By the 19th century, adequately producing, independent domestic animal breeds had developed in many regions around the globe. However, from the middle of the 20th century they have largely been replaced by high-performing, specialized, single-purpose cosmopolitan breeds. Breed maintenance is an activity aimed at rescuing old breeds from the threat of extinction. This process includes recording the valuable traits of a rare breed, specific diversity conservation selection, and utilization in the original production environment. Additionally, it deals with the history of breeds and the study of their genetic makeup. The aim of this paper is to evaluate the maternal genetic background of two autochthonous sheep breeds in Hungary.ST) calculated using the sequence-based statistics for cytochrome b gene and control region were 0.034 and 0.021, respectively (both at level p < 0.05); thus, revealing genetic differentiation in both sequences between the Tsigai and Cikta. We note that the known different history of the breeds is clearly justified by the currently studied deviations in their maternal genetic background.The aim of our research was the evaluation of the maternal genetic background of two Hungarian autochthonous sheep breeds of different geographical origin. A major argument for the preservation of endangered animal breeds is their documented past and historical importance. These also include the registration of pedigree data. This is the first study to evaluate and compare Tsigai and Cikta sheep in Hungary. Our investigation is based on two complete sequences of mitochondrial DNA (cytochrome b gene and control region). Our research was performed on these two sheep breeds with markedly different breed histories and breed characteristics to determine a possible common maternal genetic background, as ultimately the origin of both breeds can be traced back to Asia Minor. Between 2015 and 2017, a total of 203 biological samples were taken using a newly introduced founder sampling method. We found that the prevailing haplogroup B accounted for over 80% of both breeds, strengthening the common ancestral root. However, the pairwise genetic differentiation estimates (K Throughout the history of Hungarian sheep breeding, the initial primitive types were slowly replaced by new breeds. To the best of our knowledge, the development of these breeds was due to the following factors: firstly, the improvement of the old local sheep populations and secondly, the spread of foreign sheep in Hungary with the additional possibility of mixing among these. By the end of the 18th century, these breeds had become the native, autochthonous or heritage breeds of the country. Subsidies have been granted in an effort to preserve these undemanding, multipurpose fallow breeds that have been an endangered genetic resources since the middle of the 20th century. The following breeds make up our heritage breeds: Racka (in black and in white color variants), Gyimesi Racka , Cikta, Tsigai and Milking Tsigai. In this paper, we explore and compare the history and characteristics of the Tsigai ).Haplogroups D and E are the rarest groups and have only been identified in recent fossil and living samples from Turkey, the Caucasus ,20,21, EThe combined use of CR and CYTB may meet the requirements for characterizing and comparing the haplogroup and haplotype of sheep breeds. Thus, this information also gains space in the research of the genetic structure of a breed . Examining the haplotypes , alongside establishing the origin of the compared breeds , may sheThe two breeds we wish to investigate differ significantly in their external characteristics, their economically important traits, and their history. Taking into account the known differences between them, our aim is to map and compare the maternal genetic background of the two breeds originating from the same ancestral root, although developed at different geographical sites. We hypothesize that we will not find a considerable difference based on the conservative and coding cytochrome b gene sequence, however, according to the much more mutable, non-coding control region sequence there will be comparable differences between the Tsigai and Cikta coming to Hungary from the east and west, respectively.n = 134) and 20.5% (n = 69) of the seed stock of the breeds, respectively.Full pedigrees provided information to achieve the so-called “founder sampling” method during the collection of biological samples. In this case, sample collection was preceded by processing the entire pedigree for both Tsigai  and CiktOvine brucellosis). The further use of samples obtained during clinical veterinary procedures for research purposes is not considered an animal experiment under Law No. XXVIII of 1998 and Government Decree No. 40/2013, so no ethical permit is required.The live member of the families that provided the sample was selected so that as few farms as possible had to be visited for sampling in 2015 and 2017. Thus, the biological samples were obtained in nine Tsigai and three Cikta populations see . The bloBlood samples were frozen in tubes containing EDTA as soon as possible after collection. They were stored at −20 °C in a freezer until DNA was extracted.DNA was isolated using the GenElute Blood Genomic DNA Kit  according to the manufacturer’s instructions. A 1.5% agarose gel mixed with GelRedTM Nucleic Acid Gel Stain  was used for quality and for a semi-quantitative measurement of the extracted DNA. Purified DNA was stored at 4 °C until subsequent analysis.The 25 μL PCR reaction mixture prepared for each sample contained 5 μL DreamTaq™ Green PCR Master Mix , 1-1 μL forward and reverse primers (10 μM), 1 μL BSA (20 mg/mL), and approximately 10 ng DNA template and PCR grade water to volume.For both the cytochrome b gene and control region, primers ,23,31 weTo amplify the DNA sequences, a programmable Thermal Cycler 2720 PCR equipment  was chosen. For the purification of PCR products a SIGMA GenE-luteTM PCR Clean Up Kit  was used according to the protocol.® Terminator v.3.1 Cycle Sequencing Kit  was applied in the manner recommended by the manufacturer. An ABI Prism 3130XL Genetic Analyzer  was applied for sequence detection, according to the manufacturer’s guidelines. Sequence data were analysed using Sequencing Analysis Software 5.1  and aligned by SequencherTM 4.1.2 software .For the sequencing reaction, a BigDyeST). Then, we used the test developed by Fu and Li [First, we used DNAsp v6.0 software   to deteru and Li  to evaluu and Li  to clariThe resulting sequences were aligned with MEGA-X  . The numfluxus-engineering.com, accessed on 31 May 2020); [The species distribution of CR haplotypes was plotted using Network 10.2.00 software (y 2020); ). The sen = 134), OK572625-OK572692 , OK572827-OK572956  and MW427980-MW427983, MW427991, MW427993-MW428057 .All novel sequences of cytochrome b gene and control region used in this study were deposited in the GenBank public database under following accession numbers: OK572693-OK572826 .For CR, the number of nucleotide sites and number of sequences were 1180 and 199, respectively. The number of total mutations was 185 .The number of individuals sampled and polymorphic positions per breeds are shown in The total number of haplotypes in the whole study population was 40 for CYTB and 94 for CR. Of the CYTB haplotypes, 33 were distinguishable in the Tsigai and 11 in the Cikta. Of the CR haplotypes, 66 occurred in Tsigai and 29 in Cikta.−3, and 18.56 × 10−3, respectively). Only the haplotype diversity was found to be the same for both sequences .A comparison of the two breeds is presented in The average number of pairwise nucleotide differences between the two populations for CYTB and CR was 2.422 and 18.466, respectively.ST) calculated using the sequence-based statistic for CYTB and CR were 0.034 and 0.021, respectively (both at level p < 0.05); thus, the genetic differentiation in both sequences between the Tsigai and Cikta was found.The pairwise genetic differentiation estimates  was, for CYTB and CR, 2.46 × 10The number of CR haplogroups identified was three. The classification of individuals into CR haplogroups was determined in the following proportions. The most populous of the haplogroups was B (of which 126 are Tsigai and 56 are Cikta), followed by A, with 4, and 8 individuals, respectively. Furthermore, 6 Cikta individuals were included in haplogroup C.The network of the connections between the CR haplotypes and the reference CR haplogroups computed by the median-joining method illustrates well that the haplotypes are definitely located around the reference haplogroup B .p < 0.05) and for CR it was −1.480, which was statistically non-significant (p > 0.10).The Tajima D-test value for CYTB was −2.124, statistically slightly significant (p > 0.10) and 0.121 (p > 0.10), respectively, in the whole sample set investigated. The Fu and Li’s D* and F* test values for CR were as follows: 0.620 (p > 0.10) and −0.468 (p > 0.10).For CYTB, the Fu and Li’s D* and F* tests also resulted in non-significant values of 0.582 (p < 0.001) and −41.261 (p < 0.001), respectively.For both CYTB and CR, the Fu’s Fs statistic resulted in significant negative values of −33.177  than that of all breeds currently studied. In this study, the Tajima D-values of both breeds were statistically proven to be in the minus range (Gyimesi Racka −1.734 and Turcana −1.814).For the CR, in the evaluation of Kusza et al. , the ave−3 to 26 × 10−3 (higher than our), respectively. Furthermore, they observed a high common level for nucleotide diversity (π) of 21 × 10−3 in the three investigated breeds , which is higher than that of Tsigai and Cikta in our work. In the Ruda breed (which is a relative of Tsigai), they found no haplogroup C, while in the other two they detected around 15%. In the study of Niemi et al. [−3) in the Serbian Pramenka breed had similar values to ours; however, the average number of nucleotide differences (k = 8.11) was significantly lower.For Hd, Dudu et al.  found a i et al. , haplotyIn our processing of the genetic pool of the two breeds, Cikta proved to be more diverse with regard to the average number of mutations per individual, average number of nucleotide differences, and the average nucleotide diversity (JC). We can conclude that its origin is also more complex. The two peaks that appeared in the mismatch distribution of the Cikta further reinforce the more diverse maternal background of this breed. According to Alvarado-Bremer et al. , the bimFurthermore, the Tajima D-test and Fu’s Fs statistic performed on the entire study population confirmed a significant segregation of the cytochrome b gene sequence. Contrary to our expectation, we found a non-significant (Tajima D-test) differentiation in the control region. A significant positive value would be an indicator of the loss of genetic diversity in a given population (bottleneck effect) or degradation to subpopulations. Meanwhile, a significant negative value signifies an excess of low frequency polymorphisms relative to expectation or population-size expansion . A non-sp < 0.001, CR −41.261 and p < 0.001) demonstrate foreign gene flows after a spatial expansion.However, the more sensitive Fu’s Fs statistic confirmed separations according to both sequences. The significant negative values of Fu’s Fs statistic  or a gr < 0.001 ) within In our investigation, we distinguished three haplogroups. The haplogroup B  prevailed in both breeds (Tsigai and Cikta) by 97% and 81%, respectively. The Copper Age sheep in Europe were also found to belong to haplogroup B, as a link between peat sheep and its distant ancestor in Near East was found by Oliveri et al. .In our case, the haplogroup A appeared with a minor frequency. The Cikta sheep is distinguished by a proportion of haplogroup A (12%), while this haplotype is also present in the Tsigai, but much less frequently (3%).Ovis orientalis) and it is mainly present in Asian breeds, thus, it is, characteristic of the breeds in the Indian subcontinent . Ewes oDuring sheep phylogenetic development, haplogroup C was first separated from B and A. In our study, we observed the occurrence of haplogroup C only in the Cikta breed. The 9% occurrence of haplogroup C also supports the view that the Cikta has a more complex maternal background than the Tsigai. This means that ewes may have entered Hungary in the past from the central areas of Asia, from the Caspian See region , MongoliThe wild sheep found no suitable habitat and did not survive due to climatic and environmental conditions in Europe by the end of the Stone Age. The European spread of domestic sheep could have occurred through several highly probable routes, partly along the Danube Valley, via the Carpathian Basin and along the Eastern European plain . The domThe haplogroup C had dispersed from the inner core of Asia not only to central Europe. According to the literature, haplotype C can also be found in Caucasus  and GeorWe have not detected clear haplogroups E and D in our current investigation.The latest common gene pool of the two breeds can be traced back to Anatolia. We can assume that the diversification of the common gene pool took place 7500 years ago. After all, the early ancestors of the Cikta entered the central part of the European continent from the Balkans, and from here they reached the Carpathian Basin in the early 18th century. The ancestors of the Tsigai made their way from Anatolia directly to the Balkans and the Carpathian Basin over the last few centuries. Now, we conclude that the demographic history of the two breeds differs from each other, a clear sign of which is the significantly differing maternal background. Obviously, the migration of the two populations could not have been entirely independent from various genetic effects, because the genetic background of both breeds was not only modified by the occurrence of natural mutations but also by gene flow from other sheep herds depending on space and time.For the success of mitochondrial genome examinations, prior knowledge of pedigree data is essential for the identification of maternal lineages and for organized sampling. We consider the founder sampling method, which can be used to calculate the true values of mitochondrial diversity, to be a form of truly representative sampling. Furthermore, finding unique haplotypes of rare families is most likely only feasible in this way. The question arises, if a breed is considered to have a uniform genetic background, whether individuals with a haplotype that differs from their breed-mates should be excluded from preservation of native breeds, or if they are worth maintaining.Furthermore, by identifying families, our work also brings attention to the importance of the maintenance of maternal lineages and within-family selection. Increased consideration of the maternal side is also supported by the fact that the proportion of ewes and rams in breeding herds is in favor of females. Additionally, ewes stay in breeding for longer than rams. Thus, they may be better custodians of the display and transmission of genetic diversity.In the course of our research, the fact that complete sequences were analyzed in both the cytochrome b and control regions certainly provided more reliable results than the use of only one sequence or a partial sequence. In addition to these reliable results, the number of individuals in the breeds was sufficiently large considering the sample size in other similar studies referred in our paper. Furthermore, due to the introduction of the founder-sampling method as a basis for sampling, the maternal background could be characterized more realistically than by random sampling.We were faced with the fact that our working hypothesis, which was based on the breed histories and experiences with mtDNA sequences, was only partially fulfilled. Contrary to expectations, we showed a statistically significant genetic separation between Cikta and Tsigai not only in the control region but also in the cytochrome b gene. Based on the rare allele’s excess of CYTB and CR, both breeds demonstrate foreign gene flow after a spatial expansion. In addition to the European haplogroup B, the presence of haplogroup A was confirmed in both breeds, but haplogroup C only in the Cikta breed.


Capreolus capreolus) was probably extirpated from Switzerland, due to overhunting and deforestation. After a federal law was enacted in 1875 to protect lactating females and young, and limiting the hunting season, the roe deer successfully recovered and recolonized Switzerland. In this study, we use mitochondrial DNA and nuclear DNA markers to investigate the recolonization and assess contemporary genetic structure in relation to broad topographic features, in order to understand underlying ecological processes, inform future roe deer management strategies, and explore the opportunity for development of forensic traceability tools. The results concerning the recolonization origin support natural, multidirectional immigration from neighboring countries. We further demonstrate that there is evidence of weak genetic differentiation within Switzerland among topographic regions. Finally, we conclude that the genetic data support the recognition of a single roe deer management unit within Switzerland, within which there is a potential for broad‐scale geographic origin assignment using nuclear markers to support law enforcement.In the early 1800s, the European roe deer ( After 19th century extirpation, the European roe deer naturally recolonized Switzerland. Using mitochondrial and nuclear DNA markers, we demonstrate that Swiss roe deer had natural, multidirectional immigration from neighboring countries and that there is evidence of weak genetic differentiation within Switzerland among topographic regions. The situation is somewhat different in winter, when larger groups of four or more animals are most commonly observed.Roe deer do not maintain exclusive territories but live within overlapping summer and winter home ranges. In summer, which is the period of territoriality and reproductive activity, the deer which are primarily solitary are dispersed over the population range.Adult bucks establish summer territories which they have to re‐establish each year in contest with other bucks. In April and May, prior to giving birth, it does separate from their groups and occupy kidding ranges which remain same from year to year and measure several hectares.In contrast, during winter when bucks are no longer territorial, the deer mainly form groups which are concentrated on feeding ranges. Females with kids return to their winter home ranges between October and December , with animals moving freely among them. In order to address the intricacies of current fine‐scale canton‐level management and improve cooperative management plans, it is important to understand roe deer population structure and connectivity across the landscape.Even more drastic declines happened in Switzerland, where after the Napoleonic wars, crop failures and resulting famines led to uncontrolled hunting, which combined with subsequent cattle browsing competition, caused roe deer to effectively disappear from the country by around 1800  in the south and the Jura Mountains in the northwest connected by the Central Plateau with rolling hills and plains. Those three different topographic regions may influence the distribution and movement of Swiss roe deer across the country. In addition, several large lakes are scattered throughout the country which can further curb dispersal and gene flow in roe deer.1.4Conservation genetics typically uses molecular markers to enable measurement of genetic diversity, gene flow, and spatial population genetic structure. The resulting data can, in turn, be used to understand phylogeography, define evolutionary significant conservation units, and identify the origins of unknown samples. Because of its high mutation rate, haploid maternal inheritance, and lack of recombination, the hypervariable mitochondrial DNA (mtDNA) control region is often used as a marker in investigations of species’ population genetic diversity  markers may be analyzed at scale throughout the genome, the greater allelic diversity at microsatellite loci means that they remain a highly effective tool for examining neutral variation in landscape‐scale population genetic studies  in Switzerland and assess contemporary population structure in relation to landscape features, in order to understand underlying ecological processes and