Comparative analysis of the blood transcriptomes between wolves and dogs. Summary: Dogs were domesticated by human and originated from wolves. Their evolutionary relationships have attracted much scientific interest due to their genetic affinity but different habitats. To identify the differences between dogs and wolves associated with domestication, we analysed the blood transcriptomes of wolves and dogs by RNA- Seq. We obtained a total of 30.87 Gb of raw reads from two dogs and three wolves using RNA-Seq technology. Comparisons of the wolf and dog transcriptomes revealed 524 genes differentially expressed genes between them. We found that some genes related to immune function (DCK, ICAM4, GAPDH and BSG) and aerobic capacity (HBA1, HBA2 and HBB) were more highly expressed in the wolf. Six differentially expressed genes related to the innate immune response (CCL23, TRIM10, DUSP10, RAB27A, CLEC5A and GCH1) were found in the wolf by a Gene Ontology enrichment analysis. Immune system development was also enriched only in the wolf group. The ALAS2, HMBS and FECH genes, shown to be enriched by the Kyoto Encyclopedia of Genes and Genomes analysis, were associated with the higher aerobic capacity and hypoxia endurance of the wolf. The results suggest that the wolf might have greater resistance to pathogens, hypoxia endurance and aerobic capacity than dogs do. Canis lupus; Canis lupus familiaris; differentially expressed gene; RNA-Seq; transcriptome Introduction Canines are widely distributed carnivorous mammals that closely associate with human beings. They have received wide attention from investigators in the fields of ecology and molecular biology. Both the wolf (Canis lupus) and the dog (Canis lupus familiaris) are classified in the order Carnivora, family Canidae, genus Canis. Because of their similar appearance and different habitats, their evolutionary relationships have attracted considerable interest. Various studies have shown that the dog was domesticated from the wolf (Savolainen et al. [42]), and there have been remarkable changes in the morphology, habits and diet in the dog in the process of domestication. By comparing data on the population genetics of Chinese native dogs and grey wolves, investigators have found that genes within highly differentiated regions are expressed preferentially in the dog's brain, particularly in the prefrontal cortex, a specific region responsible for complex cognitive behaviours (Li et al. [24]). Wang et al. ([48]) found that parallel evolution in domestic dogs and human is very apparent in genes related to metabolism, digestion, neurological processes and cancer. The wolf is one of the most widely distributed terrestrial mammals on earth (Young & Goldman [ 52]; Wilson [49]), and its wide range and complex habitat types shows that it has great ability to adapt for survival in various environments. Previous research has shown that wild wolf populations are rarely infected by the rabies virus (Mech [32]; Busch [6]). Therefore, we speculate that wolves and dogs might have differences in some aspects of immunity. With the development of next-generation sequencing, transcriptome studies have entered a new era. Compared with hybridization technology, RNA-Seq technology has several advantages. First, due to its simplicity, with no need to design probes in advance, RNA-Seq technology can not only reduce the number of experiments required but also detect unknown genes and identify novel transcripts. Second, because of the direct sequencing of the transcripts, accurate to a single nucleotide, RNA-Seq has few problems with background noise. Third, RNA-Seq allows for the capturing of lowly expressed transcripts as long as the sequencing depth is high enough (Nagalakshmi et al. [36]; Wang et al. [46]). Some studies of the dog transcriptome have been carried out previously. By analysing the hypothalamus and cerebral cortex transcriptomes of several beagles, Roy et al. ([41]) provided some examples of novel genes that are related to domestication, including SMOC2, PRNP and ADCY8. However, transcriptome research has not been reported for the wolf, primarily because of the difficulty in collecting samples. In China, wild wolf populations are rare, and tissues other than blood is much more difficult to obtain for transcriptome research. Thus, for this study we proposed using blood for transcriptome experiments to guide the development of conservation biology. Because of increased concern for the protection of wild animals, peripheral blood is now widely used for various markers to monitor individuals (Mohr & Liew [34]). Blood is an important part of the immune system, and it is the first line of the immune defence system (Liew et al. [25]). The main blood components involved in the immune response include red blood cells, white blood cells, platelets and some active immune substances. The blood also contains many immune regulatory factors, including microRNAs, which can bind to the 3'-untranslated regions of mRNA and regulate gene expression. Blood transcriptome profiling has been accomplished in several mammalian species, including human (Luo et al. [28]; Zaatar et al. [54]), bear (Miller et al. [33]), cattle (Demasius et al. [8]), pig (Mach et al. [30]) and giant panda (Du et al. [9]). In this study, we analysed the dog and wolf blood transcriptomes using high-throughput sequencing technologies to generate the data and screen for differentially expressed genes (DEGS). We also tried to find differences in the blood immune system between wolves and dogs. Materials and methods Sample collection and storage Blood samples were collected from two dogs and three wolves during routine examinations. Among the three wolves, two individuals (a male and a female, Wolf_1 and Wolf_2) were obtained from the Luobulingka Zoo of Tibet and one individual (a female, Wolf 3) was raised in Dalailake National Nature Reserve of Inner Mongolia. Two dogs (a male and a female, Dog_1 and Dog_2) were Chinese indigenous dogs living in Zhengzhou, Henan province, in the central region of China. All of these individuals were unrelated. The experimental methods were approved by the Qufu Normal University Institutional Animal Care and Use Committee (Permit Number: QFNU2015-001). Blood samples were immediately stored in RNAprotect Animal Blood Tubes (QIAGEN) and frozen at -80 °C pending further processing. RNA extraction, library preparation and sequencing Total RNA was extracted using an RNeasy Protect Animal Blood Kit (QIAGEN), following the manufacturer's protocol, and treated with RNase-free DNase I water. RNA degradation and contamination was monitored on 1% agarose gels. RNA purity was checked using a Nanodrop 2000c (Thermo Scientific). RNA integrity was assessed using an RNA 6000 Nano Assay Kit (Agilent Technologies) with Bioanalyzer 2100 (Agilent Technologies) after checking RNA purity and concentration. A total of 1.5 μl of RNA per sample was used as the input material for RNA sample preparations. The RNA integrity numbers of the five samples were 8.9, 8.9, 8.8, 8.9 and 8.8 respectively. The transcriptome libraries were generated using the Illumina Tru-Seq RAN Low Sample (LS) protocol. First, oligo (dT) magnetic beads were used to purify the mRNA from total RNA. An elevated temperature in an Illumina proprietary fragmentation buffer was used to fragment the mRNAs. Random oligonucleotides and SuperScript II were used to synthesize first-strand cDNA and generate second- strand cDNA. Sequencing adapters were ligated to the fragments, and the products were enriched by PCR amplification. Finally, an AMPure XP system was used to purify the products, which were quantified using an Agilent High-sensitivity DNA Assay Kit on an Agilent Bioanalyzer 2100 system.

