The expression of genes related to lactogenesis in peripheral blood mononuclear cells (PBMCs) are less understood. To understand the biology of milk production in early lactating non pregnant cows in the blood, the transcriptome from PBMCs was analysed by RNA sequencing. We are able to generate a total of ~ 62.5 Gbp RNA-Seq data files (~ 24 million to ~ 35 million paired-end reads per sample) from high yielder (3) and low yielder (3) of Deoni and Hallikar breeds of Bos indicus (12 samples). The blood sample was collected from twelve early lactating non pregnant cows, the transcriptome from PBMCs was analysed by RNA sequencing. The QC passed reads were aligned against Bos taurus reference assemblies by using HiSAT2 alignment tool. An average of 79.25% and 74.28% reads were aligned in Deoni and Hallikar breeds, respectively. Amongst 85 genes were significantly differentially regulated between low yielder and high yielder of Deoni cows. On contrary, in Hallikar 492 genes were significantly differentially regulated between groups. Transcripts related to CSN gene family (CSN1S1, CSN1S2, CSN2 and CSN3) and FASN were significantly expressed in High yielders of Deoni cows. However, in low yielding Deoni cows and all Hallikar cows, these CSN gene family and FASN gene expression levels were poor or not at all expressed. However, the ATP2B4 (PMCA - Plasma membrane Ca2 + ATPase) gene is expressed only in Hallikar cows. Majority of the genes in lactating Deoni and Hallikar cows were linked to milk protein composition especially caseins, milk fat, lactose synthesis in milk and apoptosis activity in mammary glands. Our findings provide new insights into the understanding of molecular mechanism of differential milk yield among Bos indicus breeds. We believe that our study would be helpful in lactation biology and genetic selection.
Article
Transcriptomic analysis of PBMCs reveals COX3, BCL2, ATP2B4, B4GALT1, LACTB, FASN and CSN family genes regulating milk performance in lactating Bos indicus cows of Southern India
https://doi.org/10.21203/rs.3.rs-1726110/v1
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Indigenous cattle are the most important and versatile species of the Indian subcontinent. They provide milk, manure, draught power and livelihood security to the farmers. India ranks 1st in the world’s cattle population with 193.46 million, accounting for 63.65% of the total bovine population and 36.04% of the total population of livestock1. Furthermore, India is the world's largest milk producer, accounting for 187.75 million tons in the year 2019 from native cattle (10%), non-descript cattle (11%), crossbred cattle (26%), exotic cattle (1%), buffalo (49%) and goat (3%)1. Indian subcontinent is sanctified with variety of animal genetic resources, amongst them cattle genetic resource is a very important reserve where wide variety of cattle are existed. Amongst them Deoni and Hallikar are most important cattle breeds of Karnataka state. Deoni is an important dual-purpose breed found in Bidar and Gulbarga districts in Karnataka state, Latur and surrounding areas in Maharashtra state and Sangareddy, Vikarabad and Medak districts of Telangana state. The lactation yield of these animals ranged from 800–1400 kg 2. As a medium yielder, this breed has been adapted to the extremely hot conditions of its home tract. Similarly, Hallikar cattle is also an important draught purpose breed of Southern Karnataka and also said to be the progenitor of all south Indian breeds3. The lactation yield of this breed is ~ 500 kg. Furthermore, Hallikar cattle is mainly spread over plain lands of southern Karnataka (old Mysore region)4.
