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of India supported the project under grant EMR/2017/000580.
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Identiûcation of SNPs related to the resistance against

Salmonella infection in chicken using RNA-seq and
phylogenomics analysis
Mashooq Ahmad Dar 1 , Basharat Bhat Corresp., 1 , Tanveer Dar 2 , Mudasir Ahmad Syed Corresp. 1

1
Division of Animal Biotechnology, Faculty of Veterinary Sciences and Animal Husbandry, SKUAST-K, Shuhama, India
2
Department of Clinical Biochemistry/Biochemistry, University of Kashmir, Srinagar, India

Corresponding Authors: Basharat Bhat, Mudasir Ahmad Syed

Email address: bb284@snu.edu.in, mudasirbio@gmail.com

Background Deep RNA sequencing experiment was employed to detect putative single
nucleotide polymorphisms (SNP) between two chicken breeds (Kashmir faverolla and
broiler) in order to understand the variations in the coding regions that reûect diûerences
in immune response against Salmonella infection. In the present study we identiûed high
impact SNPs from both the chicken breeds in order to delineate diûerent pathways that
mediate disease resistant/susceptibility traits in both breeds. Methods Samples (liver and
spleen) were collected from Salmonella resistant (Kashmir faverolla) and susceptible
(broiler) chicken breeds. Salmonella resistance and susceptibility was checked by diûerent
pathological parameters post infection. An SNP detection analysis was performed using
RNA seq reads from 10 Kashmir faverolla and 10 broiler chicken to determine putative
polymorphisms in genes involved in disease resistance. Additionally, phylogenomics
analysis was carried to ûnd out the variation between susceptible and resistant chicken
breeds. Results A total of 1778 (1070 SNPs and 708 INDELs) and 1459 (859 SNPs and 600
INDELs) were found to be speciûc to Kashmir faverolla and broiler respectively. Based on
our results we conclude that in broiler the enriched pathways mostly included metabolic
pathways like fatty acid metabolism, carbon metabolism and amino acid metabolism
(Arginine and proline metabolism), while as in Kashmir faverolla genes with high impact
SNPs were enriched in most of the immune related pathways like MAPK signaling pathway,
Wnt signaling pathway, NOD-like receptor signaling pathway etc. which could be a possible
resistance mechanism against Salmonella infection. In Kashmir faverolla, protein-protein
interaction analysis also shows some important hub nodes which are important in
providing defense against diûerent infectious diseases. Phylogenomics analysis revealed
that indigenous poultry breeds (resistant) clearly separated from commercial breeds
(susceptible).

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1 Identification of SNPs related to the resistance against Salmonella infection in

2 chicken using RNA-seq and Phylogenomics analysis

4 Mashooq Ahmad Dar1,2,#, Basharat Bhat1,#,*, Tanveer Dar2, Syed Mudasir Ahmad1,*

5 1Division of Animal Biotechnology, Faculty of Veterinary Sciences and Animal Husbandry,

6 SKUAST-K, Shuhama, India
7 2Department of Clinical Biochemistry/Biochemistry, University of Kashmir, Srinagar, India
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23 # equally contributing authors
24 Corresponding authors
25 Syed Mudasir Ahmad, mudasirbio@skuastkashmir.ac.in
26 Basharat Bhat, bb284@snu.edu.in
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28
29

