Over the last century, genetic diversity in the cattle species has been affected by the replacement of many local, dual-purpose breeds with a few specialized, high-output dairy breeds. This replacement caused a sharp decline in the population size of local breeds. In the Netherlands, the local Dutch Friesian breed has gradually been replaced by the Holstein Friesian. This resulted in a rapid decrease in numbers of the Dutch Friesian breed with an associated risk of loss of genetic diversity due to drift. The objective of this study is to investigate genomewide genetic diversity between a group of historic and recent Dutch Friesian bulls and a group of recently used Holstein Friesian bulls. Our findings showed that a large amount of diversity is shared between the three groups, but each of them has some unique genetic identity (12% of the single nucleotide polymorphism were group-specific). The genetic diversity of the Dutch Friesians reduced over time, but this did not lead to higher inbreeding levels—especially, inbreeding due to recent ancestors has not increased. Genetically, the recent Dutch Friesians were slightly more different from Holstein Friesians than the historic Dutch Friesians. Our results also highlighted the presence of several genomic regions that differentiated between the groups.Over the last century, genetic diversity in many cattle breeds has been affected by the replacement of traditional local breeds with just a few milk-producing breeds. In the Netherlands, the local Dutch Friesian breed (DF) has gradually been replaced by the Holstein Friesian breed (HF). The objective of this study is to investigate genomewide genetic diversity between a group of historically and recently used DF bulls and a group of recently used HF bulls. Genetic material of 12 historic (hDF), 12 recent DF bulls (rDF), and 12 recent HF bulls (rHF) in the Netherlands was sequenced. Based on the genomic information, different parameters—e.g., allele frequencies, inbreeding coefficient, and runs of homozygosity (ROH)—were calculated. Our findings showed that a large amount of diversity is shared between the three groups, but each of them has a unique genetic identity (12% of the single nucleotide polymorphisms were group-specific). The rDF is slightly more diverged from rHF than hDF. The inbreeding coefficient based on runs of homozygosity (Froh) was higher for rDF (0.24) than for hDF (0.17) or rHF (0.13). Our results also displayed the presence of several genomic regions that differentiated between the groups. In addition, thirteen, forty-five, and six ROH islands were identified in hDF, rDF, and rHF, respectively. The genetic diversity of the DF breed reduced over time, but this did not lead to higher inbreeding levels—especially, inbreeding due to recent ancestors was not increased. Over the last century, genetic diversity in many European national cattle populations has been affected by replacement of traditional local breeds with just a few specialized milk producing breeds, e.g., the Holstein Friesian, Brown Swiss, and Jersey. The predominant use of these breeds caused a sharp decline in the population size of local dual-purpose breeds ,2. AlthoThe change in use from local breeds to specialized breeds has been observed in many industrialized European countries including the Netherlands ,5. UntilCurrently, single nucleotide polymorphism (SNP) chips are available for the majority of livestock species, targeting genetic variants widely spread along their entire genomes. Importantly, SNPs detected in commercial breeds were selected and used to design the chips leading to some ascertainment bias when using these chips in studies with local breeds . The latThe objective of this study is to investigate genomewide genetic diversity and loss of alleles between three groups of bulls, chosen from the historic (1961–1989) and recent (2003–2015) DF population and the recent HF (1998–2014) population. Differences in allele frequencies and in homozygosity will provide insights into the mechanisms underlying their genomic differences caused by selection or a sharp decrease in the number of breeding animals.https://www.genebankdata.cgn.wur.nl/, accessed on 27 July 2021. From this genetic material, a group of 12 historic  and a group of 12 recent  Dutch Friesian bulls were sequenced. The animals were selected based on their year of birth by taking the oldest and youngest DF sires, while avoiding closely related animals in the selection. Furthermore, sequence data from a group of 12 recently used Holstein Friesian bulls in the Netherlands  were available for this study. These bulls were a selection of unrelated animals born in different years and sequenced for a project in 2017 on efficiency and health indices of the breeding company in dairy cattle CRV.Genetic material from purebred Dutch Friesian animals born from 1961 onwards has been preserved by the Centre for Genetic Resources, the Netherlands (CGN) of Wageningen University and Research, i.e., the Dutch Gene bank . Base quality recalibration (BQSR) was performed with ‘BaseRecalibrator’ and ‘PrintReads’ of the Genome Analysis Toolkit . The known variants file  generated by the 1000 Bull Genomes Project was used to mask out positions with known variation to avoid confusing real variation with errors. The before/after BQSR reports were checked using ‘AnalyzeCovariates’ to ensure that base quality scores were corrected as expected. SNPs were called using the GATK ‘HaplotypeCaller’ with ‘-ERC GVCF’ option. The rate of genome alignment and average sequencing depth were determined with Qualimap (version 2.2.1) software [DNA was isolated form sperm using the Echolution Sperm DNA kit . Library preparation and sequencing of the DF animals were performed at the Institute national de la Recherche Agronomique (INRA), France, following their established protocols. Library preparation and sequencing of the rHF animals were performed at BGI, China, following their established protocols. Paired-end sequencing was performed on the Illumina HiSeq platform. All animals were sequenced with short reads at 10× coverage. We followed the 1000 Bull Genomes Project Run 7 guideline  to process the raw sequence data into both binary alignment map (BAM) and genomic variant call format (GVCF) files . A per-bsoftware . The ‘GeThe variants were called across all three groups combined. Only biallelic SNPs were kept, and filtration for Minor Allele Frequency was not applied at this stage. Per group , a maximum of 2 out of 12 animals were allowed to have a missing value per SNP. These criteria resulted in a total of 10,780,681 SNPs, genotyped in all three groups, for further analysis.To identify group-specific SNPs, each SNP that passed the applied filtering criteria was analyzed according to the information about the three groups. An allele was labelled as group-specific if it was only present in one of the three groups and not detected in any of the other two, also called private allele .http://www.R-project.org/, accessed on 4 November 2021).To explore the genetic distance between animals of the three groups, a Principal Component Analysis (PCA) was performed using the --pca function in PLINK (version: 1.90) . The grao) and Expected Heterozygosity (He) and Minor Allele Frequency (MAF). Observed and Expected Heterozygosity were calculated using VCFtools (version 0.1.13) (--het) [Various parameters were used to estimate genetic diversity within the groups: Observed (H (--het) . The MAFFst values, Z-transformation was performed Fst is mean Fst in a window, µFst is an average Fst over all windows, and αFst is a standard deviation of Fst values of all windows tested for a given comparison [ZFst value > 8. To reduce the number of false positives, windows with less than 5 SNPs were removed.The fixation index (Fst) was used to characterize the differentiation between the groups, i.e., to identify selection signatures. The pairwise estimates of Fst among the three groups  were calculated using VCFtools with the Weir and Cockerham approach (--weir-fst-pop) . Windowsmparison . The ZFsmparison . CandidaRuns of homozygosity (ROHs) were calculated to identify contiguous regions of the genome where an animal is homozygous across sites. ROHs were calculated individually using PLINK with adjusted parameters: --homozyg --homozyg-window-snp 50 --homozyg-snp 50 --homozyg-kb 300 --homozyg-density 50 --homozyg-gap 1000 --homozyg-window-missing 5 --homozyg-window-threshold 0.05 --homozyg-window-het 3, following Cheng et al. . No linkTo identify genomic regions most commonly associated with ROH, i.e., ROH Islands, the percentage of the occurrences of a SNP in ROH was calculated by counting the number of times the SNP was detected in those ROHs across animals  within eAmong the set of 10,780,681 SNPs, 11.98% were identified as putatively group-specific , indicatThe genetic structure of the three groups assessed with the first three principal components of PCA accounted for 13.5% (PC1), 6.4% (PC2), and 5.6% (PC3) of the total variation . The firo and He), were used to determine the levels of genetic variability in the three groups . For all three groups, Ho was lower than He. There were small differences in Ho between three groups: rHF had the lowest Ho value (0.19) and rDF had the highest Ho value (0.20).Minor Allele Frequency together with Observed and Expected Heterozygosity . The level of ancient inbreeding reached 0.05–0.13 (Froh > 2 Mb) with rDF having the highest level, whereas the recent inbreeding load was 0.01 (Froh > 16 Mb) for all three groups. So, Froh decreased as the minimum length of the ROH increased.The inbreeding coefficient derived from ROH (Froh) in different length categories differentiated past and recent inbreeding . Recent The mean Froh values for each chromosome followed the same pattern as those computed for the whole genome, but there was variation across the chromosomes . For mosThe number and length of ROHs differed among animals and across groups . The rDFTo identify the