integrated analysis of mrna expression, cpg island

Research ArticleIntegrated Analysis of mRNA Expression CpG IslandMethylation and Polymorphisms in the MITF Gene in Ducks(Anas platyrhynchos)

Ruiyi Lin 1 Weimin Lin1 Shiye Zhou2 Qiaohui Chen1 Jiahua Pan1 Yuanxin Miao 3

Mengwen Zhang1 Zhongbin Huang2 and Tianfang Xiao 1

1College of Animal Science Fujian Agriculture and Forestry University Fuzhou 350002 China2Research Center of Waterfowl Genetic Resource Protection of Shishi in Fujian Province Quanzhou 362700 China3Jingchu University of Technology Jingmen 448000 China

Correspondence should be addressed to Tianfang Xiao tfxiaofafueducn

Received 9 April 2019 Revised 29 July 2019 Accepted 5 September 2019 Published 23 September 2019

Academic Editor Diego Franco

Copyright copy 2019 Ruiyi Lin et al +is is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Microphthalmia-associated transcription factor (MITF) is a key regulator for the development and function of melanocytes inskin eye and plumage pigmentations +us the MITF was selected as a candidate gene associated with plumage coloration inducks +is study analyzed the mRNA expression promoter methylation and polymorphisms in the MITF gene in ducks withdifferent plumage colors (Putian Black Putian White Liancheng White and Longsheng Jade-green) No expression of theMITFmelanin-specific isoform (MITF-M) was detected in white feather bulbs By contrast the mRNA expression levels of MITF-Mwere high in black feather bulbs Bioinformatics analysis showed that two CpG islands were present in the promoter region of theMITF gene +e methylation level of the second CpG island was significantly lower in black feather bulbs than in white featherbulbs However the methylation level of the first CpG island was not different among the feather bulbs with various colors exceptLiancheng White feather bulbs +e methylation status of the whole CpG island significantly and negatively correlated with themRNA expression ofMITF-M (Plt 005) Furthermore four novel SNPs (single nucleotide polymorphisms) were identified in the5primeUTR exon 4 intron 7 and intron 8 of theMITF gene Allele T in g39807TgtG and allele G in g40862GgtAwere the predominantalleles only found in Putian White whereas the variant A allele in g32813GgtA exhibited a high allele frequency in LianchengWhite Collectively these results contributed to the understanding of the function of theMITF gene in duck plumage coloration

1 Introduction

Plumage color is an important characteristic of duck breedIn commercial duck meat processing white plumage duckshave an advantage over black ones because the carcasses ofwhite plumage ducks after plucking are preferred by con-sumers Plumage color variation is mainly determined bytwo kinds of melanin namely eumelanin and pheomelanin[1] In the process of melanin biosynthesis tyrosinaseencoded by the TYR gene is the rate-limiting enzyme af-fecting the production of melanin pigment [2] and de-termines which type of melanin can be synthesized [3] +etranscription of TYR family genes is activated by MITF(microphthalmia-associated transcription factor) through

binding to M-box in the upstream regulatory region [4] +egene encoding MITF is a member of the Myc superfamilywhich is part of the basic helix-loop-helix-leucine zipperfamily [5 6] Kawasaki et al [7] reported that Mitf is in-volved in the regulation of melanosome transport and thelevel of dendricity in melanophores using Xenopus laevis as amodel system A high Mitf expression is observed in SilkyFowl which is a natural mutant with hyperpigmentation invarious internal tissues [8]

MITF mutations can lead to coat color depigmentationwhite spotting or complete pigmentation loss in severalvertebrate species [9 10] In humans MITF gene mutationsare responsible for Waardenburg syndrome type II which ischaracterized by sensorineural hearing loss and depigmented

HindawiBioMed Research InternationalVolume 2019 Article ID 8512467 9 pageshttpsdoiorg10115520198512467

patches of skin and hair [11] Mitf mi-bw mutant mice harboran insertion of a long interspersed element-1 in intron 3severely affecting the expression of functional Mitf-M andresulting in a complete black-eyed white phenotype withsevere hearing loss in the homozygous genotype [12] Sub-sequent studies have also reported that variants in MITF areassociated with white spotting in dogs [13 14] piebaldism incattle [15] white markings in Spanish horses [16] and whitecoat coloring in American Standardbred foals [17] In ad-dition genome-wide analysis has revealed that MITF isregarded as a strong candidate for white spotting patterns inChinese domestic pigs [18]

MITF also plays a critical role in plumage coloration inpoultry +e causal mutation B in the MITF gene whichresults from a premature stop codon caused by a 2 bp de-letion in exon 11 is responsible for the ldquosilverrdquo plumagecolor in Japanese quail and implicated in its growth andbody composition [19] On the other hand a single nu-cleotide polymorphism (SNP) in the MITF gene has beensignificantly associated with the white plumage trait ofZhedong White geese [20] In ducks the differential ex-pression and polymorphisms of the MITF gene have beenproposed to be associated with different plumage colorphenotypes [21 22] Moreover whole-genome resequencinghas revealed that white plumage in duck is a result of theselection at the MITF locus [23] Zhou et al [24] identifiedan sim66 kb insertion between exon 1M and exon 2 in MITFthat accounts for the white feathers of Pekin duck

Although some polymorphisms of the MITF gene inducks have been investigated the mechanism that controlsthe variation in plumage color has yet to be explored In thepresent study the MITF gene was investigated to elucidateits characteristics by analyzing its mRNA expression pro-moter methylation and plumage color-related SNPs inChinese native ducks

2 Materials and Methods

21 Ethics Statement All animal procedures were approvedby the Experimental Animal Care and Use Committee ofFujian Agriculture and Forestry University (FAFU2013-0012) according to the Regulations for the Administration ofAffairs Concerning Experimental Animals (Ministry ofScience and Technology China revised in July 2013)

22 Animals and Tissues Putian Black (PTB) Putian White(PTW) Liancheng White (LC) and Longsheng Jade-green(LS) ducks are native breeds in China (Figure 1) All of themwere raised in the National Waterfowl Germplasm ResourcePool (Shishi Fujian China) Blood samples were collectedfrom 432 ducks (112 PTB ducks 107 PTW ducks 107 LCducks and 106 LS ducks) and stored at minus 20degC +e featherbulbs obtained from four ducks of each breed (a total of 16samples) were immediately frozen in liquid nitrogen andstored at minus 80degC