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30

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31 Abstract
32 Background
33 Deep RNA sequencing experiment was employed to detect putative single nucleotide
34 polymorphisms (SNP) between two chicken breeds (Kashmir faverolla and broiler) in order to
35 understand the variations in the coding regions that reflect differences in immune response
36 against Salmonella infection. In the present study we identified high impact SNPs from both the
37 chicken breeds in order to delineate different pathways that mediate disease
38 resistant/susceptibility traits in both breeds.
39 Methods
40 Samples (liver and spleen) were collected from Salmonella resistant (Kashmir faverolla) and
41 susceptible (broiler) chicken breeds. Salmonella resistance and susceptibility was checked by
42 different pathological parameters post infection. An SNP detection analysis was performed using
43 RNA seq reads from 10 Kashmir faverolla and 10 broiler chicken to determine putative
44 polymorphisms in genes involved in disease resistance. Additionally, phylogenomics analysis
45 was carried to find out the variation between susceptible and resistant chicken breeds.
46 Results
47 A total of 1778 (1070 SNPs and 708 INDELs) and 1459 (859 SNPs and 600 INDELs) were
48 found to be specific to Kashmir faverolla and broiler respectively. Based on our results we
49 conclude that in broiler the enriched pathways mostly included metabolic pathways like fatty
50 acid metabolism, carbon metabolism and amino acid metabolism (Arginine and proline
51 metabolism), while as in Kashmir faverolla genes with high impact SNPs were enriched in most
52 of the immune related pathways like MAPK signaling pathway, Wnt signaling pathway, NOD-
53 like receptor signaling pathway etc. which could be a possible resistance mechanism against
54 Salmonella infection. In Kashmir faverolla, protein-protein interaction analysis also shows some
55 important hub nodes which are important in providing defense against different infectious
56 diseases. Phylogenomics analysis revealed that indigenous poultry breeds (resistant) clearly
57 separated from commercial breeds (susceptible).
58 Conclusion
59 The SNP analysis showed that there was a significant difference between the Kashmir faverolla
60 and broiler chicken in disease resistance. In Kashmir faverolla the high impact SNP variants
61 were mainly involved in immune related pathways while as in broiler chicken, the enriched

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62 pathways were mainly involved in metabolism. These findings shall provide new insights in
63 genetic variation in chicken breeds and shall help in genomic selection of the poultry birds.

64 Keywords: Kashmir faverolla, Broiler, SNP identification, RNA sequencing

65 Introduction
66 Salmonella enterica serovar Typhimurium is a Gram-negative, facultative anaerobe, non-spore
67 forming usually motile bacilli that belongs to family enterobacteriaceae, causing severe intestinal
68 pathology in young chickens [1]. Salmonellosis is a big socioeconomic threat worldwide that
69 causes considerable mortality and morbidity in both humans and animals [2]. Salmonella is one
70 of the common pathogens causing sporadic cases or outbreaks of gastroenteritis [3]. Salmonella
71 infection is a serious concern in poultry farming. On one hand, systemic salmonellosis results in
72 considerable losses in poultry industry in terms of mortality and reduced poultry production; on
73 the other hand, poultry is a major global reservoir of non-typhoidal Salmonellae leading to
74 foodborne illnesses [4]. As none of the current vaccination programs is successful to control
75 Salmonella infections [5], antibiotics are preferred by the poultry industry. The emergence of
76 antibiotic- resistant bacteria and the accumulation of antibiotic residues in food for human
77 consumption are the two major issues associated with widespread antibiotic use [6,7]. The host
78 immune system has the ability to effectively resist microbial invasion and kill microorganisms.
79 Following bacterial identification, the bactericidal activity of host macrophages induces dendritic
80 cell maturation and migration, as well as production of inflammatory chemokines, cytokines,
81 interleukins etc. [8]. Genetic resistance is an attractive and sustainable approach for disease
82 control [9]. In this context, selection of more resistant chickens can be considered as an
83 alternative solution to decrease occurrence of the disease. In developing countries, the genetic
84 disease resistance is generally more important as indigenous breeds are more resistant to local
85 diseases [10]. The Kashmir faverolla, a well-known indigenous chicken breed from the northern
86 Indian state Jammu and Kashmir, is primarily reared for meat and egg production and is also
87 considered as the most important source of animal protein [11]. This indigenous breed has a high
88 disease resistance and is well adapted to the local climatic conditions, feed and stress
89 management [12].
90 Recent advances in molecular technology have created a new horizon for the genetic
91 improvement of quantitative traits, particularly disease-resistant traits. The identification of