differentiated genomic regions among the groups, the Z-transformed Fst (ZFst) values based on SNPs in 40-kb sliding windows with 20-kb steps were calculated. The ZFst varied markedly across the genome in all three comparisons  . We idenThe genomic distribution of ROH islands was nonuniform across chromosomes, regardless of the group . DiffereOnly one of the ROH islands identified ((BTA1: 101.26–101.30 Mb in rDF) overlapped with the genomic regions identified using pairwise Fst (hDF vs. rDF and rDF vs. rHF).https://www.fao.org/dad-is; accessed on 15 October 2021).In this study, we investigated genomewide genetic diversity within and between groups of historic and recent DF bulls, and a group of recent HF bulls. In the Netherlands, local, dual-purpose cattle breeds, including the DF breed, have gradually been replaced by the specialized dairy breed HF during the past decades. This has caused a decline in the population size of local breeds and potentially a loss of genetic diversity. The historic Dutch Friesian animals used in this study were born between 1961–1989, when the population size of DF was still large. The recent DF animals were born between 2003 and 2015. In the approximately 5–10 generations between hDF and rDF, the population size (number of adult cows) of DF has declined significantly, from 629,410 in 1970 to 3153 in 2017 [Our findings indicate that a large amount of diversity is common to the three groups. A high percentage of shared SNPs was found for the 3 groups, which is expected since all groups descend from the same ancestors. The founders of the Holstein Friesian breed originated from the Dutch Friesian breed . Furthero, an indication of a lack inbreeding has increased in rDF. This is confirmed by the inbreeding level determined by pedigree for the whole population [A decimation in numbers of a population is expected to reduce its genetic diversity and increase inbreeding levels. Indeed, diversity in the DF has reduced, e.g., rDF contains fewer specific alleles than hDF and, in the Principal Component Analysis, members of hDF are spread out across PCA2 while rDF animals cluster. However, based on manually checking the individual pedigrees of the hDF and rDF group, inbreeding levels have not increased. On the contrary, Hpulation , which ipulation . One expManual checking of individual pedigrees indicated local breeding in historic DF. Breeders generally used their own bulls and certainly no bulls from other regions. In particular, there was a separation between Friesian bulls and bulls from the North Holland region. The animals from North Holland were, for example, slightly larger and produced more milk, but had less conformation than the animals from Friesland . CurrentIn the 1990s, when the DF rapidly declined, the DF herdbook initiated a strategy called fundament breeding to counter the loss of diversity. In this strategy, the breed is divided into several fundaments, each consisting of one or a few herds. Within each fundament, 4–5 own bulls are used and rotated over groups of cows so that inbreeding is postponed for at least three generations. Bulls should not be exchanged across fundaments to safeguard their genetic distinctness. This latter point was not strictly adhered to  and our In conclusion, although the diversity has reduced, this has not led to higher inbreeding levels—particularly, inbreeding due to recent ancestors has not increased. The policy of the breeding organization has influenced inbreeding levels but has not prevented the loss of some diversity, and diversity conserved in the gene bank has been lost from the live population. Therefore, to maintain and improve the genetic diversity in the current DF population, material from historic individuals present in the gene bank, should be used in the life population. Furthermore, the current strategy of rotating bulls within the fundaments should be maintained to limit the increase in inbreeding.The pairwise Fst highlighted the presence of several genomic regions that differentiated between the groups. The Fst-based approach does not directly indicate in which group selection is operating. In this study, the region with the highest ZFst values for the comparison hDF–rDF are observed from BTA1 (96.50–96.58 and 98.80–98.86 Mb) and BTA2 (72.54–72.62 and 75.80–75.86 Mb). In two of the four regions, no genes are located, while three genes are located in the other two regions: EIF5A2, RPL22L1, and ENSBTAG00000051422. EIF5A2 is associated with fertility traits. EIF5A2 has been reported as a candidate gene for age at sexual maturity in Indian Buffalo  and for In the case of the rDF versus rHF group, we identified seven highly differentiated regions. Genes are only found in two regions on BTA22: ALS2CL, LRRC2, and TDGF1.http://www.pantherdb.org/ accessed on 24 October 2021). Some traits are associated with these candidate genes as well. For example: the PLAG1-CHCHD7 region (BTA14: 23.33–3.38 Mb) is associated with stature; body size, including height; and weight in many cattle breeds [Almost all ROH islands found in hDF partially overlapped with ROH islands found in rDF. These partially overlapped regions probably preserve segments in high homozygosis, characteristic of the ancient selection of the population. In these regions, several known candidate genes, such as HCHD7, FBXO2, MAD2L2, MOS, and PLAG1, are mapped . These ce breeds ,50,51,52The largest ROH island in the rDF group was found on BTA7 between 50.03–50.86 Mb. This region seems to coincide with an ROH island reported for taurine and indicine cattle breeds by Sölkner et al.  and for The largest ROH island in the rHF group was found on BTA8 between 105.89–106.21 Mb. This region contains one gene: ASTN2. The ASTN2 gene has been related to carcass weight of cattle  and meatOur results revealed that the Dutch national genebank has stored material containing genetic diversity that has been lost in vivo by selection and drift. Gene bank collections have been shown to capture more diversity than some in situ populations thanks to periodic resampling ,60,61. In = 10,780,681). According to Willing et al. [n = 4 to 6) if the number of markers examined is large, i.e., larger than 1000. A small sample size can lead to poor population structure estimates, which affects the ability to differentiate between loci that were under selection and neutral population structure [The accuracy with which allele frequencies and, therefore, inbreeding is estimated will depend on the sample size and number of SNPs . In thisg et al.  and Nazag et al. , Fst cantructure . Howevertructure .Through the present study with WSG data, we have described the genetic differences between historic and recent Dutch Friesian groups, and a recent Holstein Friesian group. Our findings revealed that a large amount of diversity is shared in the three groups and each of the groups has a small number of group-specific SNPs. The two DF groups are genetically distinct from the HF group. rDF is slightly more diverged from HF than hDF. We identified changes in the genetic composition of the DF population in the approximately 5–10 generations between the historic and the recent DF group. The genetic diversity has reduced and a more homogeneous group has emerged. Although diversity was reduced, this did not lead to higher inbreeding levels—especially, inbreeding due to recent ancestors has not increased.


Our previous studies suggested that the short interspersed nuclear element (SINE) retrotransposon insertion polymorphisms (RIPs), as a new type of molecular marker developed very recently, are ideal molecular markers and have the potential to be used for population genetic analysis and molecular breeding in pigs and possibly it can be extended to other livestock animals as well. However, no report is available for the application of SINE RIPs in population genetic analysis in livestock, including pigs. Here, we evaluated 30 SINE RIPs in several indigenous Chinese miniature pig breeds, including three subpopulations of Bama pigs . BM-cov is a subpopulation conserved in the national conservation farm, and BM-clo is a closed population maintained over 30 years with only 2 boars and 14 sows imported from its original area, while BM-inb herd is an 18 generation continuous inbreeding line based on the BM-clo population. To our knowledge, it is the first time to report the genetic diversity, breed differentiation, and population structures for these populations by using SINE RIPs, and which suggests the feasibility of SINE RIPs in pig genetic analysis.RIPs have been developed as effective genetic markers and popularly applied for genetic analysis in plants, but few reports are available for domestic animals. Here, we established 30 new molecular markers based on the SINE RIPs, and applied them for population genetic analysis in seven Chinese miniature pigs. The data revealed that the closed herd (BM-clo), inbreeding herd (BM-inb) of Bama miniature pigs were distinctly different from the BM-cov herds in the conservation farm, and other miniature pigs . These later five miniature pig breeds can further be classified into two clades based on a phylogenetic tree: one included BM-cov and Wuzhishan, the other included Congjiang Xiang, Tibetan, and Mingguang small ear, which was well-supported by structure analysis. The polymorphic information contents estimated by using SINE RIPs are lower than the predictions based on microsatellites. Overall, the genetic distances and breed-relationships between these populations revealed by 30 SINE RIPs generally agree with their evolutions and geographic distributions. We demonstrated the potential of SINE RIPs as new genetic markers for genetic monitoring and population structure analysis in pigs, which can even be extended to other livestock animals. Miniature pigs, due to their physiological, anatomical, and genetic similarities to human beings and the relative easy handling, are regarded as a key animal model in biomedical studies ,2. ThereRetrotransposons, as major genomic