Comparative analysis of the blood transcriptomes between wolves and dogs. Summary: Dogs were domesticated by human and originated from wolves. Their evolutionary relationships have attracted much scientific interest due to their genetic affinity but different habitats. To identify the differences between dogs and wolves associated with domestication, we analysed the blood transcriptomes of wolves and dogs by RNA- Seq. We obtained a total of 30.87 Gb of raw reads from two dogs and three wolves using RNA-Seq technology. Comparisons of the wolf and dog transcriptomes revealed 524 genes differentially expressed genes between them. We found that some genes related to immune function (DCK, ICAM4, GAPDH and BSG) and aerobic capacity (HBA1, HBA2 and HBB) were more highly expressed in the wolf. Six differentially expressed genes related to the innate immune response (CCL23, TRIM10, DUSP10, RAB27A, CLEC5A and GCH1) were found in the wolf by a Gene Ontology enrichment analysis. Immune system development was also enriched only in the wolf group. The ALAS2, HMBS and FECH genes, shown to be enriched by the Kyoto Encyclopedia of Genes and Genomes analysis, were associated with the higher aerobic capacity and hypoxia endurance of the wolf. The results suggest that the wolf might have greater resistance to pathogens, hypoxia endurance and aerobic capacity than dogs do. Canis lupus; Canis lupus familiaris; differentially expressed gene; RNA-Seq; transcriptome Introduction Canines are widely distributed carnivorous mammals that closely associate with human beings. They have received wide attention from investigators in the fields of ecology and molecular biology. Both the wolf (Canis lupus) and the dog (Canis lupus familiaris) are classified in the order Carnivora, family Canidae, genus Canis. Because of their similar appearance and different habitats, their evolutionary relationships have attracted considerable interest. Various studies have shown that the dog was domesticated from the wolf (Savolainen et al. [42]), and there have been remarkable changes in the morphology, habits and diet in the dog in the process of domestication. By comparing data on the population genetics of Chinese native dogs and grey wolves, investigators have found that genes within highly differentiated regions are expressed preferentially in the dog's brain, particularly in the prefrontal cortex, a specific region responsible for complex cognitive behaviours (Li et al. [24]). Wang et al. ([48]) found that parallel evolution in domestic dogs and human is very apparent in genes related to metabolism, digestion, neurological processes and cancer. The wolf is one of the most widely distributed terrestrial mammals on earth (Young & Goldman [ 52]; Wilson [49]), and its wide range and complex habitat types shows that it has great ability to adapt for survival in various environments. Previous research has shown that wild wolf populations are rarely infected by the rabies virus (Mech [32]; Busch [6]). Therefore, we speculate that wolves and dogs might have differences in some aspects of immunity. With the development of next-generation sequencing, transcriptome studies have entered a new era. Compared with hybridization technology, RNA-Seq technology has several advantages. First, due to its simplicity, with no need to design probes in advance, RNA-Seq technology can not only reduce the number of experiments required but also detect unknown genes and identify novel transcripts. Second, because of the direct sequencing of the transcripts, accurate to a single nucleotide, RNA-Seq has few problems with background noise. Third, RNA-Seq allows for the capturing of lowly expressed transcripts as long as the sequencing depth is high enough (Nagalakshmi et al. [36]; Wang et al. [46]). Some studies of the dog transcriptome have been carried out previously. By analysing the hypothalamus and cerebral cortex transcriptomes of several beagles, Roy et al. ([41]) provided some examples of novel genes that are related to domestication, including SMOC2, PRNP and ADCY8. However, transcriptome