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92 direct or indirect molecular markers for these traits would facilitate the use of marker-assisted
93 selection or gene introgression. Molecular markers are indispensable tools for marker assisted
94 breeding. The simple sequence repeats (SSRs) and single nucleotide polymorphism (SNP)
95 markers are two attractive and widely used because of many merits including co-dominant,
96 reproducibility, locus-specificity, and random genome-wide distribution in many organisms [13].
97 Geneticists and breeders have carried out breeding of new poultry varieties (lines) through
98 modern biotechnology, such as molecular breeding and transgenics, focusing on the selection of
99 poultry disease resistance, growth speed, meat and egg quality traits. However, these traits are
100 not only controlled at DNA level, but also regulated at mRNA level before and after transcription
101 and this level of regulation is more comprehensive, systematic and accurate.
102 The current research shall help to identify different SNPs that may be involved in disease
103 resistance against Salmonella infection in poultry and thus may lay a foundation step for future
104 in-depth studies of disease resistance mechanisms.
105 Methods
106 Samples (liver and spleen) were collected from Salmonella resistant (Kashmir faverolla) and
107 susceptible (broiler) chicken breeds. Salmonella resistance and susceptibility was checked by
108 different pathological parameters post infection [14]. Power analysis and sample size calculation
109 using RNASeqPower program v3.16[15] suggested at least 6 biological replicates should be used
110 in each group. In our study we used 9 biological replicates in Kashmir faverolla and 10
111 biological replicates in broiler chickens (from GEO accession ID: GSE168060) to determine
112 putative polymorphisms in genes involved in Salmonella resistance.
113 The sequencing data were downloaded from our previously published study [14]. The data were
114 filtered as per our previous study [14]. Briefly, sequencing read quality was assessed using
115 FASTQC program v0.11.1 [16] and adaptor sequences were filtered using Cutadapt v3.40 [17],
116 high-quality sequencing reads that passed thresholds (PhredScore > 30) were assembled for SNP
117 discovery analysis. A collection of over 40 million high-quality clean reads were obtained for
118 each sample. The cleaned reads were mapped to reference genome assembly ARS-UCD1.2.99
119 using HISAT2 [18]. The data preprocessing steps recommended in the Genome Analysis Toolkit
120 (GATK) best practices workflow was performed before variant identification [19]. PCR
121 duplicates were marked with the MarkDuplicates from Picard tools [20]. We also performed
122 local realignment around InDels, checked intron-exon junctions, and recalibrated the base quality

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123 scores with GATK. Two different variant callers were used to perform SNP and INDELs
124 discovery across eight Jersey and nine Kashmiri transcriptome samples separately: (i) GATK
125 using the HaplotypeCaller tool in multi sample calling mode (modality “GATK”); (ii) mpileup
126 from SAMtools v1.4 [21] in multi-sample calling mode using default parameters. Final set for
127 analysis contains SNPs and InDels common in both datasets. Chicken genetic variants from
128 dbSNP 2.0 build 153 dated: Aug 8, 2019, were incorporated in SNP calling to populate the
129 RS_ID column of the known SNPs. Filtering [base quality score (QScore) > 30, mapping quality
130 > 30, and minimum depth > 10] of generated variants and annotation were performed using
131 VCFtools version 0.1.8 and SnpEff program v4.1. To further evaluate the biological significance
132 of the genes with high-impact variations, the KEGG pathway enrichment analysis was performed
133 using KOBAS server version 3 [22,23].
134
135 Results
136 Quality Control, Mapping, and Post treatment
137 Sequencing of liver and spleen transcriptome libraries generated a total of 46.95 million reads
138 (range 30.51–68.56 million reads/library) and 41.74 million reads (range 26.92–63.61 million
139 reads/library) respectively. Out of 46.95 million reads of liver transcriptome, 43.76 million reads
140 (92.82%) passed QC and were aligned to the Gallus gallus genome GRCg6a. In total 41.46
141 million uniquely mapped reads were further processed while the rest of the reads were discarded.
142 In spleen out of 41.74 million reads, 38.65 million reads (92.24%) passed the quality control and
143 were aligned to the Gallus gallus CRCg6a genome. In total 35.45 million reads that uniquely
144 mapped were processed further while as the rest reads were discarded. (S1, S3 Table). A total of
145 1,141,122 and 1,151,874 variants were identified from Kashmir faverolla and broiler
146 respectively. The chromosomal distribution of SNPs and INDELs are provided in S1 Table and
147 the variant types are given in table S2 and S3 table A total of 26.036% missense, 0.138%
148 nonsense, and 73.785% silent mutation were identified in Kashmir faverolla, and 26.845%
149 missense, 0.167% non-sense, and 72.988% silent mutations were identified in broiler chicken. In
150 accordance with the previous studies, transitions to transversions ratio (Ts/Tv) for Kashmir
151 faverolla and broiler was found to be 2.7 (6080998/2236247) and 2.7 (6993925/2563136)
152 respectively. SNP distribution on different chromosomes in both Kashmir faverolla and
153 commercial broiler are shown (Figure 1). After comparison it was found that there were 758

