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Identification of Genes Showing Differential Expression Profile c Indian Academy of Sciences RESEARCH ARTICLE Identification of genes showing differential expression profile associated with growth rate in skeletal muscle tissue of Landrace weanling pig YUUTA KOMATSU1, SHIN SUKEGAWA2,MAIYAMASHITA1, NAOKI KATSUDA1, BIN TONG1, TAKESHI OHTA1, HIROYUKI KOSE3 and TAKAHISA YAMADA1∗ 1Faculty of Agriculture, Department of Agrobiology, Niigata University, Nishi-ku, Niigata 950-2181, Japan 2Research and Development Center, NH Foods Ltd., Tsukuba, Ibaraki 300-2646, Japan 3Department of Natural Sciences, International Christian University, Mitaka, Tokyo 181-8585, Japan Abstract Suppression subtractive hybridization was used to identify genes showing differential expression profile associated with growth rate in skeletal muscle tissue of Landrace weanling pig. Two subtracted cDNA populations were generated from mus- culus longissimus muscle tissues of selected pigs with extreme expected breeding values at the age of 100 kg. Three upregu- lated genes (EEF1A2, TSG101 and TTN) and six downregulated genes (ATP5B, ATP5C1, COQ3, HADHA, MYH1 and MYH7) in pig with genetic propensity for higher growth rate were identified by sequence analysis of 12 differentially expressed clones selected by differential screening following the generation of the subtracted cDNA population. Real-time PCR analysis con- firmed difference in expression profiles of the identified genes in musculus longissimus muscle tissues between the two Lan- drace weanling pig groups with divergent genetic propensity for growth rate. Further, differential expression of the identified genes except for the TTN was validated by Western blot analysis. Additionally, the eight genes other than the ATP5C1 co- localized with the same chromosomal positions as QTLs that have been previously identified for growth rate traits. Finally, the changes of expression predicted from gene function suggested association of upregulation of expression of the EEF1A2, TSG101 and TTN genes and downregulation of the ATP5B, ATP5C1, COQ3, HADHA, MYH1 and MYH7 gene expression with increased growth rate. The identified genes will provide an important insight in understanding the molecular mechanism underlying growth rate in Landrace pig breed. [Komatsu Y., Sukegawa Y., Yamashita M., Katsuda N., Tong B., Ohta T., Kose H. and Yamada T. 2016 Identification of genes showing differential expression profile associated with growth rate in skeletal muscle tissue of Landrace weanling pig. J. Genet. 95, xx–xx] Introduction Comparative analyses of expression profiles are useful in identifying the molecular differences between divergent Pig breeding programmes have focussed on growth rate and muscle phenotypes (Wimmers et al. 2004). Transcriptome leanness as major objectives in selection (Hammond and analysis of skeletal muscle tissue may be particularly valu- Leitch 1998). Selection for increased growth rate in pig has able for such studies. In recent years, the transcriptome stud- been used to develop lines with increased body and mus- ies performed in pig skeletal muscle tissue were based on the cle weight in commercial breeds (e.g. Landrace and Large analysis of difference in gene expression patterns between White) (Fredeen and Mikami 1986). Because of the impor- samples from different breeds with divergent growth tance of the growth rate on the line development of commercial rate profiles (e.g. commercial breed with higher growth breeds, there is great interest in gaining a better understand- rate versus indigenous breed with lower growth rate) (Lin ing of the networks of expressed genes and the biological and Hsu 2005; Cagnazzo et al. 2006;Murániet al. 2007; pathways controlling the growth rate in skeletal muscle tis- Tang et al. 2007;Xuet al. 2009;Kimet al. 2010; Davoli sue, which exhibits physiological changes in pig selected for et al. 2011). This experimental design is suited to under- increased growth rate (Casas-Carrillo et al. 1997). stand the between-breed component of the skeletal mus- cle transcriptome associated with the growth rate. However, ∗ the molecular network might differ among different genetic For correspondence. E-mail: [email protected]. Yuuta Komatsu, Shin Sukegawa and Mai Yamashita contributed equally to backgrounds (Cánovas et al. 2012). Therefore, the informa- this work. tion on this between-breed component seems not to result Keywords. expression profile; growth rate; Landrace pig; skeletal muscle; suppression subtractive hybridization. Journal of Genetics Yuuta Komatsu et al. in an accurate knowledge of the within-breed component of Differential screening the skeletal muscle transcriptome in a commercial pig, which To check unique expression of the subtracted cDNA may be useful for the line development in commercial