Lactogenesis is the complex process of milk secretion which involves biological alterations in the body, from mammary tissue development to milk secretion. The mammary gland anatomy and its physiological activity such as epithelial cell number, milk production potential and mammary genetics were differ between various species and breeds1. Interestingly, 1ml of milk synthesizing requires 400-500ml of blood flow to the mammary gland. Therefore, large quantity of blood flow is required for milk secretion. Milk synthesis and secretion was highly influenced by large number of genes present in the milk somatic cell as well as blood2,3. Sequencing of transcriptome offers comprehensive information of the expression of genes in a given set of tissue or organ. Somatic cell transcriptome analysis of Sahiwal cattle under different lactation stages revealed milk casein related genes and whey protein genes were differentially regulated between lactation stages4. Although, this study revealed the differential expression genes on lactation stages of Bos indicus, genes involving milk production were still not understood. In Chinese Holstein Friesian cows analysis of transcriptome reveals 100 genes were differentially regulating between high yielders and low yielders3. Further, blood is the complex of various cells and completely reflects health status of cows. The lactation mechanisms are controlled by various organs and tissues such as, liver, adipose tissue, mammary gland and blood. Hence, transcriptomes of blood is crucial for milk production potential performance and it may reflect the milk production potential capacity of cows by direct and comprehensive manner. By using microarray technology gene expression profiling has been performed in the blood related to the gene merit for milk productive traits5. Transcription profile of porcine peripheral blood mononuclear cells was generated between divergent humoral immune response and lean growth performance6. To complete understand the impact of blood transcriptome profiles on milk yield, comprehensive cataloguing of gene expression change within high yielders and low yielders is required. The aim of this study is to compare gene expression profiles in bovine whole blood of high and low milk yield cows, and to investigate potential molecular biomarkers in the blood transcriptome that relate to the productive potential of lactating cows using RNA-seq techniques.
Quality and Quantity estimation of isolated RNA
Total RNA was isolated from the PBMC pellet as per the manufacturer instruction (RNA Easy Minikit, Qiagen Netherlands). The integrity of the total RNA was analysed by using Qubit 3.0 Fluorometer and Tape station and shown in Figure 1.
RNA Sequencing Data Summary
The results indicated that many genes were expressed in the mRNA of peripheral blood mononuclear cells (PBMCs). We acquired a total of ~62.5 Gbp RNA-Seq data files (~24 million to ~35 million paired-end reads per sample) from 12 samples (six Deoni cows and six Hallikar cows). Approximately 63 million to 111 million reads were generated in both High and Low yielders of Deoni cows and 24 million to 38 million reads were generated in both High and Low yielders of Hallikar cows. Additionally, an average of 79.25% and 74.28 % reads were aligned in Deoni and Hallikar breeds, respectively to UMD3.1.1 (Bos taurus) bovine reference genome7 using HiSAT2 alignment tool8. The alignment rate is ranged from 71.42 % to 93.78 % (Table 1).
Table 1. RNA-Seq alignment report of Deoni and Hallikar cows
|
Sample name |
Total reads |
Total mapped |
unmapped reads |
Uniquely mapped |
Mapping % (Bos taurus) |
|
D418 |
81340893 |
58093768 |
23247125 |
2161069 |
71.42 |
|
D433 |
71294303 |
52034338 |
19259965 |
2149381 |
72.99 |
|
D457 |
111756969 |
94651399 |
17105570 |
2349002 |
84.69 |
|
D533 |
6308698 |
5916522 |
392176 |
3065347 |
93.78 |
|
D549 |
70563022 |
54783756 |
15779266 |
2066414 |
77.64 |
|
D571 |
65567424 |
49151310 |
16416114 |
2015024 |
74.96 |
|
HR1 |
29195662 |
21776597 |
7419065 |
2836331 |
74.59 |
|
HR2 |
38189305 |
28834340 |
9354965 |
3683477 |
75.50 |
|
HR4 |
28952695 |
21439330 |
7513365 |
2758286 |
74.05 |
|
HR5 |
28713271 |
21446523 |
7266748 |
2767310 |
74.69 |
|
HR6 |
28770768 |
21341285 |
7429483 |
2884217 |
74.18 |
|
HR8 |
24495591 |
17800739 |
6694852 |
2384605 |
72.67 |
RNA Seq data and Gene Expression
In order to explore the potential expression of genes in the whole blood associated with the milk production traits and to explore the possibility of a novel non-invasive tool to identify Differentially Expressed Genes (DEGs) for milk production traits, investigated the whole blood transcriptome profiles of high and low milk yielding cows using the RNA-seq technique. mRNA of peripheral blood mononuclear cells (PBMCs) belonging to six non-pregnant mid lactating Deoni cows and Hallikar cows were isolated. Cufflinks 2.2.1 tool9 was used to analyse DEGs between different high yielding and low yielding lactating Deoni and Hallikar cows. DEGs with log2 fold change ≥2.0 (upregulated), ≤0.05 (downregulated), an adjusted p-value (padj) and FDR < 0.05 were subjected to further analyses.