parasites of mammals, occupying for 30–45% of the genomic sequences in mammals ,11,12,13Ear or blood samples were collected from seven miniature pig populations of BM-cov, BM-clo, BM-inb, Congjiang Xiang, Wuzhishan, Tibetan, Mingguang small ear, and one Italian pig breed (Sicilian black pig/Nero Siciliano pig) and one commercial breed (Landrace pig), with a sample size of 24, 29, 22, 28, 24, 28, 20, 32, and 32, respectively. The TIANamp Genomic DNA Kit  was used for DNA isolation from the samples of each animal using the TIANamp Genomic DNA Kit . The quality of DNA was verified by using NanoPhotometer  and electrophoresis. BM-cov samples were taken from the national Bama conservation farm in Bama autonomous county, Guangxi Zhuang autonomous region. BM-clo and BM-inb samples were collected from the farm in Guangxi University pig farms in Nanning, Guangxi province. Congjiang Xiang samples were provided by Guizhou University pig farm in Guiyang, Guizhou province. Wuzhishan samples were from the national Wuzhishan conservation farm in Haikou, Hainan province. Tibetan samples were from the pig farm in the Animal Husbandry Research Institute of Ganzi Tibetan Autonomous Prefecture, Sichuan province. Mingguang small ear pig samples were taken from the national Mingguang small ear pig conservation farm in Tengchong, Yunnan Province. Sicilian black pig is an autochthonous genetic type that lives in the woods of the Nebrodi and Madonie mountains on the northern coast of the Mediterranean island of Sicily  [SINE RIPs were identified based on our recently established protocol (unpublished data). Briefly, the main process was divided into four main steps. (1) Screening SINE insertions in the genomes with a custom library which was built in advance  by usingPCR reactions were carried out in a total volume of 20 μL, composed of 1 μL 50 ng/μL genomic DNA, 10 μL 2× Taq Master Mix buffer , 1 μL of 10 μM primer F, 1 μL of 10 μM primer R, and 7 μL water. The PCR reaction conditions were set as following: 94 °C for 5 min for an initial denaturation, followed by 30 cycles  and a final extension of 10 min at 72 °C. The PCR products were detected by electrophoresis in a 1.5% agarose gel with 1× TAE buffer using a constant voltage of 130 V for 30 min. Gels were stained by ethidium bromide and visualized with UV fluorescence.IS, FST, FIT), and the Hardy–Weinberg equilibrium test was determined using Popgene [Allele frequencies, number of effective alleles per locus (Ne), observed heterozygosity (Ho), expected heterozygosity (He), fixation index . CLUMPP [Cluster analysis based on Nei’s genetic distance  was carr. CLUMPP  , which were predicted according to the protocol described in methods, were selected to evaluate their polymorphisms in 243 animals of seven Chinese miniature pig breeds, one commercial breed, and one Italian native breed (Sicilian black pig) which was selected as an outbreed control. The genomic coordinates of these markers, their PCR primers, and the predicted PCR product sizes were listed in ectively . The genectively . The Harectively .The genetic parameters, including Ne, He, Ho, PIC, and Fis for each population, are presented in ST value was 0.3780 when all loci were considered, indicating that approximately 37.80% of the total genetic variation between breeds, while the remaining 62.20% were attributed to differences among individuals within a breed  to 0.2612 (REF-13182)  . A very F-13182) . HoweverThe pairwise Nei’s distances between populations are shown in To measure the population structure and degree of admixture, we applied the STRUCTURE algorithm and principal component analysis (PCA), and the UPGMA tree was generated based on the Nei’s genetic distance. We analyzed the grouping situation when K ranged from 2 to 7, meaning that we presupposed that all individuals originated from K ancestors or breeds. The cluster results based on STRUCTURE are shown in Active retrotransposons move randomly in the genome, resulting in different types of structural variations, such as insertion, deletion, reversion, and recombination , and mayGenetic markers based on the microsatellites are widely used for the analysis of genetic diversity of Chinese miniature pigs, and the values of He and PIC are designated as important genetic parameters of genetic diversity . BotsteiBased on the SINE RIPs, we also found that most investigated miniature pigs  displayed relatively high genetic diversity compared with Sicilian black and Landrace pigs according to the genetic parameters ; while the BM-inb and BM-clo represented low genetic diversity, which generally agrees with the known genetic background and histories of these two populations. The BM-clo population, kept as a closed population in Guangxi University farm for over 30 years, was originally set up by importing 14 sows and 2 boars from the original place (Bama town) in 1987, while the BM-inb population, offspring of the 10th generation of BM-clo pigs, is a highly inbred line due to continuous inbreeding (>18 generations). However, the large genetic distances of BM-inb and BM-clo from the BM-cov pigs disagreed with their population relationships since both BM-inb and BM-clo populations were originated from the BM-cov. The exact reason is not apparent, but it may be because most detected loci (30 SINE RIPs) have been highly homogeneous in BM-inb and BM-clo pigs due to inbreeding, which resulted in an inaccurate estimation of genetic distances and population structures. The low Fis value estimation in Bov-inb may be due to the same reason. It was clear that BM-inb and BM-clo shared a large proportion of ancestry. But they did not show a close genetic relationship with the BM-cov breed. BM-cov breed was an admixture with Wuzhishan in the same clade, and Congjiang Xiang, Tibetan, and Mingguang small ear pigs formed a distinct clade, which is in good agreement with the phylogenetic analysis of 47 Chinese and European domestic breeds and wild boars based on 1.4 million SNP chip . Bama anIn summary, we identified 30 SINE RIP markers and applied them to determine the genetic diversity, differentiation, and population structure in seven Chinese miniature pig populations. Low genetic diversity, large genetic distance, and differentiation of BM-inb and BM-clo from the BM-cov and other miniature pig populations were observed. Our data revealed that the genetic distance, diversity, and breed-relationships between these populations generally agree with the evolutions and geographic distributions of these populations, and also basically agree with the population genetic analysis based on the SNP array, indicating that the SINE RIPs are reliable and applicable for population genetic analysis in pigs. In addition, our data also suggested that more SINE RIPs are required for population genetic analysis for high inbreeding populations. Overall, we demonstrated the potential of SINE RIPs in population genetic analysis, suggesting an alternative genetic marker that is simple, reliable, and high-quality. If RIP markers are analyzed in low numbers, it has the advantage of requiring no highly sophisticated instruments necessary to the capillary electrophoresis of labeled microsatellites or reading SNP chips. When a larger number of RIPs are analyzed, a labor-saving approach for genotyping is expected to be developed.


To evaluate the forensic application values of 19 autosomal short tandem repeat (STR) loci in canines. n = 200; Shiba Inus, n = 175) were analysed by the capillary electrophoresis platform. The allele frequencies and forensic parameters were calculated, and the genetic relationships between these two canine groups and a previously reported Labrador group were analysed.  The 19 STR loci in two canine groups .  The 19 STR loci had high genetic polymorphisms and forensic application values in Pembroke Welsh Corgi and Shiba Inu groups. In recent years, it has been found that the study of non-human DNA genetic polymorphism has great significance for case investigation in the forensic practice, especially for canine which is closely related to human. Canine is the most frequently kept pet in the world today. Meanwhile, canine-related cases are increasing rapidly, such as cruelty to animal, attack on people or animal, involvement in crime scene, property damage, and the identification of lost pet. In 1999, Schneider et al. used mitochondrial DNA as case evidence that were extracted from canine hair . In 2004The analyses of DNA genetic polymorphisms are helpful for the identifications and pedigree controls of canine individuals. In 2011, the International Society for Forensic Genetics (ISFG) clarified that the non-human DNA analysis was similar to human DNA testing . STRs arThe molecular genetic markers in canine DNA also need to be evaluated in different groups like the genetic markers in human DNA. However, there are still relatively few studies on genetic polymorphisms and forensic application values of molecular genetic markers in different canine groups. Pembroke Welsh Corgis, the long-bodied and short-legged canines with erect ear and fox-like head, are the small canines originated from Wales of England. Shiba Inu is a kind of canine which has ancient origin from Japan. In this study, 200 purebred Pembroke Welsh Corgis and 175 purebred Shiba Inus were used as research objects to evaluate the genetic polymorphisms of 19 STR loci. It can not only assist in breeding pure Pembroke Welsh Corgis and Shiba Inus but also provide scientific evidences for the case investigations involving Pembroke Welsh Corgis or Shiba Inus in forensic caseworks.Saliva samples were taken from 200 purebred healthy Pembroke Welsh Corgis and 175 purebred healthy Shiba Inus based on the recommendations of the ISFG on non-human DNA analysis and the ethics committees of the Xi'an Jiaotong University Health Science and Southern Medical University. All canines were registered with the National General Kennel Club. Informed consents had been obtained from the owners of the canines before the study began. Genomic DNA was extracted by using the Chelex-100 method .