Profiling of HSPB9 gene sequences in Donggala (Bos indicus) and Polled Bali (Bos javanicus) cattle breeds of Indonesia

HSPB9 gene plays important role to protect cells from heat stress in mammals. This research aim to describe the DNA sequence of HSPB9 gene in Donggala (Bos indicus) and Bali (Bos javanicus) bulls using forward sequencing method. A total of 10 blood samples from 6 Donggala and 4 Polled Bali were used in this study for DNA analysis. The bulls were kept at the breeding station at Central Sulawesi and South Sulawesi for Donggala and Bali, respectively. Results showed that a novel nonsence mutation of c.216C>T (p.L68L) was detected in the exon 1 of Bali HSPB9 gene. Commonly, the HSPB9 gene was monomorphic in Donggala and Bali cattle. In conclusion, the mutation of c.216C>T might be used as the genetic marker to discriminate Donggala and Bali cattle.


Introduction
Heat Shock Protein Family B Member 9 (HSPB9) gene is the one of the HSP's family genes [1] that plays important role for the heat stress response or other chemical stressors that influencing cellular activity [2].In addition, the HSPB9 gene also affected to adaptation and behaviour mechanism of animals [3].The bovine HSPB9 gene located at chromosome 19 (BTA19) along 518 bp with one exon region (14 th -488 th nucleotide) along 474 bp (GenBank: NC_037346.1).Recently, the effectiveness of HSPB9 gene as the molecular selection for heat stress resistance in Indonesian cattle is not reported.However, another HSP's family gene of HSP70 gene affecting to the service per conception (S/C) and heat tolerance index (HTI) in Indonesian indigenous cattle [4][5][6].Despite of that, the HSPB9 gene can be used to characterize three camel species of Camelus dromedarius, Camelus ferus and camelus bactrianus [7].Donggala (Bos indicus) and polled Bali (Bos javanicus) are two Indonesian cattle breeds that adapted well in the tropical climate area.Donggala and Bali are typical of beef cattle that kept for meat production purpose.Donggala cattle have been declared as the one of Indonesian native cattle breeds since year 2014 through the decision of Indonesian Ministry of Agriculture No: 666/kpts/SR.120/6/2014.Meanwhile, Bali cattle are one of Indonesian native cattle breeds that decided by Indonesian Ministry of Agriculture No: 325/Kpts/OT.140/1/2010.However, the Indonesian national standard (SNI) for Bali cattle (SNI 7651-4: 2020) decided that the seedstock of Bali cattle must be presence of horn.As the tropical beef cattle breed, study of profiling HSPB9 gene in Indonesian indigenous cattle is important to obtain the potential genetic marker for heat stress tolerance ability that able to affect reproductive traits.This study was aimed to describe the profile of HSPB9 gene sequence in Donggala and Bali cattle.The results of this study can be used as the basic information to obtain the genetic marker for heat stress and adaptive traits in cIndonesian tropical cattle breeds in the future.

Animals and DNA sample
A total of 10 bulls belonging of 6 Donggala (Bos indicus) and 4 polled Bali (Bos javanicus) were used for in this study for the analysis.Polled or hornless is a rarely phenotype in Bali cattle that absence of horn development.The bloods samples (5 mL) were taken in each animal from jugular vein with venoject and vacutainer tube containing EDTA.The DNA extraction was performed by using Genomic DNA Extraction Kit (Geneaid, Taiwan) following the manufacturers protocol.The collecting bloods sample protocol for Donggala and Bali bulls were performed under the Ethical Clearence Number: 077/KE.02/SK/10/2022(National Research and Innovation Agency) and 302/UN4.6.4.5.31/PP36/2021 (Hasanuddin University), respectively.