23 Bioinformatics Analysis BLAST was used to search theduck MITF promoter region sequence based on the duck

genome data (BGI_duck_10) and chicken MITF promotersequence (GenBank Accession no FJ196874) +e CpGisland of the duck MITF promoter was predicted byMethPrimer (httpwwwurogeneorgcgi-binmethprimermethprimercgi) AliBaba21 was used to identify putativetranscription factor-binding sites (TFBS) in the CpG island(httpwwwgene-regulationcompubprogramsalibaba2indexhtml)

24 DNA and Total RNA Extraction DNA samples wereextracted from the blood and feather bulb specimens via astandard phenol-chloroform method and then stored atminus 20degC until further use Total RNA was extracted fromfeather bulb specimens using TRIzol reagent (InvitrogenCarlsbad USA) according to the manufacturerrsquos in-structions +e concentrations of isolated DNA and RNAwere determined using a NanoDrop 2000 (+ermo FisherScientific Leicester UK) and denaturing gel electrophoresiswas conducted to assess the quality of RNA

25 Methylation Analysis of CpG Islands Genomic DNAfrom feather bulbs was treated with bisulfite using the EZDNA Methylation-Gold Kittrade (Zymo Research Irvine CAUSA) according to the manufacturerrsquos protocols PCR wasperformed in a total volume of 50 μL containing 150 ng ofDNA 5 μL of PCR buffer (10x) 150 μMdNTPs 3 μMof eachprimer 1 μL of Taq DNA polymerase (5UμL) and double-distilled H2O up to 50 μL +e following reaction conditionswere used 94degC for 5min 30 cycles of 94degC for 30 s 585605degC for 30 s (Table 1) and 72degC for 25 s1min and a finalextension at 72degC for 10min +e PCR products were ex-amined by 15 agarose gel electrophoresis and purifiedusing a MiniBEST Agarose Gel DNA Extraction Kit(TaKaRa Dalian China) +e expected fragment wasinserted into the pMD18-T vector and recombinant cloneswere used to transform Trans5α Chemically CompetentCells (TransGen Biotech Beijing China) +e positiverecombinant clones were selected on LBmedium containing60 μgmL ampicillin and confirmed by PCR Ten to fifteenpositive recombinant clones selected from each individualwere sequenced to identify the mutation and methylationsites +e MITF normal primer (Table 1) was designed forunmethylated DNA amplification

26 Expression of TYR and MITF in Duck Feather BulbsTYR as a marker gene of melanocyte [25] was used toconfirm the feather bulbs with different colors MITFconsists of at least five isoforms with a distinct amino-ter-minal in humans and mice and the mRNA expression ofMITF-M is exclusively expressed in melanocytes and pig-mented melanoma cells [10 26] +e specific primer ofMITF-M was designed according to Li et al [21] on the basisof the alternative 5primeexon

Total RNA was transcribed into cDNA using aPrimeScripttrade RT Reagent Kit with gDNA Eraser (TaKaRaDalian China) containing oligo (dT) random primers RTenzyme and gDNA Eraser Quantitative real-time PCR

2 BioMed Research International

(QPCR) was performed using the ABI Prism 7500 sequence-detection system (Applied Biosystems Foster City CAUSA) with SYBRGreen Real-time PCRMasterMix (Toyobo

Osaka Japan) following the manufacturerrsquos instructions AllPCR assays were performed in triplicate and β-actin wasused as an internal control +e fold change in relative gene

(a) (b)

(c) (d)

Figure 1 Ducks with different plumage colors (a) Putian Black duck (PTB) (b) PutianWhite duck (PTW) (c) LianchengWhite duck (LC)(d) Longsheng Jade-green duck (LS) PTB duck is the only indigenous breed with black plumage on the whole body It has black beaks andwebbed feet PTW duck has white plumage yellow beaks and webbed feet LC duck has white plumage black beaks and webbed feet LSduck has glossy black feathers black beaks and webbed feet Male duck has a malachite green head

Table 1 Primer pairs used for PCR amplification

Primer name Primer sequence (5prime-3prime) Product size (bp) Tm (degC)

PCR-HaeII-RFLP F TATGAGTGCCTGCTGCTACCT 485 595R GTTGATGGCAGGTGTCTGG

PCR-HaeIII-RFLP F TTGTACGGGCACCGATAT 457 526R CTTGAGGAGCAGCGATTA

PCR-ApoI-RFLP F CCTACAGAGTCAGAAGCAAGAG 329 572R TTCCAAGAAGTCCAGATAAACAG

PCR-HinfI-RFLP F GCCACGTCAACAGTCCCACA 540 565R GGCGGTCACTCACAGCATCAG

CpG island normal primer F TTCCCGGCATCTGAACAAAG 983 605R TCTCAACAGCAAAGGCACCC

CpG island 1 BSP primer F GGTTGTTTTTAAAGTGTAATTTTGT 256 585R AAACTCCCCTTAATATTTCATCCTC

CpG island 2 BSP primer F AGAGGATGAAATATTAAGGGGAGTT 222 605R ACCTACCTACCAAAAAACTCATTTC

TYR F TTACATGGTCCCCTTTATTC 182 60R CAATCACAGCTGCACCAACC

MITF F CCCAGTTCATGCAGCAGAGAGT 268 60R CCAGGCGGCATGACATGATCAC

β-Actin F AACTGGGATGACATGGAGAAGA 189 60R ATGGCTGGGGTGTTGAAGGTNote RFLP restriction fragment length polymorphism BSP bisulfite sequencing PCR Tm melting temperature

BioMed Research International 3

expression was calculated using the standard 2minus ΔΔCt method[27] +e ΔCt of the LS1 sample was arbitrarily set to 1 forthe relative quantification of the expression levels of genes inthe other groups (ΔΔCtΔCt of each group minus ΔCt of the LS1group in each experiment)

27 PolymorphismDetection of DuckMITFGene Six pairs ofprimer were designed on the basis of the genomic sequence ofthe duck MITF gene (GenBank Accession no KY114890) toscan the polymorphisms of this gene (Table S1) +e PCR assaycontained 50ng of genomic DNA 2μL of PCR buffer (10x) 1unit of Taq DNA polymerase 100μM dNTPs 1μM of eachprimer and double-distilled H2O up to 20μL Five randomlyselected DNA samples from each duck breed (a total of 20samples) were used as PCR templates+e PCR conditions wereas follows 5min initial denaturation at 94degC 35 cycles of 94degCfor 10 s 576605degC for 30 s (Table 1) and 72degC for 1min and afinal extension step at 72degC for 10min PCR products weredetected through electrophoresis in 15 agarose gels and se-quenced by a commercial service (Sangon Shanghai China)