On the basis of the Cuffdiff report, a total of 3566 DEGs (p<0.05, FDR<0.05) were identified as significantly expressed in Deoni and Hallikar cows. Therefore, in our experiment FPKM below 0.01 has been filtered. The DEGs ranged from 0.14 to 21211.74 FPKM and 0.32 to 22880.1 FPKM were kept reserved for high yielding Deoni cows and low yielding Deoni cows, respectively. Whereas, for high yielding Hallikar cows FPKM ranged from 0.33 to 12836.23 FPKM and for low yielding Hallikar cows was 0.078 to 13212.7 (Supplementary tables).
In the present study a total of 944 and 2634 DEGs were observed in Deoni and Hallikar cows, respectively. Further, 639 and 1427 genes were upregulated in Deoni and Hallikar cows, respectively; and also 305 and 1207 DEGs were downregulated in Deoni and Hallikar cows, respectively (Table 2).
Table 2. Number of significant DEGs expressed in different groups of Deoni and Hallikar cows
Among upregulated 639 DEGs in Deoni and Hallikar cows and down regulated 1427 DEGs in Deoni and Hallikar cattle, transcripts related to CSN gene family (CSN1S1, CSN1S2, CSN2 and CSN3) and FASN were significantly expressed in High yielders of Deoni cows. However, in low yielding Deoni cows and all Hallikar cows, these CSN gene family and FASN gene expression levels were poor or not at all expressed (Figure 2 and 3). Additionally, BCL2, B4GALT1 and LACTB genes were equally expressed in all animals. However, ATP2B4 (PMCA - Plasma membrane Ca2+ ATPase) expressed only in Hallikar cows as shown in Figure 2 and Figure 3. The volcano plots also showed the significant expression of all above genes in both Deoni and Hallikar high yielding and low yielding cows (Figure 4 and 5).
Functional Annotation of Expressed Genes
The DEGs associated with lactation were subjected to gene ontology and functional annotation using PANTHER10 and DAVID 6.811 tools, respectively. The functional classification of significantly expressed genes of Deoni and Hallikar cows was performed by PANTHER tool. The Gene Ontology (GO) and functional annotation of expressed genes represented in biological process and molecular functions (Figure 6, Figure 7, Figure 8 and Figure 9). In biological processes in both breeds, a major cluster associated was a cellular process (GO:0009987) followed by a metabolic process (GO:0008152), biological regulation (GO:0065007), response to the stimulus (GO:0050896), immune system process (GO:0002376), localization (GO:0051179) and developmental process (GO:0032502). In molecular functions binding (GO:0005488) showed the highest share followed by catalytic activity (GO:0003824), molecular function regulator (GO:0098772), transcription regulator activity (GO:0140110), transporter activity (GO:0005215), molecular transducer activity (GO:0060089) and structural molecule activity (GO:0005198).
Gene ontology (GO) analysis of the genes encoding DEGs transcripts showed that those in the ‘Biological process’ and ‘Molecular functions’ categories were significantly enriched for terms related to various gene binding and expression regulation processes. Protein modification processes were also significantly enriched in the ‘Biological process’ and ‘Molecular functions’ categories (Figure 6, Figure 7, Figure 8 and Figure 9). The transcripts related to binding are RNA splicing factor, ribosomal protein, kinase modulator, C4 zinc finger nuclear receptor, kinase activator and cytokines.
Gene ontology (GO) enrichment analysis of expressed genes was performed by DAVID 6.8 tool and revealed 64, 58 and 85 GO terms for Biological Process (BP), Molecular Functions (MF) and Cellular Components (CC), respectively, for Deoni cattle. Additionally, functional annotations of expressed genes in Deoni cattle unveiled signal, signal peptide, immunity, milk protein, MHC II, calcium, allergen, cytokine, RNA processing and modification, MHC I and antioxidant GO terms for a group of 28, 19, 7, 6, 5, 5, 3, 3, 2, 2 and 2 genes, respectively.