μl system, which included 1 μl DNA template, 5 μl PCR buffer, 5 μl Primer Mix, and 14 μl sterile water. The amplification was performed on a GeneAmp® 9700 PCR thermocycler. The reaction conditions were as follows: initial denaturation at 95°C for 2 min, followed by 29 cycles at 94°C for 5 s and at 60°C for 1 min, and final incubation at 60°C for 10 min. And then, amplified products were held at 10°C.The 19 autosomal STR loci  of the commercial PBG Canine Genotype STR system  were amplified in a 25 μl of the product or 1 μl of ladder were combined with 9 μl Hi-Di formamide and 0.3 μl internal size standard. The 19 STR genotypes were conducted by capillary electrophoresis on Applied Biosystems™ 3500xL genetic analyser with default instrument settings. Subsequently, the raw data and the allelic ladder were analysed by GeneMapper ID-X software. All sizes were calculated by using internal size standards on the 70, 80, 100, 125, 150, 175, 200, 233, 266, 300, 333, 366, 400, 445, 490, and 500 bp.After amplification, 1 FIS) was evaluated by genepop software v4 [https://cran.r-project.org/bin/windows/base/old/3.6.2/). The phylogenetic tree was conducted by MEGA X software [K value was calculated by the online software Structure Harvester [The Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD) were tested by the Arlequin software v3.5  and the tware v4 . The prisoftware . The gensoftware , and thearvester .A total of 165 alleles were observed at 19 STR loci in 200 purebred Pembroke Welsh Corgis with the allele frequency distributions from 0.0025 to 0.8325 , and 180P values of all 19 loci were greater than 0.0026, suggesting that the two canine groups in this study were consistent with HWE except for locus VGL3438 in the Pembroke Welsh Corgi group.In HWE tests, the correction level was adjusted to 0.0026 (0.05/19) after the Bonferroni correction. The estimated r2 values in this study. Figures r2 values of the linkage disequilibrium tests, in which 0 represented the value less than 0.01, 1 represented the value was from 0.01 to 0.02, and 2 represented the value was from 0.02 to 0.03. The results showed that all r2 values were less than 0.02, indicating that there was no linkage disequilibrium at the 19 STR loci in these two canine groups.Linkage correlation coefficient could be used to not only test the existence of LD but also evaluate the strength of LD. The correlation coefficient between the two loci was reflected by FIS was calculated to evaluate the level of inbreeding in these two canine groups. The FIS value was 0.0619 in the Pembroke Welsh Corgi group, while the FIS of the Shiba Inu group was 0.0865.The As shown in −17. The PD and PE values ranges from 0.4899 to 0.9551 and 0.0635 to 0.6462, respectively. The combined power of discrimination (CPD) and the combined power of exclusion (CPE) were 0.9999999999999999696 and 0.999889, respectively.In the corgi group, the Ho values of 19 STR loci ranged from 0.3000 to 0.8250, and the He values were from 0.2965 to 0.8426. The heterozygosity values (Ho and He) of loci PEZ01 and FH2004 were less than 0.5, and there were 7 loci  which were greater than 0.7. All PIC values were greater than 0.25, and 16 loci were greater than 0.5. The MP values ranged from 0.0450 to 0.5101, and the combined random match probability (CMP) was 3.04 × 10−19, 0.999999999999999999102, and 0.999911, respectively.In the Shiba Inu group, the He values were a range from 0.5277 to 0.8428 with the average of 0.7166. The Ho values of all loci were greater than 0.5 (except for PEZ01 and PEZ21), so were the PIC values. The MP, PD, and PE values were in the range from 0.0475 to 0.2837, 0.7163 to 0.9525, and 0.1443 to 0.5471, respectively. The CMP, CPD, and CPE were 8.98 × 10n = 214) [As shown in K) were set to 2-7 in the structure analysis, and the optimal K value was determined to be 3 after calculations. As shown in K = 3. In The PCA and genetic structure analyses were performed to reveal the genetic structure of three canine groups. As shown in In this study, we investigated the genetic polymorphisms of 19 STR loci in 200 Pembroke Welsh Corgis and 175 Shiba Inus. Canine samples were collected, stored, and conducted in the manner that were similar with corresponding human forensic DNA analysis process, and the allele frequencies and forensic genetic parameters of the 19 loci in these two canine groups were evaluated, respectively, according to the recommendations of ISFG on non-human DNA analysis in forensic cases [FIS values were calculated to evaluate the level of inbreeding in this study, and the results showed the inbreeding levels of these two studied canine groups were low (0.0619 in the Pembroke Welsh Corgi group and 0.0865 in the Shiba Inu group), respectively.The present results showed that there was no linkage disequilibrium among the 19 STR loci, and only one locus (VGL3438) deviated from Hardy-Weinberg equilibrium in the corgi group, which may be the reason for the sampling. In the canine breeding process, inbreeding is often the most common way to pursue the purity of the breed. And canine pedigrees are usually not as well documented as those of humans, so it is difficult to establish the canine family tree. Therefore, the disturbance of the inbreeding still could not be completely avoided, although we tried to track breeding to avoid collecting the biological related samples in the sampling process, and exclude possible genetic related samples after comparing the genotyping results. How to circumvent this problem is still the difficulty of non-human DNA analysis. In addition, In addition, the locus C38 with the highest heterozygosity, PIC, and PD values was high genetic diversities in the Pembroke Welsh corgi and Shiba Inu groups, which may have high potential in the applications of individual identifications of canines. The Pembroke Welsh Corgi, Shiba Inu, and Labrador groups had different genetic structures and a little genetic communications based on the results of PCA and structure analyses. The phylogenetic tree revealed that genetic relationship between the Pembroke Welsh Corgi group and Labrador group was closer in three canine groups. The Pembroke Welsh Corgi is an ancient breed of canine which originated from Wales, England. The modern Labrador is a king of breed that has been systematically bred and improved after being introduced into Britain in the 19th century, although they originally came from Newfoundland. The Shiba Inu is an ancient breed of Japanese origin. This may be the reason that the genetic relationship between the Pembroke Welsh Corgis and Labrador groups was closer than the genetic relationships between these two groups and the Shiba Inu group.In this study, we found that the 19 STR loci have high genetic polymorphisms and forensic application values in the Pembroke Welsh Corgi and Shiba Inu groups. By genetic analysis among the two studied groups and the previously reported canine group, the Pembroke Welsh Corgi, Shiba Inu, and Labrador groups had different genetic backgrounds and structures, and the genetic relationship between the Pembroke Welsh Corgi and Labrador groups was relatively closer in three kinds of canine groups.


Aotus monkeys and matched with HLA-DRβ1* molecules for use in humans. This was aimed at producing the first minimal subunit-based, chemically-synthesised, immunogenic molecules (COLSARSPROT) covering several HLA alleles. They were predicted to cover 48.25% of the world’s population for 6 weeks (short-term) and 33.65% for 15 weeks (long-lasting) as they induced very high immunofluorescent antibody (IFA) and ELISA titres against S, M and E parental native peptides, SARS-CoV-2 neutralising antibodies and host cell infection. The same immunological methods that led to identifying new peptides for inclusion in the COLSARSPROT mixture were used for antigenicity studies. Peptides were analysed with serum samples from patients suffering mild or severe SARS-CoV-2 infection, thereby increasing chemically-synthesised peptides’ potential coverage for the world populations up to 62.9%. These peptides’ 3D structural analysis (by 1H-NMR acquired at 600 to 900 MHz) suggested structural-functional immunological association. This first multi-protein, multi-epitope, minimal subunit-based, chemically-synthesised, highly immunogenic peptide mixture highlights such chemical synthesis methodology’s potential for rapidly obtaining very pure, highly reproducible, stable, cheap, easily-modifiable peptides for inducing immune protection against COVID-19, covering a substantial percentage of the human population.Thirty-five peptides selected from functionally-relevant SARS-CoV-2 spike (S), membrane (M), and envelope (E) proteins were suitably modified for immunising MHC class II (MHCII) DNA-genotyped  The WHO has emphasised that a vaccine protecting >50% of the world’s population represents the most promising methodology for halting the spread of this life-threatening disease (i.e. herd immunity). The research results described in this article show that chemically-synthesised, highly immunogenic anti-SARS-CoV-2 peptides could almost achieve such goal; the disease’s rapid spread has increased the target level to 70% nowadays regarding the urgently required herd immunity.Efficacious methods are desperately needed for controlling SARS-CoV-2-induced corona virus disease , membrane (M) and viroporin envelop (E) proteins have been the most studied and are, perhaps, the most relevant.Aotus monkeys; MHCII-DNA analysis has shown that they can be extrapolated for human use due to such molecules’ striking similarity.All ∼162 SARS-CoV-2 vaccines to date in preclinical phase, plus 212 candidate vaccines in development , vector-The S protein has broad genetic aa sequence variability. It has ~400 permanent mutations  Figure . The S pSuch approach has been refined throughout the last 34 years working on vaccine development regarding malaria and some other infectious diseases –17; funcSuch