Specific primers (listed in Table 1) were designed todetect four SNPs in a large population +e PCR conditionswere the same as those previously described For PCR-RFLP(polymerase chain reaction-restriction fragment lengthpolymorphism) 6 μL of PCR products was digested with 2units of HaeII HaeIII ApoI and HinfI (NEB IpswichMassachusetts USA) for 4 h at 37degC +e enzyme-digestedproducts were separated by electrophoresis on a 2 agarosegel with GelRed (Biotium CA USA)

28 Statistical Analysis +e sequencing results of methyl-ation analysis were examined using QUMA (httpqumacdbrikenjp) to calculate the methylation rate of CpG sites+e correlation between methylation levels and mRNAexpression was analyzed by Pearsonrsquos correlation [28]

PIC (polymorphic information content) was calculatedusing the following formula

PIC 1 minus 1113944n

i1P2i minus 1113944

nminus 1

i11113944

n

ji+12P

2i P

2j (1)

where n is the number of alleles at one locus and Pi and Pj arethe frequencies of the ith and jth alleles at one locus re-spectively and j i+ 1

3 Results

31 ExpressionofTYRandMITFGenes inDuckFeatherBulbs+e TYR expression was detected to further confirm thefeather bulbs with different colors +e expression pattern ofthe marker gene was consistent with the difference inplumage color Nearly no mRNA expression of TYR wasobserved in white feather bulbs (Figure 2(a)) It has beendemonstrated that MITF is involved in pigmentation inseveral species however whether MITF is implicated in thefeather coloration of Chinese native ducks is still unknownTo investigate that we analyzed the expression profile ofMITF-M isoform Similarly the MITF-M isoform was also

not expressed in white feather bulbs (Figure 2(b)) whereasthe B isoform of theMITF gene was expressed in both blackand white feather bulbs (data not shown) +ese findingsindicated that the melanogenesis pathway is implicated inthe plumage color in ducks

32 Bioinformatics Analysis of Duck MITF PromoterDNA methylation occurs mainly in the CpG island-richpromoter region and the sequence of the duck MITFpromoter region needs to be clear in this study firstly On thebasis of the duck genome data (BGI_duck_10) and chickenMITF promoter sequence (GenBank Accession noFJ196874) the sequence of the duck MITF promoter regionwas predicted through BLAST+e CpG island was analyzedusing MethPrimer online software Two CpG islands wereidentified in the 5primeupstream region of the MITF gene(Figure S1) +e CpG islands were located in the regionsfrom minus 1762 to minus 1661 bp and from minus 1597 to minus 1467 bpcontaining 20 CpG dinucleotides Several putative TFBSincluding Sp1 AP-1 CREB CPE_bind Oct-1 MyoD andp40x were identified in the CpG islands Two pairs of primerwere designed on the basis of the location of the CpG islandsand used for the amplification of two fragments containingthe CpG island regions (Table 1)

33 Methylation Status of Duck MITF Promoter DNAmethylation is a major epigenetic modification that regulatesgene expression To analyze the influence of promotermethylation on the expression of the duck MITF gene themethylation status of the duckMITF promoter was measuredBisulfite sequencing PCR (BSP) was performed to amplify theCpG islands +e PCR products of normal and bisulfite-treated DNA were cloned and sequenced +e sequencingresults were submitted to QUMA (httpqumacdbrikenjp)for further analysis +e average methylation rate of the CpGsites was 7507 suggesting that the CpG islands in the duckMITF promoter had a high degree ofmethylation As shown inFigure 3(a) the methylation status of the first CpG island wasnot significantly different among the feather bulbs of PTBPTW and LS However the degree of methylation was sig-nificantly higher in the white feather bulbs of LC than that inothers (Plt 005 Figure 3(a)) In the second CpG island themethylation levels differed dramatically in the white and blackfeather bulbs (Plt 005 Figure 3(b))

Pearson correlation analysis (Table 2) showed that themethylation status of the whole CpG island was negativelycorrelated with the mRNA expression ofMITF-M (Plt 005)and significant correlation coefficients were obtained forCpG_4 and CpG_6 in CpG island 2 (Plt 001)

34 Identification and Analysis of Polymorphic Loci in DuckMITF Gene To analyze the correlation between SNPs ofMITF gene and color variants in ducks we screenedpolymorphisms within whole region of the duck MITFgene through direct sequencing in twenty individualsrandomly selected from the four duck breeds We foundfour novel SNPs (g312CgtT g32813GgtA g39807TgtG


and g40862GgtA) according to the reference sequence ofPeking duck +ese SNPs located in the 5primeUTR exon 4intron 7 and intron 8 respectively (Figure 4) weremainly observed in PTB and LS ducks

PCR-RFLP was used to detect the four SNPs in a largepopulation (Figure S2) +e genotype and allele frequenciesof the identified SNPs of the MITF gene from four duckpopulations are shown in Table 3 Loci polymorphism can beconsidered high medium or low if PICgt 05 PICgt 025 orPIClt 025 respectively [29] In the present study except forPTB and LS in g312CgtTand PTB in g32813GgtA achievingmoderate polymorphism the others achieved low poly-morphism Table 3 shows that allele T in g39807TgtG andallele G in g40862GgtA were the predominant alleles andfound exclusively in PTW and that the variant A allele ing32813GgtA exhibited a high allele frequency in LC

+e four duck populations can be categorized into group1 (PTW duck) and group 2 (PTB LS and LC ducks) basedon the genotype and allele frequencies of g39807TgtG andg40862GgtA in the MITF gene Even though PTW and LCducks both have white plumage the beaks and webbed feetof PTW ducks are yellow whereas those of LC ducks areblack similar to those of PTB and LS ducks (Figure 1) Herethe correlation analysis also showed that these two loci(g39807TgtG and g40862GgtA) were significantly associatedwith beak and webbed foot colors (Plt 001)

4 Discussion

China has a rich genetic resource of domestic ducks whichare a source of meat eggs and feathers [30] +e color ofduck plumage is a breed characteristic However someducks with plumage color variation occur in the breedingprocess especially in crossbreeding [31] To obtain homo-geneous plumage color ducks breeders should have anenhanced understanding of plumage color genetics