In Hallikar cattle, the gene ontology (GO) enrichment analysis of expressed genes unveiled 181, 167 and 188 GO terms identified for Biological Process (BP), Molecular Functions (MF) and Cellular Components (CC), respectively. Further, functional annotations of expressed genes in Hallikar cows showed membrane, signal peptide, immunity, glycoprotein, inflammatory response, innate immunity, hemostasis, blood coagulation, cytokine, lipid metabolism, calcium-binding region, antioxidant, and MHC II GO terms for a group of 77, 47, 21, 39, 12, 12, 5, 5, 8, 7, 4, 3 and 3 genes, respectively.
Biological pathways
In the present study, CSNs and FASN transcripts were significantly enriched in high yielders. However, gene ontology reports of BCL2 and PMCA genes revealed that the transcripts related to these genes were taken part in the apoptotic and calcium signalling pathways, respectively. The expression of BCL2 and PMCA genes expression was highly enriched in low yielding cows of our study. The significant pathways discussed above illustrated in detail in the following Figure 10, Figure 11 and Figure 12 and Figure 13.
The isolated RNA from peripheral blood mononuclear cells (PBMCs) was intact and the quality and quantity of RNA also acceptable for further analysis12. Low expressed transcripts that is below 0.01 FPKM (Fragments Per Kilobase per Million mapped reads) per sample has been excluded from further analysis13.
Peripheral Blood Mononuclear Cell (PBMC) consists of monocytes and lymphocytes (T cells, B cells, NK cells)14. Lactogenesis is an orchestrated process where mammary gland is actively involved. This is the only organ that undergoes regular proliferation and involution cycles after maturity. Mammary epithelial cells (MECs) are unique in that they synthesize and secrete milk15. Actually, besides mammary gland epithelium, blood also plays critical roles in milk synthesis during lactation as the most important tissue where the metabolism of gluconeogenesis, lipid, amino acids and other substances takes place in dairy cows.
To gain an insight into the expression of genes during lactation, we analyzed the transcriptome of PBMCs of early lactating, non-pregnant and multiparous Deoni and Hallikar cows. The genes encoding for milk production as well as its components are already well established in ruminants16,17. The CSN family of genes (CSN1S1, CSN1S2, CSN2 and CSN3) and FASN gene encoding for milk protein18 and milk fat19, respectively are abundantly expressed in High yielders of Deoni cows. CSN genes could influence the protein content and composition in milk20 and also 75% of the milk proteins are mainly caseins21. The enhanced expression of these genes is relevant for the synthesis of milk proteins. CSN transcripts are generally called caseins and involved in the prolactin signalling pathways and one of the major gene family affecting milk production associated traits22,23. BCL2, B4GALT1 and LACTB genes were equally expressed in all Deoni and Hallikar cows. BCL2 gene is expressed and actively involved in the apoptosis activity24,25 and also B4GALT1 gene is associated with lactose synthesis in milk production26. Additionally, LACTB (B-Lactamase) gene is expressed in various tissues in conditions and is mainly involved in antitumour and apoptotic activities27,28. However, ATP2B4 (PMCA - Plasma membrane Ca2 + ATPase) expressed only in Hallikar cows. The effect of abrupt cessation of milk production on the Ca2+-ATPases and mammary calcium transport is unknown, mammary gland involution is associated with rapid downregulation of major mammary Ca2+-ATPases29.
Gene ontology of DEGs transcripts related to the casein genes is essential in prolactin signalling pathways. Furthermore, transcripts related to the FASN gene participated in forming long-chain fatty acids from acetyl-CoA, malonyl-CoA and NADPH (fatty acid synthesis pathway), and multifunctional protein has 7 catalytic activities and an acyl carrier protein30.
