

AGAGAGGCCTTCAGGGTCAA

▶ Sequence variations Hanwoo

Table 1. Sequence variations Hanwoo in DECR1 gene

SNP number and primer for detection

Location in DECR1 gene

Variations in Korean cattle

(Hanwoo) in this study

Sequence

Frequency

1 DECR1_1

Intron 1 (5634)

T/A

1.00/0.00

2 DECR1_1

Intron 1 (5636)

T/G

0.00/1.00

3 DECR1_1

Intron 1 (5624)

T/A

0.633/0.367

4 DECR1_1

Intron 1 (5658)

C/T

0.50/0.50

5 DECR1_3

Intron 1 (17651)

A/T

1.00/0.00

6 DECR1_3

Intron 1 (17652)

G/A

1.00/0.00

7 DECR1_4

Intron 1 (18318)

C/T

1.00/0.00

8 DECR1_4

Intron 1 (18240)

G/A

0.00/1.00

9 DECR1_4

Intron 1 (18243)

T/C

0.50/0.50

10 DECR1_4

Intron 1 (18251)

A/G

0.00/1.00

11 DECR1_4

Intron 1 (18261)

A/T

0.50/0.50

12 DECR1_5

Exon 2 (20788)

G/A

0.479/0.521

13 DECR1_5

Exon 2 (20796)

G/C

0.271/0.729

14 DECR1_5

Intron 2 (20830)

A/G

0.271/0.729

15 DECR1_5

Intron 2 (20986)

C/T

0.271/0.729

16 DECR1_5

Intron 2 (21053)

A/G

0.271/0.729

17 DECR1_5

Intron 2 (21093)

G/A

0.229/0.771

18 DECR1_7

Intron 4 (22982)

T/C

0.271/0.729

19 DECR1_8

Intron 4 (22991)

G/C

0.864/0.136

20 DECR1_8

Intron 4 (22982)

T/C

0.267/0.733

21 DECR1_8

Intron 4 (22984)

C/T

0.267/0.733

22 DECR1_8

Intron 4 (23070)

T/C

0.271/0.729

23 DECR1_8

Exon 5 (23201)

A/C

0.271/0.729

24 DECR1_8

Exon 5 (23262)

A/G

0.479/0.521

25 DECR1_9

Intron 5 (25760)

C/G

1.00/0.00

26 DECR1_11

Intron 5 (30149)

G/T

0.042/0.958

27 DECR1_12

Intron 6 (36064)

T/A

0.30/0.70

28 DECR1_12

Intron 6 (36115)

A/G

0.917/0.083

29 DECR1_12

Intron 6 (36184)

C/T

0.955/0.045

30 DECR1_13

Intron 6 (36538)

C/T

0.188/0.812


Table 2. Sequence variations Hanwoo in MC4R gene

SNP number and primer for detection

Location in DECR1 gene

Variations in Korean cattle

(Hanwoo) in this study

Sequence

Frequency

1 MC4R_3

709

G/A

0.646/0.354

2 MC4R_4

927

C/T

0.75/0.25

3 MC4R_4

1069

C/G

0.354/0.646

4 MC4R_5

1343

C/A

0.938/0.062

5 MC4R_6

1786

C/T

0.667/0.333

 

Table 2. Sequence variations Hanwoo in NPY gene

SNP number and primer for detection

Location in NPY gene

Variations in Korean cattle

(Hanwoo) in this study

Sequence

Frequency

1 NPY_1

3814

G/A

1.000/0.000

2 NPY_1

3869

C/T

1.000/0.000

3 NPY_2

5531

C/G

0.947/0.053

 

Table 3. Sequence variations Hanwoo in CRH gene

SNP number and primer for detection

Location in CRH gene

Variations in Korean cattle

(Hanwoo) in this study

Sequence

Frequency

1 CRH_2

234

A/C

0.789/0.211

2 CRH_2

269

G/A

0.789/0.211

 

Table 4. Sequence variations Hanwoo in POMC gene

SNP number and primer for detection

Location in POMC gene

Variations in Korean cattle

(Hanwoo) in this study

Sequence

Frequency

1 POMC

7217

C/T

1.000/0.000


Table 5. Nonsynonymous SNPs and their frequencies found in Hanwoo in this study

SNP position

Amino acid change

Frequency in Korean cattle (Hanwoo)

A23262G

Val161IIe

0.479/0.521

 

4) 9700으로 각각 PCR 진행

<PCR Composition>


Template DNA1ul
Forward Primer0.3ul
Reverse Primer0.3ul
dNTP1.2ul
10X Buffer2ul
Taq Polymerase0.1ul
Total Volume20ul


<PCR Condition>

Pre-Denaturation94℃5min
Denaturation94℃ 30sec35 cycle
Annealing
30sec
Extension72℃ 30sec
Final Extension72℃ 5min