Differences in the melanin-based coloration of feathersare caused by the relative content and distribution ofeumelanin and pheomelanin produced in melanocytes [32]MITF has emerged as an essential regulator not only formelanocyte development proliferation and survival but alsofor the expression of enzymes ensuring melanin production[33] In the present study almost no mRNA expression ofthe MITF-M isoform was observed in white feather bulbsregardless of duck breed In addition reduced TYR ex-pression in white feather bulbs might be attributed to thealtered MITF-M expression +us melanocyte developmentmight be defective in white feather bulbs

+e sequence and methylation status of the MITFpromoter region were analyzed to explore the regulatorymechanism of MITF-M expression in duck feather bulbsTwo CpG islands were predicted to be in the MITF pro-moter and the methylation level of the second CpG island

0

02

04

06

08

1

B-PTB W-PTW W-LC B-LS

lowast

Met

hyla

tion

perc

enta

ge (

)

(a)

B-PTB W-PTW W-LC B-LS0

02

04

06

08

1lowast

lowast

Met

hyla

tion

perc

enta

ge (

)

(b)

Figure 3 Methylation of CpG island in feather bulbs B-PTB black feather bulb from Putian Black duck W-PTW white feather bulb fromPutianWhite duck W-LC white feather bulb from LianchengWhite duck B-LS black feather bulb from Longsheng Jade-green duck Eachindividual was sequenced at least 10 times to identify the methylation sites and data are shown as meanplusmn standard error (n 4) from onerepresentative experiment +e asterisk (lowast) indicates a significant difference (Plt 005) (a) First CpG island (b) Second CpG island

005

115

225

335


Rela

tive m

RNA

expr

essio

nlowastlowast

lowastlowast

(a)

005

115

225

3


Rela

tive m

RNA

expr

essio

n

lowastlowast

lowastlowast

(b)

Figure 2 Gene expression in feather bulbs as determined by qPCR B-PTB black feather bulb from Putian Black duck W-PTW whitefeather bulb from PutianWhite duckW-LC white feather bulb from LianchengWhite duck B-LS black feather bulb from Longsheng Jade-green duck All assays were repeated at least three times and data are shown as meanplusmn standard error (n 4) from one representativeexperiment +e expression of each gene was normalized to β-actin +e asterisks (lowastlowast) indicate significant differences (Plt 001) (a) TYRgene (b) MITF gene


lowast

C G A G G G G G G GTTCC G A

G G G GTTT C G A

A AT

C G A G G GA AT

(a)

lowast

G G GA A A GC C C C C T T G GC A

GR C C T T G GC A

GG C C T T G GC A

T

G G GA A C C CT

G G GA A C C CT

(b)lowast

A G A A A AT T

T T

K

G C C C C T G A T A

C C C C T G A T A

C C C C T G A T A

C

A G A A A AT C

TA G A A A AT C

(c)

lowast

G G G G G G G GRC C C CA AT T T T

G G G G G G G G GC C C CA AT T T T

G G G G G G G GC C C CA A AT T T T

(d)

Figure 4 Four previously unreported SNPs in the duck MITF gene (a) g312CgtT site (b) g32813GgtA site (c) g39807TgtG site(d) g40862GgtA site Five individuals from each breed (N 20) were randomly selected for Sanger sequencing Asterisk showed mutationsites +e SNPs were mainly observed in PTB and LS ducks according to the reference sequence of Peking duck

Table 2 +e correlation analysis between methylation level and mRNA expression in the MITF gene

CpG island CpG site Correlation coefficient P value

CpG island 1

CpG_all minus 0460 0270CpG_1 minus 0830 0085CpG_2 minus 0880 0060CpG_3 0576 0212CpG_4 minus 0004 0498CpG_5 0061 0469CpG_6 0135 0432CpG_7 mdash mdashCpG_8 minus 0493 0254CpG_9 minus 0155 0422CpG_10 mdash mdash

CpG island 2

CpG_all minus 0910 0045CpG_1 minus 0109 0445CpG_2 minus 0661 0169CpG_3 minus 0269 0366CpG_4 minus 0996 0002CpG_5 minus 0714 0143CpG_6 minus 0996 0002CpG_7 minus 0661 0170CpG_8 minus 0867 0066CpG_9 minus 0753 0124CpG_10 0880 0060

CpG island CpG_all minus 0905 0047


was significantly higher in white feather bulbs than in blackfeather bulbs Pearson correlation analysis also showed thatthe methylation of the CpG islands was negatively correlatedwith MITF-M expression +ese findings indicated thatMITF-M expression is regulated by DNA methylationwhich was consistent with the results reported by Lauss et al[34] Previous studies confirmed that variants of the MITFpromoter are associated with unpigmented phenotypes inhorses and dogs [35 36] +e present study found that themethylation level of theMITF promoter was associated withduck plumage coloration To the best of our knowledge ourstudy is the first to report the potential epigenetic mecha-nisms to explain the variation in duck plumage coloration[37] However further experiments are needed to study themechanism of methylation differences in the white featherbulbs of PTW and LC ducks

In addition MITF was subjected to mutation screeningto investigate the association of MITF gene with duckplumage colors +e comparison of MITF gene among thefour Chinese native duck breeds identified four novel mu-tations (g312CgtT g32813GgtA g39807TgtG andg40862GgtA) located in the 5primeUTR exon 4 intron 7 andintron 8 respectively MITF polymorphisms in this studydiffered from those in other studies [22ndash24 38] because ofthe different duck breeds It is also worth mentioning thatthe MITF gene is large (KY114890 covers approximately486 kb) +us the partial resequencing performed in thisstudy might possibly miss other putative mutations

Interestingly the distributions of theMITF g32813GgtAg39807TgtG and g40862GgtA polymorphisms in the whiteplumage duck population were different from those in LCand PTW ducks LC and PTW are two white plumage duckvarieties LC ducks have black beaks and webbed feet

whereas PTW ducks have yellow beaks and webbed feetConsidering the similarity of appearance between PTW andCherry Valley ducks the polymorphism of these three siteswas further detected in Cherry Valley ducks As expectedthe genotype distribution of these three loci in Cherry Valleyducks was identical to that of PTW ducks +ese three SNPvariations might not only cause the differences in plumagecolor observed in this test group but also be associated withthe differences in the color of beaks and webbed feet