inform future roe deer management strategies.In this study, we use a classical conservation genetic approach to investigate the natural recolonization of the European roe deer  Where did roe deer recolonize Switzerland from?; (ii) what patterns of population contemporary genetic structure and gene flow can be observed in Swiss roe deer?; and (iii) are there any clear implications for conservation management and opportunities for development of forensic traceability tools?22.1n = 279; blood = 226 and tissue = 53) were collected in Switzerland between 2004 and 2017, by local hunters. Additional tissue samples were collected from Northern Italy close to the Swiss border  and Central Italy . Samples were grouped into six regions based on broad landscape features, primarily according to topographic variation . The additional 28 samples from Lombardy were included in the southeastern Swiss region as they originated close to the Swiss border with no barrier to free movement. We decided to split the Central Plateau into two localities, an eastern and western one because of the great distance between two ends of the Plateau and because of presence of several big lakes and highways that transect the Plateau and pose an obstacle to roe deer free movement. Lastly, we wanted to enable detection of immigration from different sides of Switzerland. The sixth group consisted of 12 individuals from central Italy, resulting in a total of 319 samples. DNA from tissue and blood samples was extracted using the DNeasy® Blood and Tissue kit (Qiagen). DNA from blood swab samples was extracted using a chelex protocol followed by a Qiagen PCR purification kit  as described in Morf et al.  of the available samples were randomly selected for mtDNA sequencing in order to ensure that each of the five regions in Switzerland and one in Central Italy was sufficiently represented . A single 704‐bp long mitochondrial DNA (mtDNA) control region fragment was amplified using two primers developed by Randi et al. (n = 76) from Randi et al.  between haplotypes in each region were calculated in ARLEQUIN, version 3.5 , observed (Ho), and expected (He) heterozygosity across all microsatellite loci and regions, were computed using the package adegenet, version 2.1.1  and inbreeding coefficients with FSTAT, version 2.9.4  based on microsatellite data. The Bayesian clustering method in STRUCTURE attempts to identify genetically distinct subpopulations on the basis of patterns of allele frequencies. A series of ten independent runs per K (ranging 1 to 10) were performed using 1,000,000 MCMC iterations after a burn‐in of 500,000 replicates, using an admixture model with correlated allele frequencies without prior sampling information.The program STRUCTURE, version 2.3.4  were performed at 500,000 MCMC iterations after a burn‐in of 200,000 replicates. The burn‐in and data collection iterations, and the number of replicates, were reduced in this phase, based on observed consistency among replicates, in order to reduce computation time. Results were summarized using STRUCTURE HARVESTER  as suggested for datasets with few markers, few individuals, or very weak structure  in our data and to compare with the prior groups (regions). The optimal clustering solution corresponds to the lowest Bayesian information criterion (BIC), and in this case, it clearly indicated 5 clusters (K = 5) that we decided to retain.To complement the STRUCTURE analysis and to assess the degree to which these five Swiss selected sampling regions differ from each other when adopting an approach without assumptions about HWE or LD, we conducted a discriminant analysis of principal components (DAPC)  in ARLEQUIN with five regional populations nested within northern  and southern (South East and South West) Swiss groupings. Finally, isolation by distance (IBD) patterns based on microsatellites genotypes were examined to determine association between matrices of pairwise codominant genotypic and geographic (Euclidean) distances by performing a simple Mantel test (1000 permutations) visualized in adegenet, version 2.1.1  and 704 nucleotides showed a total of 89 haplotypes including twelve novel sequences (H161‐H173) in Switzerland (GenBank accession nos MW916295‐MW916306) and 47 polymorphic sites.H = 0.97 ± 0.002), but nucleotide diversity (π = 0.01 ± 0.006) and pairwise divergence (k = 7.54 ± 3.52) were low  and comparable to the haplotype diversity of Serbia (0.96) which has the highest haplotype diversity among European countries observed to date. The lowest diversity among European countries was found in Portugal 0.81). On a smaller scale, haplotype diversity within Switzerland  indicating only low levels of localized inbreeding  in the Central‐West region, following sequential Bonferroni correction Rice, . No sign5 in the 3.4.2K = 3) with varying levels of genetic admixture )  North (Jura Mountains); (ii) Central West (western Swiss Plateau and west pre‐Alps); (iii) Central East (eastern Swiss Plateau and east Swiss pre‐Alps); (iv) South West (West Swiss Alps); (v) South East ; and (vi) Central Italy , there was good evidence of geographic structure, as well as introgression associated with geographic neighbors; between the Central East and Central West and to a smaller extent the South East region; and between the North and Central West regions. The Central Italian population was quite distinct with only a very small percentage of admixture with other Swiss populations  Figure a. Employ3 Figure b, with os Figure .a priori group assignment and without samples from the distinct Central Italian region. The find.clusters function retained 100 axes representing more than 88% of the total variance and covered a range of possible genetic clusters from 1 to 30. The lowest BIC value corresponded to K=5. For DAPC analysis, 40 PCA axes and 4 discriminant functions were retained. A high degree of overlap between clusters 1 and 4, and 3 and 5 was evident and showed a similar pattern to that observed in the STRUCTURE results, where North and Central West, then Central East and Central West and finally South West and South East individuals clustered together . For DAPC analysis, we retained 40 PCA axes and 2 discriminant functions. Results showed three distinct clusters that more clearly differentiated between southern (cluster one), Central East (cluster two), and Central‐West (cluster three) regions  and 1 Central Italian sampling regions indicated significant low to moderate genetic differentiation, with FST values ranging from 0.02 to 0.14 (Table FST = 0.08) with other pairwise values broadly reflecting relative geographic proximity. The results of isolation by distance testing indicated a significant correlation between genetic and geographic distances when including Central Italy , but among Swiss‐only regions this relationship was weaker (r = .48) and nonsignificant (p = .055).Population‐based gene flow estimates using pairwise 14 Table . The higp‐value = .0001). These results show that there is a level of regional genetic differentiation north and south of the Alps which is slightly greater than the level of differentiation among populations within those regions.The AMOVA results testing the hypothesis for hierarchical genetic subdivision within Switzerland, first between northern (non‐Alpine) and south (Alpine) areas of Switzerland and secondly among regions within those areas indicated that most of the variance (91.2%), were attributable to the genetic variation within individuals within sampling sites, whereas only 2.8% of the variance occurred among regions, 2.5% within regions, and 3.6% within populations   Avise, , and the) Avise, . Overall) Avise,  and show4.2Examination of current population structure within Switzerland based on five Swiss regions: North, Central East, Central West, South West, and South West, indicated no clear mtDNA structuring associated with geography within despite distinct topographical differences between the Jura Mountains, Swiss plateau, and northern and southern Alps. However, analysis of nuclear data revealed three genetic clusters representing the North and Central West, Central East, and the South displaying varying levels of admixture which corresponded to geographic proximity. The fact that only weak clustering was observed when locality information was not included as a prior in the Bayesian analysis emphasizes the lack of strong isolation among regions; this may be due to ongoing gene flow, insufficient divergence time since isolation, or insufficient power in the DNA marker panel, or a combination of these factors. These are the conditions in which prior locality information is recommended for use in STRUCTURE , it is also important to consider the influence of founder history on current population genetic patterns. Here, we discuss the possible causes of the observed weak genetic structure in Swiss roe deer, before considering the management and law enforcement implications.www.astra.admin.ch, Swiss Federal Roads Office (FEDRO)) represent other obstacles to free movement of wildlife, with fenced motorways found to cause significant restrictions to gene flow in roe deer ; Data curation (lead); Formal analysis (lead); Funding acquisition (supporting); Investigation (lead); Methodology ; Software (lead); Visualization (lead); Writing – original draft ; Writing – review & editing . Nadja V. Morf: Supervision (supporting); Writing – review & editing (supporting). Josef Senn: Resources (supporting); Writing – review & editing (supporting). Sílvia Pérez‐Espona: Writing – review & editing (supporting). Federica Mattucci: Resources (supporting); Writing – review & editing (supporting). Nadia Mucci: Resources (supporting); Writing – review & editing (supporting). Gaia Moore‐Jones: Resources (supporting); Writing – review & editing (supporting). Simone Roberto Rolando Pisano: Resources (supporting); Writing – review & editing (supporting). Adelgunde Kratzer: Funding acquisition (lead); Project administration (lead); Resources (lead); Writing – review & editing (supporting). Rob Ogden: Conceptualization ; Project administration ; Resources (supporting); Supervision (lead); Validation (lead); Writing – original draft ; Writing – review & editing .Appendix S1Click here for additional data file.