modifications must be made since conserved, functionally-relevant aa sequences are ‘immunologically silent’ , i.e. thAotus class II molecules.NetMHCIIpan-3.2  and -4.0Aotus nancymaae monkeys  were immunised with properly modified, chemically-synthesised SARS-CoV-2 peptides to test their ability to induce an appropriate immune response. Previous DNA sequencing of ~700 Aotus monkeys  found 90%-98% identity with some human immune system molecules  and 82%-92% identity (I) and 87%-98% similarity with highly polymorphic class II molecules  .MHCII genotyped Amazonian wild-caught  DP, DQ) , 28, mak DP, DQ) –22. WorkAotus) suggests that any potential vaccine can be developed  in ~2½ months. Such methodology represents an excellent procedure for safe, highly immunogenic, and fast vaccine development/improvement. This can rapidly overcome this virus’ currently observed tremendous and threatening mutability by simply adding or replacing short (16-20 mer-long) peptides where a particular mutation has appeared in an already developed mixture, rather than expressing many or several complete S proteins  long) or their domains to cover SARS-CoV-2’s tremendous genetic variability.Our experience in this field (simultaneously genotyping t-Boc protecting group was removed from α-NH2 using 40% trifluoroacetic acid (TFA) and 0.01% anisole solution in DCM for 30 minutes, washed with DCM and IPA and neutralised with 10% TEA (Triethylamine)/DCM.The peptides were synthesised by the solid-phase peptide t-Boc strategy described by Merrifield  and modiThe peptides were detached from the resin by low [25% hydrogen fluoride (HF)] and high cleavage (100% HF). Low HF was carried out at 0°C for two hours using a -10° to 0°C temperature ramp for HF.HF was evaporated from the reaction tube; the product was washed with ethyl ether, extracted with TFA, and washed once more with ethyl ether. Reverse-phase high-performance liquid chromatography (RP-HPLC) was used for characterising the peptides which were separated on Chromolith RP-18e and purified on Vydac semi-preparative C-18 columns. A 10%-46% elution gradient was used with acetonitrile (ACN) + 0.05% TFA for 9 min at 3.0 mL/min flow rate ; purification involved a 60 min gradient at 1.5 mL/min flow.A Bruker Daltonics Microflex LRF was used for MALDI-TOF mass spectrometry (MS) analysis. An α-cyano-4-hydroxycinamic acid (HCCA) supersaturated matrix solution was prepared in 40% ACN: 60% water + 0.1% TFA. The peptide samples were dissolved in water and mixed with the matrix solution in a 1:6 ratio; 2 µL of each sample mixture was then placed in a target well and analysed.HPLC and MS analysis determined that 35 peptides were >99% pure  and left to dry. Fluorescein-5-isothiocyanate (FITC)-conjugated goat IgG (goat IgG-FITC) against purified Aotus IgG (produced in our Institute) was then added at 1:100 dilution in PBS, 1:80 Evans blue, incubated in a humid chamber and in the dark for 30 minutes, washed and left to dry. A drop of 50% glycerol in saline solution was added between the slides and microarray coverslips before observing them by fluorescence microscope with 100x oil immersion objective lens.SARS-CoV-2-infected VERO cell cultures, fixed on eight-well slides, were used as antigens for evaluating native state viral protein recognition. The slides were preserved at 4°C and left to dry at RT (room temperature). They were blocked with 30 µL/well 1% bovine serum albumin for 15 minutes, washed for 5 minutes with phosphate buffered saline (PBS) and left to dry at RT. This was followed by placing 15 µL/well immunised Aotus sera were placed in wells in 1:100 dilution, incubated at 37°C for 1 hour and washed again; 100 µL/well of goat anti-Aotus IgG peroxidase conjugate were then added at 1:1,000 dilution in blocking solution . They were incubated at 37°C for 1 hour, washed and 100 µL/well developing solution was added (TMB substrate peroxidase and hydrogen peroxide: 1:1 ratio). Microplate readings were taken 30 minutes later at 620 OD (LabSystems Multiskan MS). Thirty sera from COVID-19-recovered humans were evaluated with the peptides, following the procedure explained above.ELISA involved 96-well ELISA plates (F16 MAXISORP plates) being covered with 100 µL/well test peptide at 10 µg/mL in PBS, incubated for 1 hour at 37°C and overnight at 4°C. The sensitised plates were incubated at 37°C for 1 hour, washed 5 times with washing solution , followed by a final wash with distilled water. They were blocked with 200 µL/well blocking solution  and incubated at 37°C for 1 hour. After washing, 100 µL/well immunised 5 cells per well) were seeded onto 24-well tissue culture plates the day before infection. On the next day, 80 PFU SARS-CoV-2 were incubated with or without serially diluted Aotus or human heat-inactivated sera  at 200 μL volume in microcentrifuge tubes for 60 min at 37°C in 5% CO2. The mixtures were then added to VERO E6 monolayers and incubated at 37°C for 60 min; the inoculum was removed, 1 mL semisolid medium added  and mixtures were cultured at 37°C for 72 h. Semisolid media was removed and monolayers were washed twice with PBS, fixed and stained with 4% formaldehyde/1% crystal violet for 30 min and washed twice with PBS. A 50% reduction in plaque count (PRNT50) was used as neutralising endpoint. Viral control in the absence of serum and a serum control without SARS-CoV-2 were included in the tests  using Vero E6 cells was used for testing neutralising antibodies in monkey sera. Briefly, Vero E6 cells  primate station in Leticia in Colombia’s Amazonas department.Peripheral blood samples were taken from 99 Aotus genomic DNA (gDNA) from 300 µL fresh whole peripheral blood samples, according to the manufacturer’s instructions. Extracted gDNA quality and amount were evaluated on a μDrop Plate (Thermo Scientific Multiskan GO) and visualised on SYBR Safe-stained 1% agarose gel . The mitochondrial DNA (mtDNA) D-Loop gene was amplified for determining the species.A Wizard Genomic DNA Purification Kit was used for isolating Aotus MHC DRβ class II exon 2. The primers were designed using intron 1 and intron 3 sequences from previously reported A. nancymaae and A. vociferans allele lineages. The designed primers enabled amplification without lineage bias; this is an essential requirement for successfully typing the allele repertoire in each individual analysed. B_AoDRβ-F 5′-CCTTCGTGTCCCCACAGC-3′ and B_AoDRβ-R 5′-TCACAGGGAGGCCCCG-3′ primers were used for amplifying an MHC-II DRB gene exon 2 317 bp fragment. The Barcosel high-throughput sequencing platform was used for assigning each primer a 6 base pair (bp)-long molecular identifier (MID) (PCR was used for amplifying er (MID)  (i.e. foPhusion Hot Start II High-Fidelity PCR Master Mix (Thermo Fisher Scientific) was used for amplification, using the touchdown PCR technique. Initial denaturing involved 98°C for 1 minute, 30 cycles of 72-66°C for 15 seconds and a final extension step at 72°C for 5 minutes.Amplification was confirmed on 1.5% agarose gel and a Wizard SV Gel and PCR Clean-Up System (Promega) was used for purifying PCR products, following the manufacturer’s recommendations. A NanoDrop 2000 full-spectrum UV-Vis spectrophotometer was used for quantifying the products and the amplicons were sent for sequencing using the Illumina NovaSeq 6000 system (Novogene).The Amplicon Sequencing ASsignment tool  was used for automatic genotyping of MHC genes from high-throughput sequencing data . Reads RAmpliCHECK was used for analysing MHC DRB exon II using a 1% base substitution error rate, 0.001% indel error rate (base insertion or elimination) and 1% minimum per-amplicon frequency (PAF), i.e. predetermined parameters for Illumina sequencing data . AmpliCHMinimum read depth per amplicon was set at 100 . The recommended predetermined parameters were used for grouping potential alleles together: 25% user-defined frequency threshold relative to the dominant sequence (sequences in a group having the same or greater frequency regarding the dominant sequence were classified as ‘subdominant’ and moved to a new group) and 3% PAF filtering threshold . Chimeras  were identified and removed by the software.All bioinformatics tools used here came from the AmpliSAT suite  .The Allele Frequency Net Database (AFND) was used for data mining and choosing HLA-DRβ1* alleles  occurrinPocket profiles for these loci had been previously determined, defined as a specific amino acid (aa) combination occurring in MHC pockets .The NetMHCIIpan-3.2  server . The selAotus MHC-DR binding affinities and sequence typing. A set of 200,000 non-redundant UnitProtKB/Swiss-Prot UniProt-derived peptides (13- to 21-aa-long)   was usedaa-long)  was usedNetMHCIIpan-4.0 binding affinity (BA) and eluted ligand MS (EL) strategies and Seq2Logo Kullback-Leiber (KL) and Shannon (Sh) methodologies were used for calculating the similarity index (SI) regarding binding motifs for each allele, defined as Pearson distance ) with min-max normalisation using position-specific scoring matrices. The Dist function from the amap package in the R 3.6.0 programme was used for making the calculations .The set of epitopes’ potential coverage in the human population resulted from the sum of the alleles’ frequencies in the target population for which each designed peptide had strong affinity. The frequency of the 69 previously selected HLA-DRβ1* alleles calculated from the AFND  was usedAntigenicity studies involved using 30 human sera taken from PCR-positive individuals recovered 30 days after COVID-19 onset for determining minimal subunit-based, chemically-synthesised peptide relevance regarding immunity against SARS-CoV-2. Fifteen serum samples came from patients living in Medellin (northwest Colombia) who had been hospitalised in an intensive care unit (ICU) and 15 from patients from other parts of Colombia suffering relatively mild infection. IFA titres and ELISA reactivity were determined with native, unmodified parental peptides; NAb titres were only determined in the 15 hospitalised patients.2O and 80 µM sodium trimethylsilyl trifluoromethanesulfonate for nuclear magnetic resonance (NMR) measurements. A Bruker Avance 900 MHz and 600 MHz spectrometer with cryogenic probe were used at 298K for NMR experiments. Each peptide’s resonance was assigned by 2D TOCSY  and 2D NOESY   along with 6% Dxing me) . Shorterxing me) . 