research has not been reported for the wolf, primarily because of the difficulty in collecting samples. In China, wild wolf populations are rare, and tissues other than blood is much more difficult to obtain for transcriptome research. Thus, for this study we proposed using blood for transcriptome experiments to guide the development of conservation biology. Because of increased concern for the protection of wild animals, peripheral blood is now widely used for various markers to monitor individuals (Mohr & Liew [34]). Blood is an important part of the immune system, and it is the first line of the immune defence system (Liew et al. [25]). The main blood components involved in the immune response include red blood cells, white blood cells, platelets and some active immune substances. The blood also contains many immune regulatory factors, including microRNAs, which can bind to the 3'-untranslated regions of mRNA and regulate gene expression. Blood transcriptome profiling has been accomplished in several mammalian species, including human (Luo et al. [28]; Zaatar et al. [54]), bear (Miller et al. [33]), cattle (Demasius et al. [8]), pig (Mach et al. [30]) and giant panda (Du et al. [9]). In this study, we analysed the dog and wolf blood transcriptomes using high-throughput sequencing technologies to generate the data and screen for differentially expressed genes (DEGS). We also tried to find differences in the blood immune system between wolves and dogs. Materials and methods Sample collection and storage Blood samples were collected from two dogs and three wolves during routine examinations. Among the three wolves, two individuals (a male and a female, Wolf_1 and Wolf_2) were obtained from the Luobulingka Zoo of Tibet and one individual (a female, Wolf 3) was raised in Dalailake National Nature Reserve of Inner Mongolia. Two dogs (a male and a female, Dog_1 and Dog_2) were Chinese indigenous dogs living in Zhengzhou, Henan province, in the central region of China. All of these individuals were unrelated. The experimental methods were approved by the Qufu Normal University Institutional Animal Care and Use Committee (Permit Number: QFNU2015-001). Blood samples were immediately stored in RNAprotect Animal Blood Tubes (QIAGEN) and frozen at -80 °C pending further processing. RNA extraction, library preparation and sequencing Total RNA was extracted using an RNeasy Protect Animal Blood Kit (QIAGEN), following the manufacturer's protocol, and treated with RNase-free DNase I water. RNA degradation and contamination was monitored on 1% agarose gels. RNA purity was checked using a Nanodrop 2000c (Thermo Scientific). RNA integrity was assessed using an RNA 6000 Nano Assay Kit (Agilent Technologies) with Bioanalyzer 2100 (Agilent Technologies) after checking RNA purity and concentration. A total of 1.5 μl of RNA per sample was used as the input material for RNA sample preparations. The RNA integrity numbers of the five samples were 8.9, 8.9, 8.8, 8.9 and 8.8 respectively. The transcriptome libraries were generated using the Illumina Tru-Seq RAN Low Sample (LS) protocol. First, oligo (dT) magnetic beads were used to purify the mRNA from total RNA. An elevated temperature in an Illumina proprietary fragmentation buffer was used to fragment the mRNAs. Random oligonucleotides and SuperScript II were used to synthesize first-strand cDNA and generate second- strand cDNA. Sequencing adapters were ligated to the fragments, and the products were enriched by PCR amplification. Finally, an AMPure XP system was used to purify the products, which were quantified using an Agilent High-sensitivity DNA Assay Kit on an Agilent Bioanalyzer 2100 system.

Human Heredity: Principles and Issues (MindTap Course List)

11th Edition

ISBN:9781305251052

Author:Michael Cummings

Publisher:Michael Cummings

Chapter13: An Introduction To Genetic Technology

Section: Chapter Questions

Problem 20QP: Analyzing Cloned Sequences A base change (A to T) is the mutational event that created the mutant...

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