Mitochondrial DNA T7719G in tRNA-Lys gene affects litter size in Small-tailed Han sheep
- Xiaoyong Chen†1, 2,
- Dan Wang†1,
- Hai Xiang1,
- Weitao Dun2,
- Dave O. H. Brahi1,
- Tao Yin1 and
- Xingbo Zhao1Email author
© The Author(s). 2017
Received: 7 September 2016
Accepted: 17 March 2017
Published: 1 April 2017
In farm animals, mitochondrial DNA (mtDNA) effect on economic performance remains hot-topic for breeding and genetic selection. Here, 53 maternal lineages of Small-tailed Han sheep were used to investigate the association of mitochondrial DNA variations and the lambing litter size.
Sequence sweeping of the mitochondrial coding regions discovered 31 non-synonymous mutations, and the association study revealed that T7719G in mtDNA tRNA-Lys gene was associated with litter size (P < 0.05), manifesting 0.29 lambs per litter between the G and T carriers. Furthermore, using the mixed linear model, we assayed the potential association of the ovine litter size and haplogroups and multiple-level mtDNA haplotypes, including general haplotypes, assembled haplotypes of electron transport chain contained sequences (H-ETC), mitochondrial respiratory complex contained sequences (H-MRC) and mitochondrial genes (H-gene, including polypeptide-coding genes, rRNA genes and tRNA genes). The strategy for assembled mitochondrial haplotypes was proposed for the first time in mtDNA association analyses on economic traits, although none of the significant relations could be concluded (P > 0.05). In addition, the nuclear major gene BMPR1B was significantly correlated with litter size in the flock (P < 0.05), however, did not interact with mtDNA T7719G mutation (P > 0.05).
Our results highlight mutations of ovine mitochondrial coding genes, suggesting T7719G in tRNA-Lys gene be a potentially useful marker for selection of sheep litter size.
KeywordsAssociation Haplotype Mitochondria Non-synonymous mutation Reproduction Sheep
Mitochondria are responsible for ATP production in the electron transport chain (ETC) in cells. The ETC consists of five mitochondrial respiratory complexes (MRCs), of which complex I, complex III, complex IV and complex V are constructed by both mitochondrial and nuclear encoded proteins, and complex II is entirely encoded by nuclear genes. Mitochondrial genome codes 13 polypeptides, 2 rRNAs and 22 tRNAs . Since the first report of mitochondrial DNA (mtDNA) effect on milk production traits in dairy cattle , mtDNA effects on various economic traits have been widely studied in livestock, including pigs [3, 4], dairy cattle [5, 6], beef cattle [7, 8], sheep [9, 10] and chickens [11, 12]. Litter size is one of the vital economic traits for animal breeding and production, which has been studied for decades. For sheep, the nuclear gene, BMPR1B was identified as one of causative genes for sheep prolificacy , and has been widely used in sheep breeding. For the mtDNA effect on litter size, researchers reported the association with ewe litter size among haplogroups in an Afec-Assaf flock, but did not found the interaction with BMPR1B effect . However, previous studies reported poor mtDNA effects [14–17], which made it necessary to uncover the genetic contribution of mtDNA for ewe litter size. In this study, Small-tailed Han sheep, a prolific breed of China, were used to investigate the association of litter size with mtDNA coding genes, in which non-synonymous mutations were considered as possible functional SNPs. Besides the non-synonymous mutation and haplogroup, assembled haplotypes of ETC-contained mtDNA sequences, MRC-contained mtDNA sequences, and mitochondrial genes were analyzed, respectively. The strategy for assembled mitochondrial haplotypes was proposed for the first time in association analyses. In addition, BMPR1B effect and interaction with mtDNA mutations were analyzed.
In total, 117 lambing Small-tailed Han sheep from 53 maternal lineages (families divided by female ancestors) of the same flock were performed blood sample collection, and recorded one or more times of litter size (Additional file 1: Table S1). All sheep were kept indoors year-round, and fed with mixed silage and hay to meet their nutritional requirements. Litter size (number of lambs born) was recorded, and full pedigree information was collected for all animals as reproductive management of the flock where ewes were mated with the selected rams after spontaneous estrus. All the ewes were genotyped for the BMPR1B at >2-month age.