PCR and sequencing analyses
The PCR was performed in a total volume 30 μL with the composition of 3.0 μL of DNA template; 0.6 μL of each primer; 15 μL of PCR Mastermix (Bioline, USA) and 10.8 μL of free-nuclease water.The primer pairs in the present study was designed from Bos taurus HSPB9 gene (GenBank: NM_001040577.2) using Primer3Plus program (https://www.primer3Plus.com)with the target sequence of 399 bp including of exon 1 region (Figure 1).The designed primer pairs in this study as follows: HSPB9-F: 5'-TGG CCT TTA CTG AAC GGA AC -3' and HSPB9-R: 5'-TAG AGC CAC GGC TTC TGA AT -3' (Registered on Indonesian Patent Number: P00202214077).The PCR program for HSPB9 gene amplification in this study was performed in 35 cycles of pre-denaturation at 95 °C for 5 min.;denaturation at 95 °C for 15 sec.; annealing at 52 °C for 15 sec.; extension at 72 °C for 20 sec.and final extension at 72 °C for 3 min.In addition, DNA vizualisation was performed on 1% agarose gel with electrophoresis at 100 V for 25 min using G-Box Documentation System (Syngene, UK).Therefore, the forward sequencing analysis was performed in each PCR product (amplicon) through a commercial laboratory service (First Base Laboratory, Malaysia).In addition, the obtained DNA sequences were analyzed using BioEdit [8] and MEGA-X [9] programs for the sequence alignment analysis.Therefore, detection of the amino acid changes in the mutation site was performed using BioEdit and verified with GenBank database.

Results and Discussion
Along 399 bp of the HSPB9 gene in animal study was amplified successfully on 1% agarose gel as shown in Figure 2.However, the sequencing analysis revealed that about 349 bp of the HSPB9 gene sequences in animal study can be observed clearly (Figure 2).According to Figure 2, the HSPB9 gene was monomorphic in Donggala and polled Bali samples.Interestingly, a novel nonsense mutation of c.216C>T or p.L68L was detected in the exon 1 region of bovine HSPB9 gene in pool animals (Figure 3).Hence, a mutation site of c.216C>T can be used as the genetic marker to discriminate Donggala and polled Bali cattle.In fact, both cattle breeds can be differentiated with phenotypic characteristics easily.In general, many cattle breeds can be characteized through their phenotypic characteristics and morphometrical measurements [10].In addition, the UPGMA tree with HSPB9 gene sequence revealed that polled Bali is classified into a different cluster.While, the another cluster consisted of Donggala, Bos taurus (GenBank: NM001040577) and Taurindus (GenBank: XM027516871) cattle (Figure 4).Many previous studies reporting the similar finding that Bali cattle is classified into a separated cluster and different with Bos taurus and Bos indicus cluster based on SRY [11], Cyt-b [12], D-loop [13], and COI [14] genes.
Presently, the HSPB9 gene sequence in Bos indicus and Bos javanicus from this study have been deposited on GenBank database with the accession number of OQ162040 and OQ162041, respectively.Nonetheless, the HSPB9 gene can not be used for molecular selection in Donggala and Polled Bali cattle becuse of low genetic diversity in both breeds.Nonetheless, many HSP's family genes were reported polymorphic in cattle such as HSP70 [4][5][6], HSP90A1 [15][16][17][18], HSPB6 [19], HSF1 [20,21], HSPB1 [22], HSPB7 [23] and HSPB8 [24].Interestingly, HSPB9 gene can be used to discriminate sub-species of cattle as same as camel [7].Moreover, HSPB9 gene is the main gene that associated with tropical adaptation trait in Nguni cattle (Bos indicus × Bos taurus) according to the GWAS [25].In the future, the depth study to explore the genetic marker for tropical heat stress tolerance in many Indonesian native cattle breeds is important to improve productivity.A GWAS method can be used to obtain many genetic markers or candidate gene of heat tolerance since many candidate genes for heat tolerance traits in cattle able to obtain with this method [26].

Conclusion
The HSPB9 gene in Donggala (Bos indicus) and Polled Bali (Bos javanicus) was monomorphic.Interestingly, a novel nonsense mutation c.216C>T (p.L68L) was detected in the bovine HSPB9 gene (exon 1) that able to discriminate Donggala and Polled Bali cattle breeds.