As a complex trait plumage color is determined by acomplex pathway system and multiple interactive patternssuggesting that the molecular mechanism of plumage colorformation needs further investigation [39] Our resultsprovided novel information for elucidating the MITFfunction in plumage color variations in ducks

5 Conclusion

On the basis of the results of this study we speculated thatthe differentially methylated MITF promoter is related toMITF-M expression and affects the plumage color pheno-types in ducks +e variations of duck plumage color are notonly affected byMITF-MmRNA expression but also may berelated to the genetic variants of the MITF gene

Data Availability

+e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

+e authors declare that they have no conflicts of interest

Table 3 Genotype distribution and genetic polymorphisms of the duck MITF gene

SNP position BreedGenotype frequency (number of birds) Allele frequency

PICCC CT TT C T

g312CgtT

PTB 035 (39) 054 (61) 011 (12) 0621 0379 0360PTW 099 (106) 001 (1) 0 (0) 0995 0005 0009LC 087 (93) 013 (14) 0 (0) 0935 0065 0115LS 044 (47) 046 (49) 009 (10) 0675 0325 0343

g32813GgtA

GG GA AA G APTB 036 (40) 051 (57) 013 (15) 0612 0388 0362PTW 100 (107) 0 (0) 0 (0) 1000 0000 0000LC 0 (0) 011 (12) 089 (95) 0056 0944 0100LS 078 (83) 021 (22) 001 (1) 0887 0113 0181

g39807TgtG

TT TG GG T GPTB 0 (0) 003 (3) 097 (109) 0013 0987 0026PTW 079 (85) 019 (20) 002 (2) 0888 0112 0179LC 002 (2) 0 (0) 098 (105) 0019 0981 0036LS 0 (0) 001 (1) 099 (105) 0005 0995 0009

g40862GgtA


Note PTB means Putian Black duck (N 112) PTW means Putian White duck (N 107) LC means Liancheng White duck (N 107) and LS meansLongsheng Jade-green duck (N 106) PIC means polymorphism information content PIClt 025 indicates a low level of polymorphism 025ltPIClt 05indicates a medium level of polymorphism and PICgt 05 indicates a high level of polymorphism


Acknowledgments

+e authors would like to thank Dr Nenzhu Zheng fromFujian Academy of Agricultural Sciences for providing theblood samples of Cherry Valley duck +is work was sup-ported by the National Natural Science Foundation of China(Grant no 31702109) Natural Science Foundation of FujianProvince of China (Grant no 2017J01596) Discipline De-velopment Grant from College of Animal Sciences FAFU(Grant no 2018DK001) Special Fund for Science andTechnology Innovation of Fujian Agriculture and ForestryUniversity (Grant no CXZX2016015) and Natural ScienceFoundation of Hubei Province of China (Grant no2018CFB305)

Supplementary Materials

Supplementary Figure 1 bioinformatics analysis of the CpGisland of the duck MITF gene +e blue region is the CpGisland Supplementary Figure 2 PCR-RFLP genotypingpatterns of four SNPs in the duck MITF gene Supple-mentary Table 1 primer pairs used for identifying thepolymorphisms of the MITF gene (SupplementaryMaterials)

References

[1] G Prota ldquoRecent advances in the chemistry of melanogenesisin mammalsrdquo Journal of Investigative Dermatology vol 75no 1 pp 122ndash127 1980

[2] A Sanchez-Ferrer J Neptuno Rodrıguez-Lopez F Garcıa-Canovas and F Garcıa-Carmona ldquoTyrosinase a compre-hensive review of its mechanismrdquo Biochimica et BiophysicaActa (BBA)mdashProtein Structure and Molecular Enzymologyvol 1247 no 1 pp 1ndash11 1995

[3] S Ito K Wakamatsu and H Ozeki ldquoChemical analysis ofmelanins and its application to the study of the regulation ofmelanogenesisrdquo Pigment Cell Research vol 13 no 8pp 103ndash109 2000

[4] C Goding ldquoMelanocyte development and malignant mela-nomardquo Forum (Genoa Italy) vol 10 no 3 pp 176ndash187 2000

[5] C L Hershey and D E Fisher ldquoMitf and Tfe3 members of ab-HLH-ZIP transcription factor family essential for osteoclastdevelopment and functionrdquo Bone vol 34 no 4 pp 689ndash6962004

[6] C Levy M Khaled and D E Fisher ldquoMITF master regulatorof melanocyte development andmelanoma oncogenerdquo Trendsin Molecular Medicine vol 12 no 9 pp 406ndash414 2006

[7] A Kawasaki M Kumasaka A Satoh et al ldquoMitf contributesto melanosome distribution and melanophore dendricityrdquoPigment Cell amp Melanoma Research vol 21 no 1 pp 56ndash622008

[8] Y Li X Zhu L Yang et al ldquoExpression and network analysisof genes related to melanocyte development in the Silky Fowland White Leghorn embryosrdquo Molecular Biology Reportsvol 38 no 2 pp 1433ndash1441 2011

[9] K J Moore ldquoInsight into themicrophthalmia generdquo Trends inGenetics vol 11 no 11 pp 442ndash448 1995

[10] S Shibahara K Takeda K-I Yasumoto et al ldquoMicro-phthalmia-associated transcription factor (MITF) multi-plicity in structure function and regulationrdquo Journal of

Investigative Dermatology Symposium Proceedings vol 6no 1 pp 99ndash104 2001

[11] V Pingault D Ente F Dastot-Le Moal M GoossensS Marlin and N Bondurand ldquoReview and update of mu-tations causing Waardenburg syndromerdquo Human Mutationvol 31 no 4 pp 391ndash406 2010

[12] I Yajima S Sato T Kimura et al ldquoAn L1 element intronicinsertion in the Black-EyedWhite (Mitf mi-bw) gene the loss ofa single Mitf isoform responsible for the pigmentary defectand inner ear deafnessrdquo Human Molecular Genetics vol 8no 8 pp 1431ndash1441 1999

[13] L Andersson ldquoGenome-wide association analysis in domesticanimals a powerful approach for genetic dissection of traitlocirdquo Genetica vol 136 no 2 pp 341ndash349 2009

[14] S M Schmutz T G Berryere and D L Dreger ldquoMITF andwhite spotting in dogs a population studyrdquo Journal of He-redity vol 100 no 1 pp S66ndashS74 2009