Genotyping of BMPR1B and sequencing of mitochondrial coding genes
Genomic DNA was extracted using the standard phenol/chloroform method . The BMPR1B was genotyped using PCR-RFLP assay with the Ava II restriction enzyme . Mitochondrial complete coding sequences were amplified by 17 primer pairs , and PCR products were sequenced in the Sanger method.
Haplotype and haplogroup constitution
All non-synonymous mutations were used to constitute the haplotype and haplogroup. Considering the possible action for mitochondrial function, haplotypes were furthermore assembled by mtDNA sequence of ETC, MRCs and genes respectively. Haplotypes were determined by the online software FaBox , and haplogroups were constituted based on network analysis by Network 188.8.131.52 .
In the model, the effects of lambing year-season (ys), parity number (parity), service ram (ram), BMPR1B genotype (BMPR1B), mtDNA mutations (mutations, including the effects of mtDNA non-synonymous mutations, haplotypes and haplogroups), the interaction between BMPR1B and mtDNA mutations (BMPR1B × mutations), the polygenic effect (ID), the permanent environmental effect (EP), and the random residual (e) were included. The response variable was the ewe litter size (ls). Each cell of these effects contained observations. The polygenic effect corrected the genetic background by the additive genetic relationship matrix, i.e. the pedigree information. The permanent environmental effect dealed with the repeated measurement data.
Inference about the interaction effect was made. If non-significant, that effect was dropped from the model and inference was made about the main effects. If significant, the cell means for the interaction became of interest.
When a set of statistical inferences were simultaneously considered, multiple comparisons were conducted by the false discovery rate (FDR) in the R project (R version 3.2.5) .
Mutations in mitochondrial coding genes
Non-synonymous mutations in mitochondrial coding genes and corresponding effects on litter size
Amid acid substitution
UCA → ACA
S → T
AUA → ACA
M → T
CCC → CAC
P → H
AAC → AGC
N → S
GGA → GCA
G → A
AUA → GUA
M → V
CCU → UCU
P → S
GGU → AGU
G → S
GAC → AAC
D → N
GUU → AUU
V → I
GGC → GCC
G → A
GCA → ACA
A → T
CUC → UUC
L → F
UAC → CAC
Y → H
CAU → UAU
H → Y
CAU → UAU
H → Y
UCA → CCA
S → P
UUC → CUC
F → L
AUA → GUA
M → V
For BMPR1B gene, 117 ewes were genotyped, including ++ genotype of 18 ewes, B+ genotype of 87 ewes and BB genotype of 12 ewes.
Effects of mitochondrial haplotype and haplogroup on ovine litter size
Effects of missense mutations and haplotypes in mitochondrial protein coding genes on ovine litter size
Haplotypes constituted by mitochondrial non-synonymous mutations in multiple levels and significant effects on litter size
Contained mutation number
ND1, ND2, ND4L, ND4, ND5, ND6, COII, COIII, ATP6, 12SrRNA, 16SrRNA, tRNA-Tyr, tRNA-Lys, tRNA-His, tRNA-Ser
ND1, ND2, ND4L, ND4, ND5, ND6, COII, COIII, ATP6
ND1, ND2, ND4L, ND4, ND5, ND6
Effects of BMPR1B on litter size
Litter size (Means)1
Effects of mutations and haplotypes in mitochondrial rRNA genes on ovine litter size
The 3 mutations in 12SrRNA sorted the sheep flock into 4 haplotypes (H-12SrRNA), and the 5 mutations in 16SrRNA constituted 15 haplotypes (H-16SrRNA) (Table 2 and Additional file 1: Table S7). Association analyses revealed that neither mutation nor haplotype in rRNA genes affected litter size (P > 0.05) (Tables 1 and 2). The BMPR1B genotype was remarkably associated with litter size (P < 0.05) (Table 3), however, was inconspicuously correlated to rRNA mutations (P > 0.05).
Effects of mutations in mitochondrial tRNA genes on ovine litter size
There were 4 mutations in tRNA genes, and only one variation was observed in each of them (Table 1), therefore no haplotype was constituted. Notably, T7719G in tRNA-Lys affected litter size by 0.29 lambs per litter between the G (1.79) and T (1.50) carriers (P < 0.05). Even though the BMPR1B was in prominent correlation with litter size (P < 0.05) (Table 3), there was no interaction with T7719G (P > 0.05) (Additional file 1: Table S8).