These results indicated that the regulation of metabolic and immune function is more active in high milk yielding cows. Also, high yielders were showing mammary gland development and related functional enhancement towards fatty acid synthesis. Besides, the low producing animals showed more activity of apoptosis along with the adeptness to draftability. Further research is required to explore the lactogenic traits in cattle blood transcriptomes. RNA-Sequencing results pointed towards the regulation of mammary gland, functional enhancement towards fatty acid synthesis, development metabolic and immune function is more active in high milk yielding cows, and apoptosis regulation is highly functioning in low yielding cows or draft purpose animals. Further research is required to explore the relationship between protein expressions for lactogenesis response and milk performance traits in other body fluids, including whole blood of cattle. In the present study, these CSNs and FASN transcripts were significantly enriched in high yielders. However, gene ontology reports of BCL2 and PMCA (ATP2B4) genes, revealed that the transcripts related to these genes were taken part in the apoptotic and calcium signalling pathways, respectively. These BCL2 and PMCA genes expression is highly enriched in low yielding cows. These results indicate that the regulation of metabolic and immune functions are more active in high milk yielding cows. The low producing animals showed more activity of apoptosis along with the adeptness to draftability.
Selection of cows for mRNA sequencing
All methods and experiments were carried out in accordance with relevant guidelines and regulations. Non-pregnant, early lactating Deoni cows (Figure 14) belonging to high yielding (n=3) and low yielding (n=3) maintained at Livestock Research Centre, Southern Regional Station of ICAR- NDRI, Bengaluru and also non-pregnant, early lactating Hallikar cows (Figure 15) belonging to high yielding (n=3) and low yielding (n=3) from farmers herds belonging to Kanakapura Taluk, Ramanagara district of Karnataka state were selected for RNA-Seq experiment.
The lactating high and low yielding Deoni cows were selected based on the Most Probable Producing Ability (MPPA) ranking; whereas lactating high and low yielding Hallikar cows were selected based on the predicted average lactation yield (PALY) ranking for lactation length of 260 days yield of field performance records (Table 3a and Table 3b). MPPA of the cattle was computed using a single record of the first lactation milk yield in Deoni cows31.
Evaluation of cows by using Most Probable Producing Ability (MPPA)
Most Probable Producing Ability (MPPA) of the cattle was computed using a single record of the first lactation milk yield in Deoni cows. In this method, the cows are evaluated on the basis of deviation from population mean of FLMY from its phenotypic value. The method described by J.L. Lush (1943) was used to compute MPPA as,
MPPA of cow = µ + h2 (Yi - µ)
Where, µ = Population mean
h2 = heritability of first lactation milk yield of Deoni cattle.
Yi = phenotypic value of FLMY of ithcow
Evaluation of cows by using Predicted Average Lactation Milk Yield (PALY)
Milk yield of each cow is recorded at 15 days of intervals for throughout the lactation. Subsequently, Predicted Average Milk Lactation Yield (PALY) calculated by the following method.
PALY of cow = α × LL
α = average of test day yield
LL= Lactation length of a cow
Blood collection to extract mRNA and Isolation of Peripheral Blood Mononuclear Cells (PBMC) and total RNA isolation
Aseptically blood was collected from each cow to isolate Peripheral Blood Mononuclear Cells. PBMC were isolated from early lactating, non-pregnant Deoni and Hallikar cows by the Histopaque-1077 method32. PBMCs were washed in sterile saline, placed in RNAlater (ThermoFisher, Invitrogen, Grand Island, NY), and stored at −80 °C until RNA isolation. Total RNA was extracted using the RNeasy.
mRNA Library preparation and Sequencing
The whole transcriptome sequencing experiment was conducted at Sandor Life Sciences Pvt. Ltd., Hyderabad, India. The following steps were carried out to prepare the library for mRNA sequencing.
- The total RNA sample was quantified using Quant-iT™ RiboGreen™ RNA Assay Kit (R11490), and further quality check-up done by loading 2 uLs of denatured RNA sample onto High Sensitivity RNA. ScreenTape using Agilent 4200 Tapestation system. RNA with RIN value ~5 was taken forward for library preparation, and having RIN < 4 was rejected.
- RNA libraries were prepared using NEBNext® Ultra™ DNA Library Preparation Kit for Illumina® | NEB by taking 500 ng of input RNA.