1H chemdard DSS .3JHN-Hα coupling (L and 55.7 β-sheet) , medium (2.8-3.5Å) and weak (3.5-5.0Å). Hydrogen bond constraints were introduced for low amide temperature coefficients; structure calculations only involved <5 -ΔδHN/ΔT ppb/K. Distance ranges involving likely NH···O hydrogen bonds were set at 1.8-2.5 Å between the residue acceptor oxygen (i-4) and the residue donor amide hydrogen (i). The calculations were repeated several times until a structure was obtained having minimum distance and angle restraint violations and the least root mean square deviation (RMSD). Structures having reasonable geometry and few violations were then selected. The UCSF Chimera package . The monkeys were kept in FIDIC’s field-station in Leticia, Colombia , looked after by expert primate veterinarians and workers, supervised weekly by expert biologists and veterinarians from the local environmental authorities and ethics committees. They were subcutaneously immunised at the Primate Centre with 3 mg per dose of the 35-peptide mixture in equimolecular concentrations. The first immunisation (day 0) involved the mixture being dissolved in 200 microliters of sterile double distilled water (ddH2O) emulsified with an equal volume of Freund’s complete adjuvant (FCA) containing 1 mg/mL dead Mycobacterium tuberculosis. The second immunisation (day 20) involved the same amount of COLSARSPROT mixture emulsified with incomplete Freund’s adjuvant in equal volumes. Bleeding for immunological analysis for IFA and NAb tests with individual native peptides and ELISA assays were drawn in pre-immune sera 6 and 15 weeks after initial immunisation.Ninety-nine wild-caught Aotus immunogenetic typing data had to be extrapolated to humans.Fifteen weeks (105 days) represented the longest follow-up time after immunisation for any SARS-CoV-2 vaccine by the time this trial was finished (Sep1/2020). The 75 immunised monkeys and 24 controls were DNA genotyped regarding their class II region to match human class II HLA-DRβ1* genes during the time the experiments lasted since This trial involved monkeys being maintained for an additional 4 months to determine immune response duration. They were then released back into the jungle close to their capture sites following agreement with the pertinent environmental authorities, this was accompanied by environmental authority officials (CORPOAMAZONIA) and some members of the ethics’ committees.The S-protein’s 3D structure reported by Wrapp  and Herr10 helix, some specific β-turn conformations) and immunological silence  when used in the mixture. They had strong reactivity against SARS-CoV-2 (Human/Medellín/UdeA1/2020 strain) as determined by bright immunofluorescent (green) dot recognition of the virus inside infected VERO cell cytoplasm   to 75% 85 days later  (Completely negative IFA titres in isation) , non-humisation) –56 and cisation) .Aotus sera reactivity with native (unmodified) parental peptides; Aotus sera was used in triplicate for ELISA at 1:100 dilution, showing that (22/35) S-, M- (42768 and 42796) and E (42806) -derived peptides were highly immunogenic. The results have been organised according to HLA-DRβ1* frequency in the human population (numbers in bold indicate modified ones), demonstrating also E and M subunit-based protein peptide immunogenicity for the first time  and 4501N alleles  . The absence of these alleles in this Aotus trial represented ~30% of the human population (Aotus population (Some peptides (10/35) designed to have high HLA-DRβ1* and Aona-DRβ core-binding probability (<1.0%) and rank (<1.0) did not induce any Abs by week 6 (This led to classifying 17/35 (48.25%) of the peptides inducing high and low ELISA Ab titres against unmodified parental molecules –24, 45. Aotus MHCII allele % similarity indexes strongly suggested the potential of 4 of the COLSARSPROT peptides most frequently recognised by Aotus monkeys for direct use in humans: 42766 re HLA-DRβ1*0101 (10.4%), 42796 re HLA-DRβ1*0401 (8.7%), 42816 re HLA-DRβ1*1501 (17.0%) and 42768 re HLA-DRβ1*0301 (17.0%). This would cover ~23.5% of the world’s population for ~6-15 weeks while ~½ of humankind could be protected for ~15 weeks if the other 12 LL-LiViNAb-inducing peptides strongly recognised by other alleles were included: 42780 re HLA-DRβ1*0404 (5.5%), 42772 re HLA-DRβ1*0302 (2.2%), 42792 re HLA-DRβ1*0403 (5.5%), 42812 HLA-DRβ1*0102 (4.1%), 42794 re HLA-DRβ1*0101 (10.4%), 42824 re HLA-DRβ1*1104 (6.5%), 42838 HLA-DRβ1*1302 (9.6%), 42806 re HLA-DRβ1*1501 (17.0%), 42820 re HLA-DRβ1*1301 (9.3%), 42804 re HLA-DRβ1*0901 (5.1%) and 42782 re HLA-DRβ1*1402 (1.0%)  (HLA-DRβ1*1101) and 42816(S) (HLA-DRβ1*1501) by 2/8 and 42768(M) (HLA-DRβ1*0301) by 1/8 in the aforementioned trial. This was associated with long-lasting live virus Nab (LL)-LiViNAb, thereby strongly supporting our appropriate peptide selection for modifications regarding COLSARSPROT development.Peptide 42766(S) (HLA-DRβ1*0101) was recognised by 7/8 Aotus seroconverting (by IFA) in week 6 developed NAb in LiViNAb assays at ≥1:20 dilution, some having a 1:160 titre (36.6% (15/41) of 60 titre ; howeverSamples were always run in triplicate (starting at 1:10 dilution up to 1:640) to avoid non-specific and noisy results (which would have increased the percentage of LiViNAb activity), unlike other groups reporting NAbs using plain, undiluted serum or 1:2, 1:4, 1:5 dilutions, thereby increasing their seroconversion frequencies.Aotus LiViNAb titres were equal to or higher than those induced by the leading vaccines now being injected into millions of people using the complete Wuhan S protein in mRNA-1273 , stabilising hinge conserved region (42830) and membrane fusion (42808)  taken from COVID-19-recovered individuals  were taken 30 days after disease onset (their IFA and NAb titres having been determined) (ELISA results regarding COVID-19 recovered (PCR-positive) people’s sera reactivity also provides strong support for this methodology since many HLA-DRβ1* allele-related peptides  were included in these trials to select the best one for a particular/target allele or to cover minimal allele variations in different ethnic groups.Aotus monkeys having the Aona-DRβ3802 analogue were found in this trial) since 42814 parental peptide had 10/30 COVID-19 recovered human sera reactivity (33.3%) above the threshold level and 42800 9/30 (30%), higher, close to HLA-DRβ*0701 world frequency . Something similar happened with HLA-DRβ1*0102 (42812) where few monkeys had the allele related to these two peptides but 9/30 (30%) of human sera has high reactivity with its parental peptide. Such allele frequency is high in Jews (19.3%) and Berbers (16.2%) and average in the worlds’ populations (4.1%). High sera reactivity frequency (30%) was found for HLA-DRβ1*0302 (42802)  was derived from the E protein, clearly suggesting this viroporin protein’s great relevance regarding SARS-CoV-2 infection, which has otherwise been ignored in the immune response against this deadly and life-threating disease. Something similar occurred with HLA-DRβ1*1101 (42776) also being recognised by recovered individuals’ sera .These results also stress the importance of designing multiple peptides for the same HLA-DRβ1* allele; this enabled us to identify the best peptides for inclusion in COLSARSPROT or complement minimal allele variations in different populations. Regarding peptides designed for HLA-DRβ1*1501, 5/30 (16.6%) reacted with peptides 42806 and 42816, giving a percentage similar to that for the world population (17%). Native parental peptide 42806  did not react with COVID-19 recovered sera due, perhaps, to tremendous virus-related genetic variability in this and some other RBD region residues. The lack of peptide reactivity with sera from recovered people having matching HLA-DRβ1* 0901, 1104, 0402, 0403, 1502, 1503, 1001 and 0103 could have resulted from the few samples analysed  induced the highest reactivity against its parental wild-type (Wuhan strain) peptide in analysed  and thesanalysed .Aotus alleles Aona DRβ3802 and 4501N. Sera from individuals treated in an ICU had some weak reactivity with HLA-DRβ1*1101 (42790), HLA-DRβ1*1601 (42808) (both involved in membrane fusion) and HLA-DRβ1*1503 (42830) (highly immunogenic but unable to induce LiViNAb activity in Aotus). This highlighted some HLA-DRβ1* alleles’ association with mild infection or partial protection and others with severe infection; however, such data must be confirmed by using a larger group of patients involving different ethnic groups. It is interesting that none of these peptides had cross-reactivity with a large group of common human pathogens and vaccines , HLA-DRβ1*0301 (42826) and HLA-DRβ1*0102 (42812) which were not immunogenic in this monkey trial, due to the absence of vaccines .It should be mentioned that none of the aforementioned individuals were HLA-DRβ1* genotyped; however, ongoing work involves using their samples as they are accessible for genotyping the different Colombian ethnic groups .1H-NMR (600 to 900MHz) . This confirmed our extensive malarial vaccine development results showing that highly immunogenic protection-inducing modified peptides tended to have a propensity for PPIIL formation  and 42826(S) had β-turn type IV3 formation propensity  between high activity binding peptides (HABPs) distantly located in aa sequences but conformational very close in a molecule’s 3D structure , 71. CleSome examples of COLSARSPROT LiViNAbI linear epitope immunogenicity and/or NAb activity aa location in the S protein sequence, followed by our peptide number , would be: S487-498 42766) , 73, S116 , 73, SThese first results clearly suggested that multi-epitope, multi-protein, minimal subunit-based