Lactiplantibacillus plantarum 75 (L75), Leuconostoc pseudomesenteroides 56 (L56) and its combination (L56 + 75) on the quality, bioactive and volatile compounds of mango juices (MJ) from three cultivars  were investigated. Fermented and unfermented MJ were evaluated for LAB growth, physicochemical parameters, volatile compounds, antioxidants activities , total phenolic content (TPC) and sensory properties. The unfermented juices served as a control. Twenty-four-hour fermentation was ideal for MJ based on LAB growth profiles. Generally, titratable acidity, TPC, FRAP, DPPH and ABTS scavenging activities significantly increased with fermentation by the L75 strain and were highest in the L75-fermented ‘Sabre’ MJ, while L75-fermented ‘Peach’ MJ had higher ABTS activity (p < 0.05). In contrast, the L56 strain enhanced β-carotene retention, with improved colour properties in L56-fermented ‘Peach’ MJ. Fermentation with L75 in ‘Sabre’ and ‘Peach’ MJ aided the synthesis of new volatile compounds . A PLS-DA scatter plot showed two clusters separating the ‘Peach’ and ‘Sabre’ mango juice fermented with L75 from the rest. Based on the variable importance of the projection value (VIP) scores, pentadecane, 8-hexyl and butyl isobutyrate were shown as marker candidates to distinguish ‘Peach’ and ‘Sabre’ MJ fermented with L75 from the other treatments, whereas ethyl octanoate and isobutyl acetate differentiated the ‘Sabre’ MJ fermented with L75 from the other treatments. ‘Sabre’ and ‘Peach’ MJ fermented with L75 and L56 could provide antioxidants, meeting the recommended daily requirements for ascorbic acid and carotenoids in adults and teenagers. Hence, lactic acid fermentation of these local cultivars is a way to benefit consumers.The effects of lactic acid fermentation using  Lactiplantibacillus plantarum (Ltp. plantarum)-fermented tomato juice in an in vitro transepithelial electrical resistance assay [Lactic acid bacteria (LAB) have been employed for decades in food fermentations. The fermentation process involves the use of carbohydrates to make organic acids, most notably lactic acid, and results in metabolites that have the potential to promote health . In the ce assay . Fermentce assay . Additioce assay . Fruit jce assay . Accordice assay  LAB breaMangifera indica) fruit contains a range of antioxidants, including carotenoids, ascorbic acid, omega 3 and 6 polyunsaturated fatty acids, lutein, quercetin, mangiferin and flavonoids [Ltp. plantarum is the most widely used strain for lactic acid fermentation [Lactobacillus acidophilus on the quality of beetroot juice and carrot juice [Mango  56 were tested for fermentation of local mango cultivars  juices singly or in combination and compared with the commercial export cultivar ‘Tommy Atkins’ mango juice.Our study tested the hypothesis that cultivar variation and different LAB strains do not affect the physicochemical properties, functional compounds, antioxidant properties, sensory properties or aroma compounds after fermentation. The mango cultivars studied were ‘Peach’ and ‘Sabre’, which are popular local mango varieties widely consumed by the rural communities in South Africa, and they were compared with the commercially available cultivar ‘Tommy Atkins’ mangoes. Physicochemical properties, functional compounds, antioxidant properties, sensory properties and aroma volatile compounds were determined at 24 h of fermentation after optimising the fermentation duration and were compared to the unfermented juice. It is anticipated that the results of this study will provide food manufacturers with information about the suitability of a specific cultivar to produce fermented MJ with good nutritional properties. It is the first study in which the LAB strains v/v) sodium hypochlorite solution, washed thoroughly in distilled water and peeled. The juice was extracted using a Russell Hobbs (700 W) household blender and diluted with sterile Valpre spring water  at a ratio of 1:2 . Mango juice (500 mL) was pasteurized at 85 °C in a water bath  for 15 min and cooled to room temperature (25 °C) before fermentation [The ripe ‘Peach’, ‘Sabre’ and ‘Tommy Atkins’ mango cultivars used in the study were purchased at an edible maturity stage from Tshakhuma market, Venda, Limpopo province, South Africa. All mango cultivars were disinfected by dipping in 1% (entation .Ltp. plantarum 75 (L75) and Leu. pseudomesenteroides 56 (L56) were isolated from cabbage [g for 10 min and suspended in sterile water, washed twice with sterile distilled water, and suspended in the same. The turbidity of LAB cultures was determined at 720 nm using a spectrophotometer  and bacterial population was adjusted to 0.05 McFarland standard (5 × 108 CFU/mL) [L56 + 75) was developed at a ratio . Each fermentation was replicated five times, with unfermented juice as a control.LAB strains used in the study were from the culture collections held by the University of La Réunion and QualiSud, France.  cabbage . ‘Peach’ cabbage , with mo CFU/mL) . The juiEscherichia coli, Salmonella spp., Shigella spp., mold and yeast and aerobic bacteria count were used to ascertain the safety of juices using the European commission regulation (EC 1441) [The total counts of coliform, EC 1441) . SamplesEC 1441) .Sensory analysis was determined using a qualitative descriptive analysis , with moTo determine the titratable acidity, MJ was titrated with 0.1 N NaOH, 1%, and 3–4 drops of phenolphthalein indicator were used . A refraL* describes the lightness , a* refers to the intensity of redness (+) and greenness (−), b* refers to the intensity of yellowness (+) or blueness (−), and total colour differences (Δε) were calculated [The colour values of raw and fermented mango juice were measured using a colorimeter (CR-400 chroma Meter) calibrated with standard white tile  and measThe ascorbic acid (Vitamin C) was determined using 2, 6-dichloroindophenol titration method . The ana2CO3 solution (7.5%) was added to the sample and incubated for 2 h. The absorbance of the sample was measured at 750 nm using a spectrophotometer . The blank was distilled water. The concentration was calculated using a calibration curve prepared with 1 µM gallic acid and the result was expressed in mg 100 mL−1 of juice.The Folin–Ciocalteu assay method with modifications was used to determine the juice’s TPC . A 200-µw/v) butylated hydroxytoluene (BHT) and kept in the dark overnight as described by Panfili et al. [g for 15 min at 25 °C. The residue was rinsed thrice with 5 mL of the extraction solvent and centrifuged. Afterwards, the supernatants were pooled, dried in anhydrous sodium sulphate, filtered through Whatman paper (No. 1) and evaporated under a stream of nitrogen until dry. Prior to analysis, the extracts were re-dissolved in 1 mL of isopropyl alcohol (10%) in n-hexane and filtered through a 0.45-m PTFE syringe filter. A Shimadzu Prominence-I LC-2030C 3D liquid chromatograph fitted with an LC-2030 autosampler and LC-2020/2040 PDA detector  was used for the characterisation and quantification of β-carotene. The column was a 250 mm × 4.6 mm id, 5-µm Shim pack GIST NH2 column. The wavelength was set at 460 nm at 30 °C and the injection volume was 10 µL with a flow rate of 0.6 mL min-1. β-carotene was identified by comparing retention times and absorption spectra with a pure standard. The standard curve was constructed from a range of pure β-carotene standard (0–100 g mL−1) at LOD (19.16) and LOQ (6.323) values and was used for quantification.Briefly, an aliquot of mango juice (10 mL) was extracted using 5 mL of acetone: hexane (1:1) containing 0.1%  and the results were expressed in mg mL−1 of juice.The antioxidants’ radical-scavenging activities of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals were assayed. The DPPH solution in methanol  was dispensed into 96-microplate wells along with various concentrations of the juice extract (0.5–15.0 mg in 150 mL methanol). After vigorous mixing, the samples were incubated for 30 min in the dark. Absorbance was measurements at 593 nm on a spectrophotometer  . The ant+) was prepared by mixing ABTS stock solution (7 mM) with 4.9 mM potassium persulphate in a 1:1 ratio and the mixture was stored at 25 °C for 12–16 h [+ solution, 40 μL of the sample  was added. The mixture was incubated in a 96-well microplate reader at 37 °C for 10 min, under dark conditions. A spectrophotometer  was used to measure the decrease in absorbance at 593 nm at a concentration of 40 μM of the sample (ABTS+). To calculate the IC50, the formula in Equation (1) was used, where: A = absorbance of control reaction; B = absorbance of the test sample. The results were expressed as mg mL−1.The ABTS radical cation decolorization assay was used to assess the free radical scavenging activity. The ABTS radical cation (ABTS 12–16 h . In 1500A ferric reducing antioxidant power (FRAP) assay was conducted by combining TPTZ dissolved in 40 mM HCl with 20 mM of FeCl3 in a 1:1:10 ratio with a 20 mM acetate buffer (pH 3.6) . The samg for 10 min and the supernatant was separated. The residue was rinsed thrice with n-hexane (2 mL) and centrifuged as above. The supernatants were pooled and dried with sodium sulphate, filtered with a white filter paper number 1, concentrated to 250 µL under a gentle stream of nitrogen, and, thereafter, stored at 5 °C before analysing using an Agilent 7890. A gas chromatograph  hyphenated to an Agilent 5975 C MSD with a triple-axis detector was used to analyse the volatile compounds present in non-fermented and fermented MJ. The machine was equipped with an auto sampler (Agilent Technologies GC Sampler 80). Helium was used as the carrier gas at a constant flow rate of 1 mL/min. Separation was done on a Zebron