[15] L Fontanesi E Scotti and V Russo ldquoHaplotype variability inthe bovine MITF gene and association with piebaldism inHolstein and Simmental cattle breedsrdquo Animal Geneticsvol 43 no 3 pp 250ndash256 2012

[16] S Negro F Imsland M Valera A Molina M Sole andL Andersson ldquoAssociation analysis of KIT MITF and PAX3variants with white markings in Spanish horsesrdquo AnimalGenetics vol 48 no 3 pp 349ndash352 2017

[17] N Durig R Jude V Jagannathan and T Leeb ldquoA novelMITF variant in a white American Standardbred foalrdquo An-imal Genetics vol 48 no 1 pp 123-124 2017

[18] C Wang H Wang Y Zhang Z Tang K Li and B LiuldquoGenome-wide analysis reveals artificial selection on coatcolour and reproductive traits in Chinese domestic pigsrdquoMolecular Ecology Resources vol 15 no 2 pp 414ndash424 2015

[19] F Minvielle B Bedrsquohom J-L Coville S Ito M Inoue-Murayama and D Gourichon ldquo+e ldquosilverrdquo Japanese quailand the MITF gene causal mutation associated traits andhomology with the ldquobluerdquo chicken plumagerdquo BMC Geneticsvol 11 no 1 2010

[20] Y Wang S-M Li J Huang S-Y Chen and Y-P LiuldquoMutations of TYR and MITF Genes are Associated withPlumage Colour Phenotypes in Geeseltgtltgtltgtltgtrdquo Asian-Australasian Journal of Animal Sciences vol 27 no 6pp 778ndash783 2014

[21] S Li CWangW Yu S Zhao and Y Gong ldquoIdentification ofgenes related to white and black plumage formation by RNA-seq from white and black feather bulbs in ducksrdquo PLoS Onevol 7 no 5 Article ID e36592 2012

[22] H Sultana D Seo N-R Choi et al ldquoIdentification ofpolymorphisms in MITF and DCT genes and their associa-tions with plumage colors in Asian duck breedsrdquo Asian-Australasian Journal of Animal Sciences vol 31 no 2pp 180ndash188 2018

[23] Z Zhang Y Jia P Almeida et al ldquoWhole-genome rese-quencing reveals signatures of selection and timing of duckdomesticationrdquo GigaScience vol 7 no 4 2018

[24] Z Zhou M Li H Cheng et al ldquoAn intercross populationstudy reveals genes associated with body size and plumagecolor in ducksrdquoNature Communications vol 9 no 1 p 26482018

[25] S Vijayasaradhi P M Doskoch J Wolchok andA N Houghton ldquoMelanocyte differentiation marker gp75the brown locus protein can be regulated independently oftyrosinase and pigmentationrdquo Journal of Investigative Der-matology vol 105 no 1 pp 113ndash119 1995


[26] T Udono K-I Yasumoto K Takeda et al ldquoStructural or-ganization of the human microphthalmia-associated tran-scription factor gene containing four alternative promotersrdquoBiochimica et Biophysica Acta (BBA)mdashGene Structure andExpression vol 1491 no 1ndash3 pp 205ndash219 2000

[27] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minus ΔΔCt methodrdquo Methods vol 25 no 4 pp 402ndash408 2001

[28] K Pearson ldquoNotes on the history of correlationrdquo Biometrikavol 13 no 1 pp 25ndash45 1920

[29] D Vaiman D Mercier K Moazami-Goudarzi et al ldquoA set of99 cattle microsatellites characterization synteny mappingand polymorphismrdquo Mammalian Genome vol 5 no 5pp 288ndash297 1994

[30] Y Huang Y Li D W Burt et al ldquo+e duck genome andtranscriptome provide insight into an avian influenza virusreservoir speciesrdquoNature Genetics vol 45 no 7 pp 776ndash7832013

[31] Y Gong Q Yang S Li et al ldquoGrey plumage colouration inthe duck is genetically determined by the alleles on twodifferent interacting locirdquo Animal Genetics vol 41 no 1pp 105ndash108 2010

[32] G Emaresi A-L Ducrest P Bize H Richter C Simon andA Roulin ldquoPleiotropy in the melanocortin system expressionlevels of this system are associated with melanogenesis andpigmentation in the tawny owl (Strix aluco)rdquo MolecularEcology vol 22 no 19 pp 4915ndash4930 2013

[33] J Vachtenheim and J Borovansky ldquoldquoTranscription physiol-ogyrdquo of pigment formation in melanocytes central role ofMITFrdquo Experimental Dermatology vol 19 no 7 pp 617ndash6272010

[34] M Lauss R Haq H Cirenajwis et al ldquoGenome-wide DNAmethylation analysis in melanoma reveals the importance ofCpGmethylation inMITF regulationrdquo Journal of InvestigativeDermatology vol 135 no 7 pp 1820ndash1828 2015

[35] EKKarlsson I BaranowskaCMWade et al ldquoEfficientmappingof mendelian traits in dogs through genome-wide associationrdquoNature Genetics vol 39 no 11 pp 1321ndash1328 2007 httpswwwnaturecomarticlesng200710supplementary-information

[36] R Hauswirth B Haase M Blatter et al ldquoMutations inMITFand PAX3 cause ldquosplashed whiterdquo and other white spottingphenotypes in horsesrdquo PLoS Genetics vol 8 no 4 Article IDe1002653 2012

[37] A Roulin and A-L Ducrest ldquoGenetics of colouration inbirdsrdquo Seminars in Cell amp Developmental Biology vol 24no 6-7 pp 594ndash608 2013

[38] L Yang C Mo W Shen et al ldquo+e recessive C locus in theMITF gene plays a key regulatory role in the plumage colourpattern of duck (Anas platyrhynchos)rdquo British Poultry Sciencevol 60 no 2 pp 105ndash108 2019

[39] J-S Ran X-Y You J Jin et al ldquo+e relationship betweenMC1R mutation and plumage color variation in pigeonsrdquoBiomed Research International vol 2016 Article ID 30597566 pages 2016


Hindawiwwwhindawicom

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Submit your manuscripts atwwwhindawicom

patches of skin and hair [11] Mitf mi-bw mutant mice harboran insertion of a long interspersed element-1 in intron 3severely affecting the expression of functional Mitf-M andresulting in a complete black-eyed white phenotype withsevere hearing loss in the homozygous genotype [12] Sub-sequent studies have also reported that variants in MITF areassociated with white spotting in dogs [13 14] piebaldism incattle [15] white markings in Spanish horses [16] and whitecoat coloring in American Standardbred foals [17] In ad-dition genome-wide analysis has revealed that MITF isregarded as a strong candidate for white spotting patterns inChinese domestic pigs [18]