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154 common genes with SNPs (Figure 2). The common SNPs were filtered out and further analyses
155 were carried out on high-impact SNPs and INDELs specific to broiler 1778 (1070 SNPs and 708
156 INDELs and Kashmir faverolla 1459 (859 SNPs and 600 INDELs) (Supplementary File 1 and
157 2).
158 To gain insights into the genetic variations with respect to salmonella resistance the
159 phylogenomic analysis of boiler chickens, Korean commercial chicken, and Kashmir faverolla
160 were performed using transcriptome sequencing (RNA-Seq) data. The phylogenomic analysis
161 revealed that commercial breeds are grouped in a single clad, and native Kashmir breed shows
162 significant difference than commercial breeds (Figure 3). We could only find a single
163 transcriptome data of Korean commercial chicken that we included in analysis.
164 Analysis of Genes with SNPs and INDELs
165 Functional annotation suggests the enrichment (p-value < 0.05) of signaling pathways like
166 Wnt signaling pathway, FoxO signaling pathway, Cellular senescence, NOD-like receptor
167 signaling pathway in Kashmir faverolla (S4 Table, Figure 3). In broiler chicken, the enriched
168 pathways included Metabolic pathways, Herpes simplex virus 1 infection, Fatty acid
169 biosynthesis, Influenza A, Fatty acid metabolism, Carbon metabolism, Citrate cycle (S5 Table;
170 Figure 4). Gene ontology (GO) analysis strongly suggests (p-value < 0.05) genes with SNPs in
171 Kashmir faverolla and broiler chickens were mainly involved in the binding process (enzyme,
172 ribonucleotide and histone binding) and antigen processing (S6 and S7 Tables; Figure 5).
173 Protein -Protein Interaction
174 Salmonella infection affects every chicken breed; however, every breed has its own defense
175 mechanism to counter the infection. In both the chicken breeds we found similar hub genes
176 however in the resistant breed (Kashmir faverolla) PLK1, MK1671P gene mutations were
177 hyperactive suggesting a possible role in this particular breed. Plk-1 which is the member of the
178 serine/threonine polio like kinase family has a vital role in immune signalling [24]. Further PLK-
179 1 interacts with BRCA-2 and MLF1P which regulate autophagy, antigen presentation, immune
180 response, angiogenesis and apoptosis [25.26] (Figure 6).

181 Discussion
182 Indigenous chickens appear to be more genetically diverse than the commercial breeds, as they
183 have been improved and established through a long breeding history, by processes remarkably
184 different from those used for commercial breeds. Therefore, the conservation of local chicken

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185 breeds, as a genetic resource, is crucial to satisfy future unanticipated breeding demands [27,28].
186 The Kashmir faverolla, a well-known indigenous chicken breed from the northern Indian state
187 Jammu and Kashmir, is primarily reared for meat and egg production and is also considered as
188 the most important source of animal protein [11]. This indigenous breed has a high disease
189 resistance and is well adapted to the local climatic conditions, feed and stress management [12].
190 For understanding the disease resistance mechanism, we have analysed RNA sequencing data
191 and performed a comparative study between Kashmir faverolla and broiler chicken breeds [15].
192 An SNP detection analysis was performed using RNA seq reads from 10 Kashmir faverolla and
193 10 broiler chicken to determine putative polymorphisms in genes involved in disease resistance.
194 A total of 1,141,122 and 1,151,874 variants were identified from Kashmir faverolla and broiler
195 respectively. A total of 1778 (1070 SNPs and 708 INDELs) and 1459 (859 SNPs and 600
196 INDELs) were identified in broiler and Kashmir faverolla respectively. The KEGG analysis and
197 gene ontology showed that the genes were involved in many important immune related
198 pathways. It was observed that MAPK signaling pathway, ECM-receptor interaction, Wnt
199 signaling pathway, FoxO signaling pathway, cellular senescence was highly enriched in Kashmir
200 faverolla. These pathways stimulate the immune response against Salmonella infection [29,30].
201 WNT signalling is essential for maintaining tissue homeostasis, epithelial barrier functions,
202 inflammatory cytokine production modulation, host cell innate defence mechanisms, and the
203 integration of innate and adaptive immunity [31]. Further enrichment of WNT signalling
204 pathway in response to Salmonella infection in Kashmir faverolla may help in either increasing
205 B cell proliferation or B cell survival [32]. We found variations in different genes that regulate
206 Wnt signalling (S1 Figure) (TCF7, LRP5, CaMKII, WNT5A, NLK etc.). TCF7 plays a vital role
207 in tissue repair, remodelling and disease pathogenesis [33]. LRP5 has been found to possess a
208 novel role in IL-10 signalling, thereby exerting a protective role during inflammation [34]. CaM-
209 dependent proteins (CaMKII) have a critical role in infectious diseases through involvement in
210 inflammatory processes, apoptosis and necroptosis [35]. Wnt5A facilitates the killing of several
211 bacterial pathogens by bringing alterations in actin assembly in macrophages and finally leading
212 to bacterial phagocytosis [36]. Nemo-like kinase (NLK) has a potential role in modulating
213 immune responses through regulation of NF-»B signalling by interfering with different
214 signalling molecules [37]. In broiler the enrichment analysis revealed that genes with high
215 impact SNPs were involved mainly in metabolic pathways, fatty acid metabolism, carbon