- The library preparation started with pulling out the mRNA by using Oligo dT beads, the resulted mRNA was fragmented by targeting 200nt fragments and the resultant fragments were converted to First Strand cDNA followed by 2nd strand cDNA.
- The 2nd strand cDNA reaction was purified by using 1.8X AgencourtAMPure XP Beads, and the pure 2nd cDNA will be eluted in 55.5 uL of 0.1X TE buffer.
- The resultant 2nd strand cDNA undergoes end repair wherein the mix converts the overhangs resulting from fragmentation into blunt ends. The 3’ to 5’ exonuclease activity of end repair mix removes the 3’ overhangs, and polymerase activity fills in the 5’ overhangs. The blunt ended fragments are cleaned up using AMPure XP Beads.
- To the blunt ended fragments adenylation was performed by adding single ‘A’ nucleotide to the 3’ ends. To the adenylated fragments ligation of Illumina universal adapters is performed.
- Adapter ligated fragments was amplified using Illumina universal primers and indexing primers which ligates multiple indexing adapters to the ends of DNA fragments. A single ‘T’ nucleotide on the 3’ end of the adapters provides a complementary overhang for ligating the adapter to the fragment. This strategy ensures a low rate of chimera formation. Each adapter index has different index sequences. Then two rounds of clean-up process are performed by using AM pure beads.
- The final transcriptome library was quantified by using Qubit™ dsDNA HS Assay Kit (Q32851), and quality check is done by using Agilent DNA 7500 kit either on Agilent 2100 Bioanalyzer system. The peak size and concentration of the sample was determined and the sample was further normalised to 10 nMol followed by serial dilutions to 2 nMol.
- The normalised samples were sequenced on Illumina HiSeq platform by taking 10 pMol denatured libraries.
mRNA Sequencing and Quality control
The Illumina HiSeq platform used for the sequencing of the normalised RNA library. Sequence quality of each RNA-seq sample was assessed by FastQC version 10.1 (www.bioinformatics.bbsrc.ac.uk/projects/fastqc). Trimmomatic version 0.3259 was used to trim out low quality sequences, ambiguous sequences and artificial sequences such as residual adaptors and Illumina specific sequences as described elsewhere. The Trinity program constructed four de novo transcriptome assemblies with default settings. Because de novo transcriptome assemblies involve many contigs in a contig cluster, we required a minimum expression filter of one fragment per kilobase of exon per million fragments mapped (FPKM) and filtered the contigs with FPKM value smaller than one; the contig with the highest expression level in a contig cluster was selected to be a unique gene for downstream analyses.
mRNA Alignment
The raw reads were aligned against reference genome UMD 3.1 (Bos taurus) using HiSAT2 tool. SAMtool is used to convert the sequence alignment map file to BAM format33. Then by using Cufflinks 2.2.1 tool Differential Expression of Genes (DEG) was performed.
Differential Gene Expression
Differential gene expression analysis was done using the Cufflinks 2.2.1 tool34, between different high yielding and low yielding lactating Deoni and Hallikar cows. Differential Expressed Genes (DEG’s) with log2 fold change ≥2.0 (upregulated), ≤0.05 (downregulated), an adjusted p-value (padj) and FDR < 0.05 were subjected to further analyses.
Functional Annotation of Expressed genes and Identification of Biological Pathways
All functional analyses and gene ontology of the genes were done against UMD 3.1 (Bos taurus) genome. DAVID35 and Panther10 tools were used for gene ontology, enrichment in pathways and functional annotation of the differentially expressed genes which is based on database source including KEGG PATHWAY Database online tool.
Heatmap and Volcano plot
Heat map of differentially expressed gene was generated by web based tool called usegalaxy (https://usegalaxy.org/)36 and volcano plot was also generated by web based tool VolcaNoseR37.