MITF also plays a critical role in plumage coloration inpoultry +e causal mutation B in the MITF gene whichresults from a premature stop codon caused by a 2 bp de-letion in exon 11 is responsible for the ldquosilverrdquo plumagecolor in Japanese quail and implicated in its growth andbody composition [19] On the other hand a single nu-cleotide polymorphism (SNP) in the MITF gene has beensignificantly associated with the white plumage trait ofZhedong White geese [20] In ducks the differential ex-pression and polymorphisms of the MITF gene have beenproposed to be associated with different plumage colorphenotypes [21 22] Moreover whole-genome resequencinghas revealed that white plumage in duck is a result of theselection at the MITF locus [23] Zhou et al [24] identifiedan sim66 kb insertion between exon 1M and exon 2 in MITFthat accounts for the white feathers of Pekin duck

Although some polymorphisms of the MITF gene inducks have been investigated the mechanism that controlsthe variation in plumage color has yet to be explored In thepresent study the MITF gene was investigated to elucidateits characteristics by analyzing its mRNA expression pro-moter methylation and plumage color-related SNPs inChinese native ducks

2 Materials and Methods

21 Ethics Statement All animal procedures were approvedby the Experimental Animal Care and Use Committee ofFujian Agriculture and Forestry University (FAFU2013-0012) according to the Regulations for the Administration ofAffairs Concerning Experimental Animals (Ministry ofScience and Technology China revised in July 2013)

22 Animals and Tissues Putian Black (PTB) Putian White(PTW) Liancheng White (LC) and Longsheng Jade-green(LS) ducks are native breeds in China (Figure 1) All of themwere raised in the National Waterfowl Germplasm ResourcePool (Shishi Fujian China) Blood samples were collectedfrom 432 ducks (112 PTB ducks 107 PTW ducks 107 LCducks and 106 LS ducks) and stored at minus 20degC +e featherbulbs obtained from four ducks of each breed (a total of 16samples) were immediately frozen in liquid nitrogen andstored at minus 80degC

23 Bioinformatics Analysis BLAST was used to search theduck MITF promoter region sequence based on the duck

genome data (BGI_duck_10) and chicken MITF promotersequence (GenBank Accession no FJ196874) +e CpGisland of the duck MITF promoter was predicted byMethPrimer (httpwwwurogeneorgcgi-binmethprimermethprimercgi) AliBaba21 was used to identify putativetranscription factor-binding sites (TFBS) in the CpG island(httpwwwgene-regulationcompubprogramsalibaba2indexhtml)

24 DNA and Total RNA Extraction DNA samples wereextracted from the blood and feather bulb specimens via astandard phenol-chloroform method and then stored atminus 20degC until further use Total RNA was extracted fromfeather bulb specimens using TRIzol reagent (InvitrogenCarlsbad USA) according to the manufacturerrsquos in-structions +e concentrations of isolated DNA and RNAwere determined using a NanoDrop 2000 (+ermo FisherScientific Leicester UK) and denaturing gel electrophoresiswas conducted to assess the quality of RNA

25 Methylation Analysis of CpG Islands Genomic DNAfrom feather bulbs was treated with bisulfite using the EZDNA Methylation-Gold Kittrade (Zymo Research Irvine CAUSA) according to the manufacturerrsquos protocols PCR wasperformed in a total volume of 50 μL containing 150 ng ofDNA 5 μL of PCR buffer (10x) 150 μMdNTPs 3 μMof eachprimer 1 μL of Taq DNA polymerase (5UμL) and double-distilled H2O up to 50 μL +e following reaction conditionswere used 94degC for 5min 30 cycles of 94degC for 30 s 585605degC for 30 s (Table 1) and 72degC for 25 s1min and a finalextension at 72degC for 10min +e PCR products were ex-amined by 15 agarose gel electrophoresis and purifiedusing a MiniBEST Agarose Gel DNA Extraction Kit(TaKaRa Dalian China) +e expected fragment wasinserted into the pMD18-T vector and recombinant cloneswere used to transform Trans5α Chemically CompetentCells (TransGen Biotech Beijing China) +e positiverecombinant clones were selected on LBmedium containing60 μgmL ampicillin and confirmed by PCR Ten to fifteenpositive recombinant clones selected from each individualwere sequenced to identify the mutation and methylationsites +e MITF normal primer (Table 1) was designed forunmethylated DNA amplification

26 Expression of TYR and MITF in Duck Feather BulbsTYR as a marker gene of melanocyte [25] was used toconfirm the feather bulbs with different colors MITFconsists of at least five isoforms with a distinct amino-ter-minal in humans and mice and the mRNA expression ofMITF-M is exclusively expressed in melanocytes and pig-mented melanoma cells [10 26] +e specific primer ofMITF-M was designed according to Li et al [21] on the basisof the alternative 5primeexon

Total RNA was transcribed into cDNA using aPrimeScripttrade RT Reagent Kit with gDNA Eraser (TaKaRaDalian China) containing oligo (dT) random primers RTenzyme and gDNA Eraser Quantitative real-time PCR




(a) (b)

(c) (d)





















i1P2i minus 1113944

nminus 1

i11113944

n

ji+12P

2i P

2j (1)


3 Results











4 Discussion




0

02

04

06

08

1


lowast

Met

hyla

tion

perc

enta

ge (

)

(a)


02

04

06

08

1lowast

lowast

Met

hyla

tion

perc

enta

ge (

)

(b)


005

115

225

335


Rela

tive m

RNA

expr

essio

nlowastlowast

lowastlowast

(a)

005

115

225

3


Rela

tive m

RNA

expr

essio

n

lowastlowast

lowastlowast

(b)



lowast


G G G GTTT C G A

A AT

C G A G G GA AT

(a)

lowast


GR C C T T G GC A

GG C C T T G GC A

T

G G GA A C C CT

G G GA A C C CT

(b)lowast

A G A A A AT T

T T

K

G C C C C T G A T A

C C C C T G A T A

C C C C T G A T A

C

A G A A A AT C

TA G A A A AT C

(c)

lowast




(d)