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216 metabolism and amino acid metabolism (Arginine and proline metabolism). Salmonella utilizes
217 these metabolites as energy source for its intracellular survival and proliferation [38].
218 The phylogenomic analysis revealed the exhaustive similarity between commercially available
219 chicken breeds and possible similar mechanism of weak resistance against Salmonella infection.
220 Resistance to salmonellosis in chicken greatly varies among the chicken line [39]. Large genetic
221 variation in poultry for resistance to S.Typhimurium has been confirmed due to the breed
222 variations [40].
223
224 Conclusions
225 The SNP analysis showed that there was a significant difference between the Kashmir faverolla
226 and broiler chicken in disease resistance. In Kashmir faverolla the high impact SNP variants
227 were mainly involved in immune related pathways while as in broiler chicken, the enriched
228 pathways were mainly involved in metabolism. These findings shall provide new insights in
229 genetic variation in chicken breeds and shall help in genomic selection of the poultry birds.

230

231 Ethical approval

232 The tissue collection was carried out only after receiving approval from institutional animal
233 ethics committee on ethical standards in animal experimentation (AU/FVSc/PS-57/16021).
234 Data availability:
235 The sequencing data is available in NCBI under accession number GSE168060.The database is
236 accessible through web URL https://skuastk.org/pif/index.html
237
238 Conflict of interest:
239 The authors declare no conflict of interest.
240
241 Funding
242 The Science and Engineering Research Board (SERB), Ministry of Science and Technology,
243 Government of India supported the project under grant EMR/2017/000580.
244
245 Author contributions:

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246 SMA & BB designed and supervised the experiments. MAD performed the experiment and
247 wrote the manuscript. BB performed the data analysis. TAD summarized results and helped in
248 proof reading of manuscript.
249

250 Figure legends

251 Figure 1: Chromosomal distribution of high impact SNPs and Indels in Kashmir faverolla and
252 broiler chicken.
253 Figure 2: Venn diagram showing common genes with SNPs between Kashmir faverolla and
254 broiler chicken breeds.
255 Figure 3: Comparative Phylogenetic analysis between commercial and native chicken breeds.
256 Figure 4: KEGG enrichment of genes with high impact variations. (a) Broiler (b) Kashmir
257 faverolla.
258 Figure 5: GO enrichment analysis of genes with high impact variations. (a) Broiler (b) Kashmir
259 faverolla.
260 Figure 6: Protein-protein interaction network of genes with high impact variations. (a) Broiler
261 (b) Kashmir faverolla.
262
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Figure 1
SNP Chromosomal distribution

Chromosomal distribution of high impact SNPs and Indels in Kashmir faverolla and broiler

chicken.

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Figure 2
VENN Diagram

Figure 2: Venn diagram showing common genes with SNPs between Kashmir faverolla and

broiler chicken breeds.

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Figure 3
Phylogenetic analysis

Figure 3: Comparative Phylogenetic analysis between commercial and native chicken

breeds.

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Figure 4
Pathway Enrichment

Figure 4: KEGG enrichment of genes with high impact variations. (a) Broiler (b) Kashmir

faverolla.

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Figure 5
GO Enrichment

Figure 5: GO enrichment analysis of genes with high impact variations. (a) Broiler (b)

Kashmir faverolla.

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Figure 6
PPI Network

Protein-protein interaction network of genes with high impact variations. (a) Broiler (b)

Kashmir faverolla.

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