PCR 산물을 3130 Genetic Analyzer 장비로 Sequencing 진행하여 SNP확인

4. 실험결과


4. 실험 결과

총 4개의 유전자에서 SNP 40곳이 발견


- DNA STAR 프로그램을 이용하여 각각의 SNP 분석

- 유전자 내의 SNP를 이용하여 경제형질과의 관계를 추가 분석 필요

- 추후 DNA Marker 조합을 만들어서 경제형질 관련 DNA 분자표지 개발 가능

▶ Distribution of genotypes and alleles for each gene

Table 1. Distribution of genotypes and alleles for DECR1 gene in this study

SNP position

Genotype

Frequency

Allele

Frequency

T5624A

TT

0.267

T

0.633

TA

0.733

A

0.367

AA

-

T5634A

TT

1.00

T

1.00

TA

-

A

-

AA

-

T5636G

TT

-

T

-

TG

-

G

1.00

GG

1.00

C5658T

CC

-

C

0.50

CT

1.00

T

0.50

TT

-

A17651T

AA

1.00

A

1.00

AT

-

T

-

TT

-

G17652A

GG

1.00

G

1.00

GA

-

A

-

AA

-

G18240A

GG

-

G

-

GA

-

A

1.00

AA

1.00

T18243C

TT

-

T

0.50

TC

1.00

C

0.50

CC

-

A18251G

AA

-

A

-

AG

-

G

1.00

GG

1.00

A18261T

AA

-

A

0.50

AT

1.00

T

0.50

TT

-

C18318T

CC

1.00

C

1.00

CT

-

T

-

TT

-

 

SNP position

Genotype

Frequency

Allele

Frequency

G20788A

GG

0.25

G

0.479

GA

0.458

A

0.521

AA

0.292

G20796C

GG

0.125

G

0.271

GC

0.292

C

0.729

CC

0.583

A20830G

AA

0.125

A

0.271

AG

0.292

G

0.729

GG

0.583

C20986T

CC

0.125

C

0.271

CT

0.292

T

0.729

TT

0.583

A21053G

AA

0.125

A

0.271

AG

0.292

G

0.729

GG

0.583

G21093A

GG

0.125

G

0.229

GA

0.208

A

0.771

AA

0.667

T22982C

TT

0.133

T

0.271

TC

0.267

C

0.729

CC

0.600

C22984T

CC

0.133

C

0.267

CT

0.267

T

0.733

TT

0.600

G22991C

GG

0.864

G

0.875

GC

-

C

0.125

CC

0.136

T23070C

TT

0.125

T

0.271

TC

0.292

C

0.729

CC

0.583


SNP position

Genotype

Frequency

Allele

Frequency

A23201C

AA

0.125

A

0.271

AC

0.292

C

0.729

CC

0.583

A23262G

AA

0.292

A

0.479

AG

0.375

G

0.521

GG

0.333

C25760G

CC

1.00

C

1.00

CG

-

G

-

GG

-

G30149T

GG

0.042

G

0.042

GT

-

T

0.958

TT

0.958

T36064A

TT

0.300

T

0.300

TA

-

A

0.700

AA

0.700

A36115G

AA

0.917

A

0.917

AG

-

G

0.083

GG

0.083

C36184T

CC

0.909

C

0.955

CT

0.091

T

0.045

TT

-

C36538T

CC

0.083

C

0.188

CT

0.208

T

0.812

TT

0.709

 

Table 2. Distribution of genotypes and alleles for MC4R gene in this study

SNP position

Genotype

Frequency

Allele

Frequency

G709A

GG

0.292

G

0.646

GA

0.708

A

0.354

AA

-

C927T

CC

0.625

C

0.75

CT

0.25

T

0.25

TT

0.125

C1069G

CC

0.208

C

0.354

CG

0.292

G

0.646

GG

0.5

C1343A

CC

0.875

C

0.938

CA

0.125

A

0.062

AA

-

C1786T

CC

0.417

C

0.667

CT

0.5

T

0.333

TT

0.083

 

Table 3. Distribution of genotypes and alleles for NPY gene in this study

SNP position

Genotype

Frequency

Allele

Frequency

G3814A

GG

1

G

1

GA

-

A

-

AA

-

C5531G

CC

1

C

1

CG

-

G

-

GG

-

C3869T

CC

0.895

C

0.947

CT

0.105

T

0.053

TT

-

 

Table 4. Distribution of genotypes and alleles for CRH gene in this study

SNP position

Genotype

Frequency

Allele

Frequency

A234C

AA

0.631

A

0.789

AC

0.316

C

0.211

CC

0.053

G269A

GG

0.631

G

0.789

GA

0.316

A

0.211

AA

0.053


Table 5. Distribution of genotypes and alleles for POMC gene in this study

SNP position

Genotype

Frequency

Allele

Frequency

C7217T

CC

1

C

1

CT

-

T

-

TT

-

5. 주의사항


5. 주의 사항

- 개체 수 증가 필요

- 경제형질과의 상관관계 분석 추가 필요



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