properly modified chemically-synthesised peptides form part of an excellent methodology for inducing an Ab-mediated immune response against SARS-CoV-2, i.e. IFA reacting with complete virus particles .Aotus monkeys was induced by 17/35 (48.25%) modified peptides having strong binding affinity, targeting specific HLA-DRβ1* alleles   which are completely different to humans where results could have been biased due to their immunogenetic characteristics.NetMHCIIpan-3.2 and -4.0 showed that LiViNAb activity during week 6 post-initial immunisation of wild-caught eptions) . This sueptions) , with HLeptions)  and HLA-eptions) , i.e. COThe fundamental difference regarding other vaccines lies in our methodology for developing minimal subunit-based (16-20 aa long) multi-epitope, multi-protein, highly immunogenic chemically-synthesised modified peptides able to induce LL-LiViNAb in DNA-Aona-DRβ genotyped monkeys designed to match human class II HLA-DRβ1* molecules (LiViNAb-inducers were subdivided into long-lasting (LL), LiViNAb-inducers (LL-LiViNAbI) and short-term (ST) NAb-inducers (ST-LiViNAbI) according to reactivity induced 15 weeks after the first immunisation. Very strong synergistic association was found in LL-LiViNAbI for some HLA-DRβ1* binding peptides  and ST-LiViNAbI , suggesting a key role for peptides associated with these HLA-DRβ1* alleles in this protection associated immunity. ST-LiViNAbI peptides were associated with inducing very short-term NAb immunological memory, in turn, being strongly associated with their 3D structure.Aotus monkeys immunised with 35 SARS-CoV-2 modified peptides and 24 controls) strongly suggested that close HLA-DRβ1* allele association with LL and ST immunity should be seriously considered when developing vaccines for the world’s population . This clearly suggested that more peptides are needed to cover the WHO’s actual goal of achieving 70% coverage to ensure herd immunity (ongoing work at our institute).Such solid experimental data  where some vaccines’ potential effectiveness is by-passed or diminished by VOC strains .Potential 48.25% COLSARSPROT coverage of the human population worldwide in week 6 was based on HLA-DRβ1*-binding peptide frequency for inducing LiViNAb activity in Aona-DRβ/HLA-DRβ* matched alleles  formation in the region fitting into the PBR , and complete protection-inducing peptides   . ConversFurther tracking studies/genetic sequence information published by GISAID regarding 235,611 sequences  regarding high‐quality protein annotation revealed that while S peptide 42766 has multiple genetic variations, E peptide 42806 only has the V58F and V62F genetic variants that were properly replaced; 42824> and 42816 are variable in the first residue and 42870 is variable in the second residue outside the MHCII-PBR. This means that they can be used in humans without any further modification as they are conserved in this critical MHCII-PBR segment.No cellular immune response studies were performed re the above due to the small amount of blood (1.5 mL) allowed to be taken from the monkeys. This approach cannot be informative, since performing assays with the complete 35-peptide mixture will not provide reliable information regarding individual peptides where each one should have been used for blastogenesis, IFNγ and multiple cytokine determination since cellular immune responses are so exquisite regarding aa sequence recognition, so much so that rotamer orientation of the 6 TCR contacting residues determines the immune response  , 23.Aotus data. It is clear that if Aotus reactivity is added to human sera reactivity, especially with peptides 42814 and 42800 HLA-DRβ1*0701 (22.3%) and 42776 1101 (11.2%) peptide mixture, worldwide coverage could be 62.9%, close to the WHO’s proposed ~70% coverage to reach desired herd immunity.The peptides’ antigenicity results regarding sera from COVID-19 recovered patients showed some association with HLA-DRβ1* allele frequency and further studies are in progress to complement human and There is no doubt that combining the data concerning immunogenicity in MHCII (Aona-DRβ) genotyped monkeys with antigenicity in COVID-19 recovered patients regarding the different forms of the disease’s presentation and different ethnic HLA-DRβ1* genotyped groups will provide a very logical and rational methodology for a second generation of minimal subunit-based, chemically-synthesised peptides. The aim is to obtain >70% coverage to benefit the world population’s livelihood and welfare, i.e. our institute’s goal for almost four decades.L formation to properly fit into MHCII-PBR antigen-presenting cells to allow an appropriate HLA-DRβ1*-peptide-TCR complex formation for inducing high NAb production . Our results have highlighted this concept and methodology as an outstanding tool for combating the COVID-19 pandemic and shows a logical approach to selecting functionally-relevant peptides having no or a minimum amount of genetic variation for developing COLSARSPROT, i.e. a highly immunogenic synthetic peptide mixture.Selected SARS-CoV-2 structures were modified and chemically-synthesised for developing COLSARSPROT; such modified peptides had a propensity for PPIICOLSARSPROT peptides are extremely pure (>99%), as assessed by physicochemical techniques (HPLC and MS), harmful-contaminant free , which is conformed by members of Instituto Colombiano Agropecuario (ICA), Corpoamazonia, and Municipal Board of Animal Defenders.Aotus genotyping and classification with AC. YS and MF were responsible for the immunological tests. CA and EG did the Aotus experiments. All authors contributed to the article and approved the submitted version.MEP and MAP conceived, designed, and developed the project. LP and JS did the peptide synthesis and HPLC and MS analysis. MTR, WA-J, and LF performed the neutralisation tests. MPA, AB, JA-C, and CR did the 3D structural calculations and analysed them. CG and AKR did the NMR studies. CFS was responsible for immunoinformatic procedures and did the Funding was provided by the Universidad de Ciencias Aplicadas y Ambientales (U.D.C.A) and the Universidad Santo Tomás. Funding to CG by Max Planck Society is gratefully acknowledged.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Tlaloc hildebrandi is an endemic freshwater species that lives in four subbasins of the Grijalva and Usumacinta basins, and one of the most geographically restricted species of the Produndulidae family. The species was originally described as endemic to springs in the high limestone plateau in San Cristóbal de Las Casas in the Río Amarillo subbasin . However, it was recently recorded in the Jataté and Tzaconejá subbasins in the upper Usumacinta basin, thereby expanding its known distribution range. The discovery of these populations is relevant not only for the conservation of the species but also for a better understanding of its evolutionary history. Currently, the scarce populations of T. hildebrandi, found in only a few localities in the Grijalva and Usumacinta basins, are fragmented and living under unfavorable conditions. Here, we analyzed three mitochondrial (mt-atp8&6 and mt-nd2) and one nuclear (nuc-s7) marker in order to assess the genetic diversity and population structure of T. hildebrandi. We found that, in comparison with other endangered freshwater fish species from Mexico, T. hildebrandi showed a lower level of genetic diversity . Moreover, the analyzed populations exhibited a strong genetic structure in accordance with their geographic distribution, and can be placed into three genetic clusters: (1) Amarillo plus Chenhaló in the upper Grijalva basin, (2) Jataté, and (3) Tzaconejá, both in the upper Usumacinta basin. On the basis of our results, we propose the recognition of at least three evolutionarily significant units (ESUs) for the species and the urgent implementation of ex situ and in situ conservation and management efforts that consider the genetic background of the species.The endangered Chiapas killifish  Profundulus and Tlaloc, during the early Miocene (26 Mya), and the second was a diversification event during the upper Miocene to early Pliocene (from 5 to 10 Mya) associated with divergences within each of these two genera , in particular, taken into consideration. This metric is important as it reflects the number of individuals breeding, rather than the total number of individuals in a population. When the Ne is small, genetic variation is lost rapidly due to genetic drift and the deleterious effects of inbreeding . With respect to the Poeciliidae, Xiphophorus cortezi and Poecilia sulphuraria have been two of the more well studied species. A low to moderate level of haplotype diversity (mt-d-loop: h = 0–0.530) was observed for X. cortezi, an endemic of the southern Panuco river system  analyses of the three known populations of the species revealed a positive correlation between population size and genetic diversity   .T. hildebrandi necessitates a characterization of the species’ genetic diversity, which will improve our knowledge of its current situation and inform conservation efforts. As previously shown, populations with limited ranges can be highly vulnerable to environmental changes, and those impacted by human-induced range reductions often lack genetic diversity information  and one nuclear (nuc-s7) markers. With this information, we can then establish Evolutionary Significant Units (ESUs) that may prove valuable in the management and conservation of the species.The importance of genetic diversity studies for conservation and the lack of genetic data for the endangered ormation . We hypoThe care and use of animals complied with the SEMARNAT animal welfare laws, guidelines and policies as approved by SEMARNAT-SGA/DGVS/01283/20 collection permit.T. hildebrandi on the basis of the taxonomic key of T. hildebrandi is known to occur  and ATP synthase 8 and 6 (mt-atp8&6) genes were amplified via polymerase chain reaction (PCR), as were the first and second introns of the nuclear S7 ribosomal protein gene (nuc-S7). Information on the PCR protocols and primers used are provided in 2 and dNTPs standardized, 0.2 μl of each primer (10 μM), 0.1 μl of MyTaq DNA Polymerase (Bioline), and 2 μl (10–100 ng) of DNA template. The PCR amplification conditions used followed mt-nd2, MW438870–MW438902; mt-atp8, MW438903–MW438924; mt-atp6, MW438925–MW438946; nuc-S7, MW438947–MW438978 .To evaluate the geographic correspondence of the haplotype distribution, network estimations were constructed for each gene fragment using the median-joining algorithm, as implemented in PopArt v1.7 (S), and nucleotide (π) and haplotype diversity (h), were estimated for each gene in Arlequin v3.5.1  and (2) groups considered as the three genetic clusters obtained from the haplotype network analyses, with the two subbasins of the upper Usumacinta basin (Tzaconejá and Jataté subbasins) each comprising a separate group and the Amarillo plus Chenalhó subbasins of the upper Grijalva comprising the third.Genetic diversity parameters, including the number of haplotypes (H) and polymorphic sites  groups considered as the two basins in which T. hildebrandi is distributed  and (2) the three major groups indicated by the haplotype networks, which are the Amarillo plus Chenalhó subbasins, the Tzaconejá subbasin, and the Jataté subbasin.To quantify the genetic differences among the sampled populations of ΦST) for each gene, with Bonferroni correction  were conducted using Arlequin v3.5.1.3 , 22 of mt-atp8&6 (788 bp), and 32 of nuc-S7 (819 bp), from the samples collected from 10 localities across the distribution range of T. hildebrandi in the upper Grijalva and upper Usumacinta basins , followed by nuc-S7  and mt-atp8&6 . When genetic diversity was calculated by basin , the upper Usumacinta  showed a higher level of genetic diversity for all loci, with mt-atp8&6  having the highest level, followed by nuc-S7 (h = 0.530 π = 0.001) and mt-nd2 . The low level of genetic diversity in the upper Grijalva basin  is mainly reflected by the lack of diversity found for mt-atp8&6 and nuc-S7 , Jataté showed a higher level of genetic diversity for the three genes  was recovered from samples from the Amarillo subbasin  in San Cristóbal de Las Casas, Chiapas ), those from the Chenalhó subbasin , and some from the Tzaconejá subbasin of the upper Usumacinta . One mutation step separated the most common haplotype of mt-nd2 from the rest of the haplotypes, except for one haplotype from the Chenalhó subbasin, which was separated by two mutation steps. The peripheral haplotypes, separated by one mutation step, included samples from the Amarillo and Chenalhó subbasins and those from the Jataté subbasin (upper Usumacinta). The Ocosingo and Las Lajas populations (upper Usumacinta), which did not have any shared haplotypes, were separated by two mutation steps (mt-atp8&6 was separated by one mutation step from the one in the Jataté subbasin (upper Usumacinta). Like for mt-nd2, the populations of Ocosingo and Las Lajas were separated by two mutation steps, and did not share any haplotypes  was found in samples from the Amarillo subbasin of the upper Grijalva , those from the Chenalhó subbasin, two from Ocosingo (Jataté subbasin) and one from the Tzaconejá subbasin. This haplotype was separated by one mutation step from the one found in the El Naranjal and Tzaconejá (Tzaconejá subbasin) and Ocosingo (Jataté subbasin) populations in the upper Usumacinta basin. Additionally, an exclusive haplotype separated by three mutation steps from the nearest haplotype was found in the El Naranjal population  between the two basins  was 0.1% for mt-nd2 and 0.2% for mt-atp8&6 and nuc-S7 , whereas the highest distance was observed between the Jataté and Amarillo + Chenalhó subbasins (0.3%). Results for mt-atp8&6 were the same as for mt-nd2, except that a relatively high genetic distance was also found between the Jataté and Tzaconejá subbasins (0.3%). By contrast, for nuc-S7, the lowest genetic distance was observed between the Jataté and the Amarillo + Chenalhó subbasins (0.1%), and the highest was between the Tzaconejá and the Amarillo + Chenalhó subbasins (0.2%), though the Tzaconejá and Jataté subbasins also showed a distance of 0.2%  resulted in ΦST values of 0.117 for mt-nd2, 0.480 for mt-atp8&6, and 0.692 for nuc-S7, all of which were significant. However, pairwise comparisons among the three main groups identified by the haplotype analyses showed a lack of genetic differentiation between Tzaconejá and the Amarillo + Chenalhó subbasins for mt-nd2 (ΦST = 0), though significant differentiation between Jataté and the Amarillo + Chenalhó subbasins, and also between Jataté and Tzaconejá . Significant differentiation between the same subbasins for mt-nd2 was observed for mt-atp8&6, though with higher ΦST values . Finally, for nuc-S7, significant differentiation was only observed between Tzaconejá and the Amarillo + Chenalhó subbasins (ΦST = 0.823); moderate but non-significant differences were found between Jataté and the Amarillo + Chenalhó subbasins ; however, differences between populations and within populations were significant , with ΦST as the only significant factor. When the samples were grouped according to the three groups indicated by the haplotype analyses, the percentage of variation among groups was as follows: for mt-nd2, 40.02% ; for mt-atp8&6, 66.70% ; and for nuc-S7, 76.94% , and only the ΦST value was significant for all three markers . Migration rates for mt-atp8&6  were lower than those for nuc-S7 . The results for the mitochondrial genes indicate a greater magnitude and direction of migrants from Grijalva to Usumacinta than from the other direction. By contrast, the rates for nuc-S7 indicate greater migration from Usumacinta to Grijalva , mt-atp8&6 , and nuc-S7 . The genetic diversity values obtained for T. hildebrandi are considerably lower than those reported for some other endangered Cyprinodontiformes in Mexico. For instance, in the viviparous goodeine Z. quitzeoensis, which still has at least 10 population across the Lerma-Chapala basin, high levels of haplotype and nucleotide diversity  were observed, and on the basis of these results, seven operational conservation units (OCUs) were identified for the species  and A. meeki   .Ameca splendens, moderate values of genetic diversity (He ranging from 0.34 to 0.94) were found  were similar to those founded for T. hildebrandi; also, in this case, the population with the largest size had the highest level of genetic diversity (a proxy for population size) for T. hildebrandi and some of aforementioned species. Based on estimates using Google Earth, the goodeines Z. quitzeoensis  and N. bilineata  have the largest geographic distributions, followed by the poeciliid X. cortezi , T. hildebrandi , and P. sulphuria (0.24 km2). The poeciliids, which appear to be more geographically restricted, all present a similar low level of genetic diversity compared with the goodeines, consistent with the idea that genetic diversity is correlated with distribution area. Although there are no specific studies on population size in T. hildebrandi, the low number of individuals reported in previous ecological studies  suggests the distinctiveness of populations within the two upper Usumacinta subbasins and the upper Grijalva basin  and the Amarillo and Chenalhó subbasins , given their geographic proximity and the geographic features of the region. These features, possibly related to geological events, such as reversal flow and collapse of the stream bed, could have allowed underground connectivity among the subbasins .T. hildebrandi. According to our Migrate analysis, migration occurred in both directions between the two basins, supporting their historical geographic connection. The genetic differentiation observed in the AMOVAs and the patterns observed in the haplotype networks , indicating its colonization by species from both regions . Likewise, pairwise ΦST values were higher when comparing the three groups as opposed to the two basins   .Tlaloc hildebrandi is cataloged as an endangered species by both the IUCN  a restricted geographic distribution  of the species, despite its crucial importance for conservation efforts. In recent works demonstrating the wider than previously thought distribution of T. hildebrandi, very few specimens were collected from the different sampling localities with the exception of Chenalhó (T. hildebrandi at this site.the IUCN  and NOM- species . The lowribution  and (2) ribution . UnfortuChenalhó . The watIn recent decades, there has often been a lack of connection between the genetic information of a species and the conservation policies enacted for its protection . This reTlaloc hildebrandi represents one of the most geographically restricted species of the family Profundulidae . Another threat to the species is the introduction of exotic species. Parasites of the exotic species Cyprinus carpio have been shown to negatively affect larvae and juveniles of T. hildebrandi . One of the most significant in situ conservation projects involves the reintroduction of Zoogoneticus tequila (Goodeidae) (once considered extinct in the wild) into the Teuchitlán River in Jalisco  and the establishment of protected areas in its distribution range, habitat restoration, and the reintroduction of the species to some rivers (in situ strategies). Finally, we recommend the promotion of societal activities related to environmental education, and improved communication between the scientific community and the authorities and decision makers who enact policies at the local and state levels.Genetic diversity plays an important role in the conservation management of populations of endangered species. 10.7717/peerj.11952/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.11952/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.11952/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.11952/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.11952/supp-5Supplemental Information 5Click here for additional data file.