Guardian Capillary GC column (USA) with dimensions (30 mm × 0.25 mm i.d and 0.25-µm film thickness). The chromatographic oven program was as follows: 70 °C for 1 min then 3 °C/min to 142 °C for 0 min then 5 °C/min to 225 °C for 3 min, then 25 °C/min to 320 °C for 3 min; the total run time was 51.4 min. The split less injection was carried out at a 61.6 kpa pressure and 280 °C inlet temperature. The mass detector was operated with an electron energy of 70 eV in electron ionisation mode. The ion source and quadrupole temperatures were 230 °C and 150 °C, respectively, at an injection volume of 1 µL. Compounds were identified using the mass spectral library . Alkanes were quantified using the pentadecane reference standard.Volatile constituents were extracted according to the method of Hijaz et al.  with a fAnalysis was carried out in five replicates and the analysis of variance (ANOVA) was performed using statistical software . Two-way ANOVA was employed to evaluate the effect of fermentation hour, LAB’s strains and cultivar and the means were separated using Fischer’s least significant difference at 95% significance. Results were presented as mean ± SD. MetaboAnalyst was used to establish the correlation pattern of juice volatile compounds using the PCA, PLS-DA, heat map and clustering analysis.L75, L56, or L56 + 75 decreased the TSS compared with their unfermented counterparts. Meanwhile, ‘Sabre’ MJ fermented with L75 was significantly different from the unfermented juices (at 0 and 2 h) in terms of TSS, while it was not the case in L56 and L56 + 75-fermented juices. The ‘Peach’ MJ fermented with L75 had the lowest TSS content, which represented a 50% decrease of TSS due to fermentation. Our observations agree with the decline in TSS of mango juice and sapota juice fermented with Ltp. plantarum. NCDC LP 20 for 24 h, 48 h and 72 h [Ltp. plantarum fermented cashew apple juice proceeded at a much faster rate than other LAB’s during the fermentation process [TSS is largely responsible for determining the sweetness of fruit juices. The TSS of fresh ‘Peach’ MJ (3.42 °Brix) was higher than un-inoculated ‘Sabre’ (2.40 °Brix) and ‘Tommy Atkins’ (1.76 °Brix) juices . Fermentand 72 h . AdditioL75 > L56 + 75 > L56) in all cultivars, ranging from 3.66 to 4.85 and was lowest in L75-fermented ‘Sabre’ MJ (3.66). According to Mashitoa et al. [Leu. pseudomesenteroides 56 and Ltp. plantarum 75 decreased after fermentation. In the same way, Leu. mesenteroides MPL18 and MPL39 decreased the pH of mango juice after fermentation [Overall, fermentation of juices from ‘Peach’, ‘Sabre’ and ‘Tommy Atkins’ mango cultivars with different LAB strains in combination or stand-alone resulted in a reduction in pH compared with their corresponding unfermented juices at 0 or 24 h . The pH a et al.  the pH oentation . The degL75, L56 and L56 + 75 compared with the unfermented juices, except the ‘Tommy Atkins’ cultivar (L75-fermented juices had a significant higher TA compared to other LABs’ fermented and unfermented mango cultivar juices (p ≤ 0.05). On the other hand, L56 or L56 + 75-fermented ‘Tommy Atkins’ juice showed no significant changes in the TA (p ≥ 0.05). Higher TA was observed in fermented ‘Sabre’ mango juice than in ‘Peach’ and ‘Tommy Atkins’ mango juices. A similar observation of increased TA was reported in sapota juice fermented with Ltp. plantarum NCDC LP 20 for 24 h [Consistently, fermentation led to an increase in titratable acidity (TA) in ‘Peach’ and ‘Sabre’ MJs fermented with cultivar . The TA for 24 h . Therefob*), and chroma (c*) of the yellow colour ‘Peach’ mango juice fermented by L75 or L56 were similar to that when unfermented at 0 h (ε) value was lower with LAB fermentation for 24 h compared with the unfermented condition. The colour difference was lowest in L56-fermented juices across mango cultivars and, furthermore, it was lowest in ‘Peach’ MJ (1.41). This observation could be due to increased acidity of the fermented juices which help protect against enzymatic browning. Food products with dark colours are less likely to be accepted by consumers and colour change equal to or greater than two is considered a significant colour difference in samples [Indicators of quality include colour intensity. Luminosity, brightness, colour coordinate , significant reductions in colour attributes  were observed in ‘Peach’ and ‘Sabre’ MJs relative to the L56 and L75-fermented juices, possibly due to synergistic oxidative activities of the homo-fermentative LAB’s (L75 or L56) causing the fast triggering of an enzymatic colour degradation process. In ‘Tommy Atkins’ juice, the L56 + 75 had a non-significant decline in colour properties compared with L75 and L56 juices but significantly different from the unfermented juices (p > 0.05).The observed colour change in un-inoculated (U 24 h) mango juices from all cultivars could be due to an enzymatic browning activity . HoweverSalmonella, E. coli, coliforms or Shigella spp. were detected in the fermented or unfermented MJ from the selected cultivars. The total fungi count in fermented and unfermented MJ were in the range; 0.02–4.43, 0.02–0.43 and 0.01–3.46 Log CFU/mL, while the total aerobic bacterial count ranged from 1.6–9.33, 2.56–9.83 and 0.63–8.4 Log CFU/mL in ‘Peach’, ‘Sabre’ and ‘Tommy Atkins’ MJs, respectively. The yeast and total aerobic bacterial count was highest in L75, 24-h-fermented juices across all cultivars. With respect to cultivars, ‘Tommy Atkins’ had the lowest aerobic bacteria count while ‘Sabre’ had the highest bacterial and yeast counts in fermented MJ. According to Managa et al. [p ≤ 0.05), while no growth was obtained in the non-fermented samples, thus signifying a contamination-free and safe production process. No LAB growth was observed in the unfermented juice across the mango cultivars (L75 samples relative to the L56 and combined cultures (L56 + 75) in all mango juices. This corresponded to the beginning of the growth phase for the LAB cultures, while, at 24 h, they ended the logarithmic growth phase. At 24 h, the LAB’s count ranged from 8.99 to 9.17 log CFU/mL in ‘Peach’, ‘Sabre’ (8.79–9.31 log CFU/mL) and ‘Tommy Atkins’ (8.31–8.62 log CFU/mL). The highest population was observed in L75-fermented juices  compared with L56- and L56 + 75-fermented MJs from different cultivars.The absence of growth in fermented and unfermented juices plated on XLD, MCA, PCA and PDA media  is an ina et al. , LAB areultivars . After 2p ≤ 0.05) was observed in all cultivars with the smallest decrease in ‘Peach’ fermented with L75 (0.5%). L56 + 75 reached the stationary phase with a 4.78% decline at 48 h (8.56 Log CFU/mL) and thereafter maintained stability at 72 h. A significant decline in the LAB population of L56 + 75-fermented MJs from all cultivars could be due to LAB’s competing for the potential use-up of nutrients and the build-up of metabolites which could constitute a toxic system. Probably the lower pH and increased acidity could have caused the slight reduction in the survival of LAB strains at 48 h. Acids inhibit bacterial growth and viability by acidifying the cytoplasm, increasing energy consumption necessary for the maintenance of intracellular pH, and inhibiting enzyme reactions [L75 could be due to the versatile nature of Ltp. plantarum 75 in fermentation [However, a decline in LAB population at 48 h before reaching a stationary phase  to bright yellow (8) in the fresh juice. Fermentation and storage caused a decrease in the bright yellow colour of MJs from different cultivars, however, the L56 strain aided the preservation of the yellow colour in fermented mango cultivar juices, which probably related to the inhibition of enzymatic colour change and preservation of the carotenoids. The decrease in the yellow colour perception could be due to an autoxidation process of the poly-phenolics in the juice thereby causing browning [L75 strain increased the aroma perception in fermented mango juices of all cultivars. The highest aroma perception was reported by the panellists in ‘Sabre’ MJ fermented with L75. Consistency and sweetness were reduced in fermented mango juices of all cultivars, while bitter and sour taste increased in fermented juices. The degree of sourness was highest in ‘Peach’ MJ fermented with L56 + 75, while a higher bitter taste was observed in ‘Tommy Atkins’ and ‘Peach’ MJs fermented with L56 + 75. The lower sour taste in ‘Tommy Atkins’ MJ could be due to the lower sugar content in this cultivar [L75 and sensory quality was comparable to the commercial juice. The higher acceptability of ‘Sabre’-fermented juices by the panellists could be due to their mild acid taste compared to ‘Peach’ MJs.The sensory attributes of LAB fermented and unfermented mango juices from ‘Peach’, ‘Sabre’ and ‘Tommy Atkins’ cultivars are presented in browning . L75 strcultivar  thereby −1) and L75-fermented ‘Sabre’ mango juice had the highest AA retention (11.23 mg/100 g) of the three mango cultivars  content . In contrast, L56 + 75 increased the TPC of ‘Peach’, and ‘Sabre’ MJs by 16.48% and 3.79%, respectively, while a 10.8% decrease was observed in ‘Tommy Atkins’ juice. The decrease in L56 + 75-fermented ‘Tommy Atkins’ juice could be due to a low sugar matrix in the juice and lower concentration of the versatile fermentative L75 strains used relative to L75 singly inoculated juices.The TPC in un-inoculated MJs stored for 24 h decreased in the juices of ‘Peach’ and ‘Sabre’ mango cultivars, and the greatest decrease was obtained in ‘Sabre’ (13.7%) . This obLtp. plantarum during fermentation, thus releasing soluble conjugated or insoluble bound phenolic compounds from plant cell walls. Further, the differences in the TPC concentrations among the LAB