CpG island 1


CpG island 2









5 Conclusion


Data Availability






PICCC CT TT C T

g312CgtT

PTB 035 (39) 054 (61) 011 (12) 0621 0379 0360PTW 099 (106) 001 (1) 0 (0) 0995 0005 0009LC 087 (93) 013 (14) 0 (0) 0935 0065 0115LS 044 (47) 046 (49) 009 (10) 0675 0325 0343

g32813GgtA


g39807TgtG


g40862GgtA




Acknowledgments




References













































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




(a) (b)

(c) (d)





















i1P2i minus 1113944

nminus 1

i11113944

n

ji+12P

2i P

2j (1)


3 Results











4 Discussion




0

02

04

06

08

1


lowast

Met

hyla

tion

perc

enta

ge (

)

(a)


02

04

06

08

1lowast

lowast

Met

hyla

tion

perc

enta

ge (

)

(b)


005

115

225

335


Rela

tive m

RNA

expr

essio

nlowastlowast

lowastlowast

(a)

005

115

225

3


Rela

tive m

RNA

expr

essio

n

lowastlowast

lowastlowast

(b)



lowast


G G G GTTT C G A

A AT

C G A G G GA AT

(a)

lowast


GR C C T T G GC A

GG C C T T G GC A

T

G G GA A C C CT

G G GA A C C CT

(b)lowast

A G A A A AT T

T T

K

G C C C C T G A T A

C C C C T G A T A

C C C C T G A T A

C

A G A A A AT C

TA G A A A AT C

(c)

lowast




(d)




CpG island 1


CpG island 2









5 Conclusion


Data Availability






PICCC CT TT C T

g312CgtT

PTB 035 (39) 054 (61) 011 (12) 0621 0379 0360PTW 099 (106) 001 (1) 0 (0) 0995 0005 0009LC 087 (93) 013 (14) 0 (0) 0935 0065 0115LS 044 (47) 046 (49) 009 (10) 0675 0325 0343

g32813GgtA


g39807TgtG


g40862GgtA




Acknowledgments




References













































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018








i1P2i minus 1113944

nminus 1

i11113944

n

ji+12P

2i P

2j (1)


3 Results











4 Discussion




0

02

04

06

08

1


lowast

Met

hyla

tion

perc

enta

ge (

)

(a)


02

04

06

08

1lowast

lowast

Met

hyla

tion

perc

enta

ge (

)

(b)


005

115

225

335


Rela

tive m

RNA

expr

essio

nlowastlowast

lowastlowast

(a)

005

115

225

3


Rela

tive m

RNA

expr

essio

n

lowastlowast

lowastlowast

(b)



lowast


G G G GTTT C G A

A AT

C G A G G GA AT

(a)

lowast


GR C C T T G GC A

GG C C T T G GC A

T

G G GA A C C CT

G G GA A C C CT

(b)lowast

A G A A A AT T

T T

K

G C C C C T G A T A

C C C C T G A T A

C C C C T G A T A

C

A G A A A AT C

TA G A A A AT C

(c)

lowast




(d)




CpG island 1


CpG island 2









5 Conclusion


Data Availability






PICCC CT TT C T

g312CgtT

PTB 035 (39) 054 (61) 011 (12) 0621 0379 0360PTW 099 (106) 001 (1) 0 (0) 0995 0005 0009LC 087 (93) 013 (14) 0 (0) 0935 0065 0115LS 044 (47) 046 (49) 009 (10) 0675 0325 0343

g32813GgtA


g39807TgtG


g40862GgtA




Acknowledgments




References













































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





4 Discussion




0

02

04

06

08

1


lowast

Met

hyla

tion

perc

enta

ge (

)

(a)


02

04

06

08

1lowast

lowast

Met

hyla

tion

perc

enta

ge (

)

(b)


005

115

225

335


Rela

tive m

RNA

expr

essio

nlowastlowast

lowastlowast

(a)

005

115

225

3


Rela

tive m

RNA

expr

essio

n

lowastlowast

lowastlowast

(b)



lowast


G G G GTTT C G A

A AT

C G A G G GA AT

(a)

lowast


GR C C T T G GC A

GG C C T T G GC A

T

G G GA A C C CT

G G GA A C C CT

(b)lowast

A G A A A AT T

T T

K

G C C C C T G A T A

C C C C T G A T A

C C C C T G A T A

C

A G A A A AT C

TA G A A A AT C

(c)

lowast




(d)




CpG island 1


CpG island 2









5 Conclusion


Data Availability






PICCC CT TT C T

g312CgtT

PTB 035 (39) 054 (61) 011 (12) 0621 0379 0360PTW 099 (106) 001 (1) 0 (0) 0995 0005 0009LC 087 (93) 013 (14) 0 (0) 0935 0065 0115LS 044 (47) 046 (49) 009 (10) 0675 0325 0343

g32813GgtA


g39807TgtG


g40862GgtA




Acknowledgments




References













































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


lowast


G G G GTTT C G A

A AT

C G A G G GA AT

(a)

lowast


GR C C T T G GC A

GG C C T T G GC A

T

G G GA A C C CT

G G GA A C C CT

(b)lowast

A G A A A AT T

T T

K

G C C C C T G A T A

C C C C T G A T A

C C C C T G A T A

C

A G A A A AT C

TA G A A A AT C

(c)

lowast




(d)




CpG island 1


CpG island 2









5 Conclusion


Data Availability






PICCC CT TT C T

g312CgtT

PTB 035 (39) 054 (61) 011 (12) 0621 0379 0360PTW 099 (106) 001 (1) 0 (0) 0995 0005 0009LC 087 (93) 013 (14) 0 (0) 0935 0065 0115LS 044 (47) 046 (49) 009 (10) 0675 0325 0343

g32813GgtA


g39807TgtG


g40862GgtA




Acknowledgments




References













































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018







5 Conclusion


Data Availability






PICCC CT TT C T

g312CgtT

PTB 035 (39) 054 (61) 011 (12) 0621 0379 0360PTW 099 (106) 001 (1) 0 (0) 0995 0005 0009LC 087 (93) 013 (14) 0 (0) 0935 0065 0115LS 044 (47) 046 (49) 009 (10) 0675 0325 0343

g32813GgtA


g39807TgtG


g40862GgtA




Acknowledgments




References













































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Acknowledgments




References













































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



















Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


integrated analysis of mrna expression, cpg island

Documents