Table 3a. Lactation performance of Deoni cow’s used for transcriptomic analysis
ALY: Average Lactation Yield
MPPA: Most Probable Producing Ability
Table 3b. Lactation performance of Hallikar cows used for transcriptomic analysis
PALY: Predicted Average Lactation Yield
Author Information
Affiliations
Nizamuddin Azharuddin
Southern Regional Station, ICAR-National Dairy Research Institute, Bangalore-560030, India
Email: [email protected]
ORCID ID: 0000-0001-7020-1674
Kerekoppa P. Ramesha
Southern Regional Station, ICAR-National Dairy Research Institute, Bangalore-560030, India
Email: Kerekoppa P. Ramesha: [email protected]
ORCID ID: 0000 0003 1828 2192
Ekta Rana
Southern Regional Station, ICAR-National Dairy Research Institute, Bangalore-560030, India
Email: [email protected]
Sandeep Kasargod
Centre for Systems Biology and Molecular Medicine, Yenepoya Research Center, Yenepoya (Deemed to be University), Mangalore-575018, India
Email: [email protected]
Santosh Kumar Behera
Centre for Systems Biology and Molecular Medicine, Yenepoya Research Center, Yenepoya (Deemed to be University), Mangalore-575018, India
Email: [email protected]
M. Ashokan
Southern Regional Station, ICAR-National Dairy Research Institute, Bangalore-560030, India
Email: [email protected]
Arun H. Patil
Centre for Systems Biology and Molecular Medicine, Yenepoya Research Center, Yenepoya (Deemed to be University), Mangalore-575018, India
Email: [email protected]
Sakthivel Jeyakumar
Southern Regional Station, ICAR-National Dairy Research Institute, Bangalore-560030, India
Email: [email protected]
A. Kumaresan
Southern Regional Station, ICAR-National Dairy Research Institute, Bangalore-560030, India
Email: [email protected]
M. A. Kataktalware
Southern Regional Station, ICAR-National Dairy Research Institute, Bangalore-560030, India
Email: [email protected]
D. N. Das
Southern Regional Station, ICAR-National Dairy Research Institute, Bangalore-560030, India
Email: [email protected]
Naveen Kumar G.S.
Associate Professor and Head, Department of Animal Genetics and Breeding, KVAFSU- Veterinary College, Hassan-573201, India
ORCID ID: 0000 0002 6990 9103
Thottethodi Subrahmanya Keshava Prasad
Centre for Systems Biology and Molecular Medicine, Yenepoya Research Center, Yenepoya (Deemed to be University), Mangalore-575018, India
Email: [email protected]
ORCID ID: 0000-0002-6206-2384
Author Contributions
K.P.R. and T.S.K.P. conceptualized and designed the study; N.A., K.P.R., T.S.K.P., S.J., A.K., M.A.K., D.N.D., E.R. and A.M. managed the study; N.A., N.K.G.S, collected the biological samples N.A., E. R. and M.A. and isolated the PBMCs and RNA. N.A, S.K. and S.K.B carried out transcriptome assembly, QC and alignment. K.P.R., T.S.K, N.A. and A.H.P designing of analysis pipeline. N.A., E.R., M.A., S.K., and S.K.B., involved in the analysis of transcriptomic data. K.P.R, S.J., N.A., E.R., M.A., S.K., S.K.B, N.K.G.S and T.S.K wrote the initial manuscript with all authors contributing to subsequent draft versions.
Competing interests
The author(s) declare no competing interests.
Data availability
Genome data has been deposited to NCBI SRA (project id: PRJNA844925; https://dataview.ncbi.nlm.nih.gov/object/PRJNA844925).
Ethics declaration
The work was approved by the Institutional Animals Ethics Committee of CPCSEA vide No. CPCSEA/IAEC/LA/SRS-ICAR-NDRI-2018/No.1, 2017/No.1 and 2021/No.15
Acknowledgments
We are highly grateful to the Karnataka Livestock Development Agency of Department of Animal Husbandry and Veterinary Services, Government of Karnataka for partial funding of the study under the project “Proteo-Genomic approach to elucidate productive and reproductive performance of Malnad Gidda, Amrit Mahal and Hallikar breeds of cattle and field performance recording of Malnad Gidda cattle” to Southern Regional Station of ICAR-NDRI, Bengaluru under the Ministry of Forest and Environment Climate Change under Adaptation Fund, Government of India.
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No competing interests reported.