In human diseases, mutations in mitochondrial coding genes led to changes of oxidative phosphorylation enzyme complexes . It is rational to image the possible effects of mtDNA on livestock traits. In this study, the Small-tailed Han sheep were used to explore the correlation of mtDNA and ewe litter size.
In order to investigate the overall mtDNA effects, a novel strategy of assembling mitochondrial haplotypes based on biology functions is proposed. Extensively, there are four levels of mitochondrial haplotypes for mitochondrial biological functions. The general haplotype is assembled by all non-synonymous mutations to reflect the integrated characteristics of mtDNA coding regions. Subsequently, the ETC based haplotype (H-ETC) represents the general feature of all ETC-contained mtDNA sequences. The MRC based haplotype infers the integrated signal of particular MRC-contained mtDNA sequences. H-MRC includes four types, i.e. MRC-I, MRC-III, MRC-IV and MRC-V. The last is the gene level, which indicates the information of a particular gene-contained mtDNA sequence.
In the mixed linear model, unnecessary factors interfere with the estimation of interested factors, as the degree of freedom is wasted. Therefore, based on the thoughts of multiple models  and variable selection in a mixed linear model , a two-step method of environmental variable selection was put forward in the association analyses, in which non-significant environmental factors were ignored to improve accuracy of genetic estimation. In this study, the environmental factors included lambing month, parity of dam, and service ram, and the genetic factors, which we were interested in, included BMPR1B genotype, mtDNA mutations and their interaction. We started a full model (Model I) with all the environmental and genetic factors to test if environmental factors were noises. Results revealed that the effects of parity of dam and service ram were not significant on litter size. Subsequently, these two factors were excluded in the aims to construct an optimized model (Model II), which was used to test the significance of genetic factors, especially the effects of mtDNA mutations on litter size.
As a conclusion, we summarize the research procedures for mtDNA effects. Firstly, sweeping mitochondrial variations on maternal lineages; secondly, constituting the haplotype, haplogroup and assembled haplotypes; lastly, analyzing the association between mtDNA mutations (individual mutations, haplotypes and haplogroups) and interested traits. The present study discovered the mtDNA T7719G was linked with ewe litter size. For traits of low heritability, the marker assisted selection could increase the accuracy of breeding and selection. For the further study, the mtDNA T7719G should be put into post-association validation, and may become a genetic marker in the sheep breeding programs.
Electron transport chain
Mitochondrial respiratory complex
The haplotype constituted by all non-synonymous mutations in mitochondrial coding sequences
The haplogroup classified by H
The assembled haplotypes of mtDNA electron transport chain contained sequences
The assembled haplotypes of mtDNA mitochondrial respiratory complex contained sequences, including H-MRCI, H-MRCIII, H-MRCIV, and H-MRCV, corresponding to complex I, III, IV and V, respectively
- H-gene :
The assembled haplotypes of mitochondrial genes, including polypeptide-coding genes, rRNA genes and tRNA genes
The authors thank J. C. Jiang (China Agricultural University) for data analysis. The authors also thank Dr. J. M. Zhong and Ms. D. Miller (Auburn University) for language editing.
The research was supported by the Project of Science and Technology of Hebei Province (15226308D) and the National Key Basic Research Program of China (2014CB138500). The former collected the sample and fed the experimental sheep, and the latter designed the study, performed the experiments, analyzed the data, interpreted the data, and drafted the manuscript.
Availability of data and materials
All data generated or analysed during this study are included in this published article and its supplementary information files.
XZ designed the study. WD and XC collected the sample and fed the experimental sheep. the XC, HX, DB and TY performed the experiments. DW, HX and XC analyzed the data. DW, HX, XC and XZ interpreted the data and drafted the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
All experimental sheep were maintained under the standard farm management practices according to the guidelines of the experimental animal management of China Agricultural University (CAU), and the study was approved by the Experimental Animal Care and Use Committee of CAU. Blood samples were collected followed the guideline of Animal Ethical Committee of China.
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