breeds due to selection. sequences, all transcript clones were subjected to differential The molecular events resulting in increased growth rate screening. Subtracted cDNA inserts were amplified by PCR are likely to occur in part around the weanling age when fat- using plasmid DNA as a template and nested PCR primer tening is started (Gross 1986). Thus, in this study, we have 1 and nested PCR primer 2 as primer, with Advantage 2 investigated difference in skeletal muscle gene expression polymerase mix (Clontech). PCR products were denatured profiles between two groups of Landrace weanling pigs with in 0.5 mol/L NaOH, transferred onto two identical Hybond divergent genetic propensity for growth rate. N membranes (GE Healthcare, Little Chalfont, UK) and UV cross-linked. Macroarrays were subsequently hybridized with the different probes (cDNA obtained from high-growth group and low-growth group). Probe labelling was per- Materials and methods formed using DIG high prime DNA labelling and detection Samples starter kit (Roche, Mannheim, Germany) as described by the manufacturer. The screening was performed visually. In this study, we used six weanling males of Landrace (three animals with genetic propensity for extremely high growth rate and three animals with extremely low growth rate, Analysis of differentially expressed clones named as high-growth group and low-growth group, respec- tively). These animals were selected from the extreme tails of The insert DNA containing checked differentially expressed the distribution, based on the expected breeding value for the cDNA sequence was sequenced using the 3730xl DNA ana- age at 100 kg calculated as (sire’s predicted breeding value + lyzer following standard Big Dye protocols (Applied Biosys- − − − dam’s predicted breeding value)/2 ( 5.03, 5.50 and 6.50 tems, Foster City, USA). The basic local alignment search for the high-growth group and 4.10, 4.71 and 5.91 for the tool (BLAST) (Altschul et al. 1990) was used to identify low-growth group), in a conventional commercial Landrace = sequence homology of each differentially expressed cDNA population (n 140). This population has been maintained sequence with publicly available pig genome data in dif- by mating between Landrace pig breed, consisting of a diver- ferent partially overlapping databases: GenBank Nucleotide sity of lines. There was no bias for a specific line in these nr/nt, GenBank Genome Pig (Sscrofa10.2 release, Septem- animals. Animals had been reared in the same conditions, ber 2011), and Ensembl Pig Genome (Sscrofa10.2). The and musculus longissimus muscle tissues were taken from these animals at the age of 23 days. This study conformed sequence homology was considered ‘true’ only if: (i) e-value − to the guidelines for animal experimentation of the Gradu- was higher than 20, (ii) more than 80% of the sequence ate School of Science and Technology, Niigata University length was homologous to the target and (iii) the homo- (Niigata, Japan). logy percentage was higher than 90% for pig sequences and higher than 75% for nonpig sequences. To identify the chromosomal position of each differentially expressed Generation of subtracted cDNA population cDNA sequence in this study, the GenBank pig genome database (Sscrofa10.2) was searched to detect the match We extracted mRNA from the muscle tissue sample using Poly(A) Purist kit (Ambion, Austin, USA). Both quality and with sequences already integrated in the genome assembly. quantity were evaluated using the 2100 BioAnalyzer (Agi- The map information of the matched sequences obtained lent, Santa Clara, USA). Two mRNA pools, each was created was integrated into QTL map available at Pig QTL database from three animals of the high-growth group or low-growth (Hu and Reecy 2007) in NAGRP Pig Genome Coordination group. Suppression subtractive hybridization (Diatchenko Program (http://www.animalgenome.org/pig/). et al. 1996) was performed using a PCR Select cDNA Subtraction kit (Clontech, Mountain View, USA) and Advan- tage 2 Polymerase Mix (Clontech) as described by the man- Real-time PCR analysis ufacturer. Forward and reverse subtractions were carried out Total RNA was extracted from the muscle tissue using using 2 μg of the pooled mRNA samples. A control exper- RNeasy fibrous tissue kit (Qiagen) according to the man- iment was included following the manufacturer’s instruc- ufacturer’s instructions. Total RNA (2 μg) was reverse- tions. The two subtracted cDNA populations were cloned into the pCR2.1-TOPO vector of the TOPO TA Cloning Kit transcribed into cDNA using iScript advanced cDNA syn- (Invitrogen, Carlsbad, USA) and transformed into E. coli thesis kit for RT-qPCR (Bio-Rad, Hercules, USA) accord- TOP10 (Invitrogen). Following overnight growth on selec- ing to the manufacturer’s instructions. Real-time
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