strains-fermented MJs suggest the ability of the strains to produce more hydrolytic enzymes is highly dependent on the matrix.LAB biotransformation of phenolics is matrix- and LAB-strain-dependent in mango juices, thus supporting the findings of  in fermeL75, or L56 or L56 + 75 when compared to the unfermented juices at 0 or 24 h  and lowest in ‘Tommy Atkins’ (0.15 µg mL−1). As suggested by Venil et al. [L75 relative to L56-fermented juices from all cultivars supports the report of slight carotenoid degradation in Ltp. plantarum- and Lb. fermentum-fermented carrot juice [L56 fermented ‘Peach’ mango could deliver the daily requirement of β-carotene for teenagers.The β-carotene content in all three mango juices increased significantly after fermentation with  or 24 h . Howeverl et al.  the incrot juice . Lactic ot juice . It is pot juice . As 9–13L75 fermented juices across cultivars and was highest in L75-fermented ‘Sabre’ mango juice (558.28 µM TEAC/mL) compared with the unfermented and un-inoculated juices (p < 0.05). In contrast, L56-strain-fermented ‘Peach’ and ‘Sabre’ mango juices and L56 + 75-fermented ‘Tommy Atkins’ MJ had significantly lower FRAP activities relative to the fermented juices. The positive influence of L75 on the antioxidant activity of the ‘Peach’ and ‘Sabre’ mango juices could be due to their hydrolytic capacity to liberate active compounds during fermentation. In addition, the antioxidant scavenging activities (DPPH) were greater in all three mango cultivar juices fermented with L75 compared with other fermented juices. The highest IC50 DPPH activity was in L75-fermented ‘Sabre’ juice (44.7 mg/mL), while L75-fermented ‘Tommy Atkins’ cultivar juice was not significantly different from the control (p > 0.05). The antioxidant ascorbic acid is a free radical scavenger and it can scavenge DPPH radicals [L75-fermented ‘Sabre’ juice. Additionally, lowering the pH increases the stability of ascorbic acid and slows its transformation to dehydroascorbic acid, which supports DPPH scavenging [L75, with ‘Peach’ mango juice showing the highest IC50 level (14.51 mg/mL).As shown in radicals , thus, cavenging . Moreoveavenging . ABTS raavenging . ABTS raL75 strain increased the total volatile compounds in MJs from different cultivars compared with L56 and L56 + 75 juices. ‘Sabre’ mango juice fermented with L75 after 24 h had the highest volatile compounds content (27) relative to other juices, comprising alkanes (6), ketones (2), esters (14), aldehydes (4), alcohols (3) and a terpene hydrocarbon. In order to identify potential discriminant variables, multivariate data analyses, such as principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were employed to reduce the complexity of the data set, as shown in L75 and ‘Sabre’ mango juice fermented with the combined starter (L56 + 75) contained high amounts of pentanal. Pentanal correlated positively on PC1 and separated the ‘Peach’, and ‘Sabre’ mango juices fermented with L75 and L56 + 75 from other treatments.A total of 171.71, 169.19 and 84.14 µg/mL of volatile compounds were found in the raw juice of ‘Peach’, ‘Sabre’ and ‘Tommy Atkins’ mango cultivars, respectively . The totL75 from the rest  ethyl octanoate (30.60 µg/mL), decane-3,7-dimethyl (26.2 µg/mL), butyl acetate (22.6 µg/mL), 2,6-di-t-butylmethylphenol acetate (84.9 µg/mL) ethyl butyrate (49.02 µg/mL) and penten-3-ol (36.7 µg/mL), as shown in The ‘Sabre’ MJ fermented with eatments . The maiL75 with a higher proportion of butyl acetate, amyl acetate, 2-heptanone, nonanal, 2-pentanone, and methyl benzoate. The second cluster included all other cultivars such as ‘Peach’ or ‘Tommy Atkins’ mango juices fermented with different LAB cultures and controls. Results suggest that fermentation of ‘Sabre’ mango juices with L75 significantly improved aroma volatile compound formation. Additionally, ethyl acetate, ethyl hexanoate and methyl acetate were only detected in ‘Peach’ MJ fermented with L75 at higher concentrations compared with other treatments and was not found in ‘Sabre’ MJ fermented with L75.As shown in 3D, the mango juices fermented with different LAB cultures were grouped into two clusters in the heat-map. The first cluster was found in ‘Sabre’ MJ fermented by L75 in this investigation. The major contributor to the green taste was (E)-3 hexanol, which was found in higher concentration in ‘Peach’ (24.46%), ‘Sabre’ (36.79%) and ‘Tommy Atkins’ (31.55%) MJs fermented by L75 and in ‘Sabre’ MJ fermented by L56 + 75 (36.68%) compared with their raw counterpart. Equally, the percentage increase in 1-penten-3-ol  was highest in L75-fermented ‘Sabre’ MJ (80.23%) followed by ‘Peach’ MJ (74.99%) and was significantly different from other juices relative to the raw juice (U 0 h).Esters positively influence the fruity flavour of the juices and enhance the sensory profile . In unfeL75. Nonanal was not detected in ‘Sabre’ MJ fermented by L56, L56 + 75, nor in fermented ‘Peach’ and ‘Tommy Atkins’ mango juices. ‘Peach’ and ‘Sabre’ MJ fermented with L75 had the highest levels of newly formed pentanal (fruity note), followed by ‘Sabre’ MJ fermented with L56 + 75. Newly formed volatile  was detected only in ‘Peach’ (1.71 µg/mL) and ‘Sabre’ (2.70 µg/mL) mango juices fermented by L75. L75 fermented ‘Peach’ and ‘Sabre’ MJs had a higher E-2 hexanal (green note) than L56, L56 + 75 or ‘Tommy Atkins’ MJ fermented with any of the three LAB strains. Aldehydes are typically unstable compounds that are either reduced to alcohol or oxidized to acids in food matrices [L56 and L56 + 75-fermented mango juices. ‘Sabre’ MJ fermented with L75 was the most abundant in ketones (2-pentanone and 2-heptanone), which were not detected in the unfermented nor other fermented juices of ‘Peach’ and ‘Tommy Atkins’ cultivars. All alkanes  increased significantly in ‘Sabre’ mango juices fermented with L75 (p ≤ 0.05).Differences in the genetic make-up of the strains, metabolism type and the type of available sugars (carbohydrate) in substrate could be the reason for the observed differences in aroma volatile compounds observed with these different LAB strains . Nonanalmatrices . DiverseL75 produced higher levels of β-myrcene compared to other fermented and raw mango cultivar juices. A similar increase in β-myrcene was reported in Ltp. plantarum POM1- and LP09-fermented pomegranate juices [p ≤ 0.05) levels of terpene (β-myrcene) found in ‘Sabre’, ‘Peach’, and ‘Tommy Atkins’ mango juices. Acid hydrolysis changes the monoterpene aglycones and dramatically reduces their aroma, but enzymatic hydrolysis does not alter the monoterpene aglycones, and it appears to be a better method of liberating volatile monoterpenes [L75 .Furthermore, ‘Sabre’ MJ fermented with e juices . Monotere juices . Due to terpenes . SeveralL75, L56 and L56 + 75 for 24 h in mango cultivar  juices was considered suitable for quality mango juice. L75-fermented ‘Sabre’ mango juice showed the potential to function as a probiotic due to its higher LAB survival. The L56 strain helped to retain the carotenoid content in the juices of mango cultivars and influenced the yellow colour perception. Approximately a glass of L56 fermented ‘Peach’ mango juice could provide the daily requirement of β-carotene for teenagers. Therefore, frequent intake of ‘Peach’ mango juice fermented by L56 could be an ideal strategy to manage cases of night blindness in rural communities. Furthermore, the L75 strain improved the physiochemical, volatile compounds, ascorbic acid, phenolic content and antioxidants activities of fermented mango cultivar juices. Fermentation of ‘Sabre’ mango juice by L75 aids the synthesis of new alcohols, esters, ketones and aldehydes. Due to the improved physicochemical and bioactive properties, L75 fermented ‘Sabre’ mango juice is considered most preferred and able to deliver a functional benefit to health. The lower pH and TSS contents in L75-fermented juices made them suitable for people with diabetic conditions, thereby aiding the limiting of sugar intake. However, further investigations on the organic acids produced during the fermentation process should be further investigated. Hence, the fermentation of local mango cultivar ‘Sabre’ with L75 strain is acceptable and could be recommended to local and food processors in South Africa for better quality mango juice production.Fermentation of ‘Sabre’, ‘Peach’ and ‘Tommy Atkins’ mango juices improves the quality its functionality. Lactic acid fermentation of mango cultivar juices is dependent on the type of strain causing the fermentation, mango cultivar and duration of fermentation. From this study, fermentation by


Danio rerio, has been a valuable model for understanding genetics and development of pigment pattern formation due to its genetic and experimental tractability, advantages that are shared across several Danio species having a striking array of pigment patterns. Here, we use the sister species D. quagga and D. kyathit, with stripes and spots, respectively, to understand how natural genetic variation impacts phenotypes at cellular and organismal levels. We first show that D. quagga and D. kyathit phenotypes resemble those of wild-type D. rerio and several single locus mutants of D. rerio, respectively, in a morphospace defined by pattern variation along dorsoventral and anteroposterior axes. We then identify differences in patterning at the cellular level between D. quagga and D. kyathit by repeated daily imaging during pattern development and quantitative comparisons of adult phenotypes, revealing that patterns are similar initially but diverge ontogenetically. To assess the genetic architecture of these differences, we employ reduced-representation sequencing of second-generation hybrids. Despite the similarity of D. quagga to D. rerio, and D. kyathit to some D. rerio mutants, our analyses reveal a complex genetic basis for differences between D. quagga and D. kyathit, with several quantitative trait loci contributing to variation in overall pattern and cellular phenotypes, epistatic interactions between loci, and abundant segregating variation within species. Our findings provide a window into the evolutionary genetics of pattern-forming mechanisms in Danio and highlight the complexity of differences that can arise even between sister species. Further studies of natural genetic diversity underlying pattern variation in D. quagga and D. kyathit should provide insights complementary to those from zebrafish mutant phenotypes and more distant species comparisons.Vertebrate pigmentation is a fundamentally important, multifaceted phenotype. Zebrafish,  Danio quagga and D. kyathit, respectively. We show that these patterns arise late in development owing to alterations in the development and arrangements of pigment cells. In the closely related model organism zebrafish (D. rerio) single genes can switch the pattern from stripes to spots. Yet, we show that pattern differences between D. quagga and D. kyathit have a more complex genetic basis, depending on multiple genes and interactions between these genes. Our findings illustrate the importance of characterizing naturally occurring genetic variants, in addition to laboratory induced mutations, for a more complete understanding of pigment pattern development and evolution.Pigment patterns of fishes are diverse and function in a wide range of behaviors. Common pattern themes include stripes and spots, exemplified by the closely related minnows  How diverse adult forms arise across species is a classic question in evolutionary biology. Modern evolutionary and population genetic methods have provided insights into the allelic underpinnings of differences between populations and closely related species, yet a fuller understanding of trait evolution requires knowledge of the cellular and developmental mechanisms that translate gene activities into particular morphological outcomes. Achieving such an integrative perspective demands a system accessible to genetic analysis, but also developmental observation and experimental manipulation.Danio rerio . During ontogeny, interstripe elements of D. quagga and interspot elements of D. kyathit became populated with xanthophores, though these cells were somewhat fewer and less densely arranged in D. quagga. By adult stages, however, xanthophores in D. quagga were somewhat more numerous than in D. kyathit . Visible xanthophore pigment was similar between species during larval through juvenile stages but slightly greater in D. kyathit adults than D. quagga adults .Adult xanthophores first became apparent at similar stages in the two species . A complete understanding of pattern variation in these species will thus need to account for evolutionary alterations at multiple loci, potentially having direct or indirect effects on the differentiation and morphogenesis of multiple cell types.We identified developmental and cellular features of pigment pattern formation in striped D. rerio, single loss-of-function mutations were sufficient to move the pattern through a substantial region of a morphospace defined by dorsoventral and anteroposterior pattern variation. Yet, the loss-of-function phenotypes most often isolated in forward or reverse genetic screens are unlikely to represent the full spectrum of naturally-occurring allelic variation influencing pigment cells and the patterns they form. Pattern mutants isolated in the laboratory are typically chosen and recovered because they have marked and consistent pattern defects. Alleles of small effect are more likely to be missed, and these, as well as alleles that are difficult to characterize molecularly  may be less likely to be isolated. By contrast, naturally occurring variation might, or might not, be under selection, and alternative alleles would be expected to have varying degrees of penetrance and expressivity. Moreover, our analysis of the broader morphospace occupied by Danio species  illustrates that some phenotypes—like vertical bars—appear to be relatively inaccessible in D. rerio, even after decades of genetic screening.By studying natural phenotypic variation, our study uncovered complexities of pigment pattern evolution in a way that cannot be achieved solely from laboratory-induced mutations. We found that in D. quagga and D. kyathit, we were able to find several QTL affecting phenotypes of hybrid, backcross individuals. The QTL we identified are notable for being several in number, in some instances epistatic to one another, and different between crosses derived from different parents. Some of the associated regions harbored genes known to have roles in pigment pattern formation in D. rerio  that are plausible candidates for variation in D. quagga and D. kyathit. Nevertheless, some QTL corresponded to genomic regions not previously associated with pigmentation or pigment pattern in D. rerio. That such regions were identifiable, even with mapping crosses having modest numbers of individuals, suggests that further efforts to identify segregating variation within and between species of Danio, combined with mapping or association studies at larger scale, could provide mechanistic and evolutionary insights complementary to what can be learned from zebrafish.In focusing here on naturally occurring variation in D. quagga and D. kyathit is complex developmentally and genetically. Like some D. rerio mutants, D. kyathit initially formed two primary stripes but these broke into spots as development proceeded. This failure of stripe integrity was not the result of a single alteration, for example the absence of a single class of pigment cell, or to pleiotropic changes owing to mutation at a single, major effect locus, as can be the case for mutants of D. rerio Please let us know if you have any questions while making these revisions.Yours sincerely,Robert N. KelshGuest EditorPLOS GeneticsGregory BarshEditor-in-ChiefPLOS GeneticsThis is a detailed, thorough and well-illustrated study, examining the genetic basis for naturally-evolved pigment patetrn differences in an experiemtnally accesible model system. Parichy and colleagues take advantage of phylogenetic proximity of two Danio sp. with distinct pigment patterns, and which form fertile hybrids, to begin to dissect the basis for natural pigment pattern evolution, in the context of the experimental studies in zebrafish which examine single locus mutations affecting coding regions and which generate large effects.They begin by developing a quantitative measure of Danio pigment pattern, enabling ready comparison of spotted v striped patterns. Whilst in many ways this simply confirms the obvious visual differences seen in the 3 species, it has the advantage of giving an objective measure of the pattern differences, albeit restricted to melanophores, for both the DV and AP axes, and so comes into its own when examining rerio mutants and quagga x kyathit hybrids. The description in the Results of why they do this (p.4 Results second paragraph) is clear, but the description would benefit from slight expansion to explain the metrics used to define melanophore pattern; I am struggling to pin down what exactly the DV:AP ratio is measuring. The S3 Fig is helpful for explaining how images are processed, but doesn’t quite explain this final step.Subsequently they perform a characteristically thorough and highly quantitative assessment of pigment pattern formation in both species. This section is very well documented, but somewhat overwhelming as presented. A summary table clarifying the observed similarities and distinctions in the evolution of pattern in the two species, with direct comparison to WT (and mutant?) zebrafish, would be helpful in highlighting clearly to the reader which features are candidates for being relevant to the switch between spots and stripes.F1 hybrids between the 2 species were generated and patterns quantitated for comparison with parents, before backcrossing to kyathit for genetic characterisation by QTL mapping. Two independent backcrosses identify broad candidate regions on multiple chromosomes associated with a set of pigment pattern differences between the two species, although none were shared between the two backcrosses. None of these are directly characterised, but several are associated with known rerio pigmentation genes; however, the linked domains are very extensive, so it remains unclear if these known loci are contributing.The authors main conclusion is that natural pigment pattern evolution in these two species appears to result from a complex genetic mechanism, involving multiple loci, similar to observations in other species.Minor point:p.11 ‘This situation contrasts with spotted mutants of D.rerio, in which melanophores are often fewer   or of similarabundance   to that of wild-type.’ For this comparison will be useful, it seems that comparison of quagga and hyathit melanophore density to WT zebrafish is required.Reviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: Nicely conducted, and clearly reported, study. I have no significant concerns, as the data collection, and statistical analysis, have been carried out to a high standard. The general conclusion - that natural variation has a different, and more complex genetic basis than discovered in laboratory mutants - is not surprising to me, but is still not generally appreciated. This paper lays out a quantitative methodology that should be broadly applicable to other studies/systems.Just a few minor typos remaining, for example:Fig 6. Chromosomal regions associated with pattern and size metrics in F2 backross individuals. (backcross)Reviewer #2: In their manuscript "A complex genetic architecture in zebrafish relatives Danio quagga and D. kyathit underlies development of stripes and spots", McCluskey and co-authors provide an extremely thorough analysis of the genetic, cellular and developmental underpinnings interspecies variation in pigmentation pattern in Danio species.The authors first document the pigmentation phenotypes of the three Danio species, Danio rerio, Danio quagga (both striped) and Danio kyathit (spotted) at a cellular level. Next, they quantified dorso-ventral and anterior-posterior variation of melanic patterns across the Danio species complex and in mutant lines of the zebrafish, Danio rerio. This allowed the authors to compare the pattern variation by presenting the data in a 2-dimensional morphospace. The rest of the analyses focuses on the two sister species Danio quagga (striped) and Danio kyathit (spotted). A detailed analysis of pigment pattern development in the two species shows how the two phenotypes diverge during development. An analysis of the genetic basis using quantitative trait loci mapping provides insights into the (very polygenic) genetic basis of the phenotypic variation in pigmentation patterns between these two species.I very much liked this manuscript for three reasons:i. The study investigates a phenotype (pigmentation patterns) from three completely different angles. It investigates the phenotypes from an evolutionary viewpoint by comparing pattern across species, provides a very detailed analysis of the developmental differences between two sister species that differ in their pigmentation and lastly and most importantly gives insights into the genetic basis underlying this variation. It therefore provides a uniquely complete and integrative analysis.ii. The analyses conducted in this study have a very high quality and are very elaborate.iii. I could imagine that it might be initially disappointing to realize that the identification of target genes (or even mutations) turned out to be so difficult because of the complex genetic basis. Nevertheless, I found the take-home message a very important one: That the genetic basis of natural variation is often much more complex, even if phenocopies could be generated by a single mutation.I would like to congratulate the authors to their work. I have only a few, mostly minor suggestions.Additional comments:1) I initially thought that the F2 have been generated through inbreeding of F1 individuals. I think it would be helpful to indicate earlier that those are backcrosses. Also, a crossing scheme could be helpful, especially to more easily follow the results in Fig. 6D that are very complex. Also, the method/concept of QTL mapping could be maybe already introduced earlier .2) I enjoyed reading the introductory paragraph of the species. One information that would be nice to have there too, is the divergence time between Danio quagga and Danio kyathit and both to Danio rerio, also to know what “closely related” means in this fish family.3) Although the schematics are cute and helpful, I would consider adding actual photographs to Figure 2 on top. Also, seeing photographs of some of the Danio rerio mutants that are close to the D. kyathit morphospace would be a helpful visualization of one of the key messages . Please ignore the request in case it is difficult because of copyright issues.4) While I found Figure 5 to be really interesting to understand the correlation between the cellular characteristics and pigmentation quantifications. This is now only a suggestion, but I kept wondering where the parental species and F1s would be in this PCA. If measurements for these are available a helpful visualization would be to perform a PCA with the parental species only and then project the F1s and BC individuals on the original PCA.5) It would be great if the authors could expand a bit more on the method part of the QTL . Also, is there any evidence for larger rearrangements between these two species and the Danio rerio genome that could have affected the analysis ?6) In case genomic sequences of Danio quagga and Danio kyathit are available: Are there any coding differences between the target genes within the target intervals in Fig. 6 . I assume the responsible mutations are rather non-coding, but as comparing the coding sequences might be relatively straight-forward it might be worth checking.Reviewer #3: McCluskey and colleagues have submitted a manuscript describing an approach to quantify morphometric features for stripe/spot patterns in closely related danio fish species. They then used this quantification scheme to map QTL's of two closely related species of danio, one with stripes the other with spots that when intercrossed produced fertile offspring. They were able to determine regions on several chromosomes that had significant lod scores resulting in their being able to reject the single gene hypothesis for pattern differences. Further selective crosses identified other loci with variation both between species and within each species.Honestly the paper is well written, clear, the data of high quality. I have no significant critiques for improving the paper.minor:-fig 2 is not referenced in the text.-I do wonder if the significant regions could have been narrowed rapidly and more finely by doing low coverage whole genome sequencing on pools of classified offspring looking for regions with loss of hetrozygosity. Not required for the paper, just curious.Reviewer #4: In this manuscript, McCluskey et al. make use of two closely-related Danio species with divergent pigment patterns to begin explore the genetics underlying these differences. This approach relies upon the ability to generate fertile hybrids of the two species, D. quagga and D kyathit, and a morphometric approach to quantify various elements of the adult pattern for the purpose of identifying QTL’s. The study represents an alternative to the standard genetics of the Danio rerio model, by interrogating the nature of phenotypic differences in natural populations as opposed to those generated via laboratory-induced mutations.The data in the paper are complex but presented with great clarity and rigor. Although the basic result that the stripe vs. spot difference between the two species is polygenic, and that while several genomic regions of interest are identified there are no “smoking guns” might be seen as slightly disappointing, in fact it validates the approach. This result is of great importance as biologists increasingly try to expand beyond just the traditional model systems.My only suggestions:Fig 5A presents shows a typical example of the F1 hybrid pattern, but I’d also be curious to see examples of the extremes in the backcross generation used for the Fst scan . Also, are the individual male and female hybrids used for the backcrosses represented here? It would be interesting to know where each lies in the morphospace.If I understand correctly, the differences observed between the results of the two backcrosses  are likely due to interspecies variation as opposed to the sex of the parent-of-origin per se having some functional role? Perhaps that could just be clarified with a sentence somewhere.Small points:p. 5 fourth line from the bottom: word “adult” is repeatedp. 6 last line before “Genetics of pigment pattern variation” subheading: “each having more specific effects”S1 Fig legend third line from bottom: presumably should be “~3.5 cm SL”, not 3.5 mmReviewer #5: The manuscript by McCluskey et al. describes their approach to identify genetic bases determining ‘stripes’ or ‘spots’ adult pigment pattern in Danio species. Their study is very intriguing and challenging: they used two close relatives of Danio species which can produce fertile F1 progeny, thus enabling them to use F2 for mapping natural genetic variants between the two species, responsible for the different pigment patterns of ‘stripes’ or ‘spots’. Unfortunately, their genetic analyses haven’t reached a clear conclusion: instead of finding a simple main effect locus involved, they revealed a complex genetic basis with several loci contributing to pattern variation. In overall, the results are convincing and the strategies described are valuable for future research by others.Comments:Their genetic analyses pointed out some loci, where the candidate genes known to be involved in pigment pattern formation are located. Are any of these responsible for the formation of stripe or spot?They don’t mention about the difference across species in sequence there, nor in their expression . Are there any nucleotide alterations? If any, are they all non-coding?Their imaging analyses for chromatophore behavior upon pigment pattern formation in the two species revealed different timing of melanophore differentiation is key to generate distinct pattern, ‘stripes or spots’, suggesting that heterochrony should explain for this interspecies difference. Are the above loci related to the heterochrony?‘Morphospace’ part is hard to understand, especially Fig 5B. Where is Fig 5C?In Fig. 7, is A consistent with B in genotypes? Is the maternal allele always indicated first (left)? Why are the genotypes different between 7A and 7B?SL appears prior to spelling-out .Duplicate ‘adult’ .**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: YesNo: No, but the authors have declared that data will be available upon publication through public repositories.Reviewer #2: Reviewer #3: YesReviewer #4: YesReviewer #5: Yes**********what does this mean?). 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