Globally, fermented beverage and condiments are made by using different conventional practices, raw materials, and microorganisms. This paper presents the available literature review on the technology and microbiology of traditional Ethiopian beverages and condiment products. Traditional fermented beverage and condiment products have essential vitamins, minerals, enzymes, and antioxidants that are all enhanced through the process of traditional fermentation practices. In Ethiopia, fermented beverage and condiment products have practiced in a long history. During the production of traditional fermented beverage and condiment products, controlled natural fermentation process with the absence of starter cultures are used to initiate it. Moreover, the preparation of many traditionally fermented beverage and condiment products is still practiced in a household art, thereby a wide variety of fermented beverages and condiments are consumed in Ethiopia. In conclusion, the review discusses the nature of the beverage and condiment preparation, poor traditional household processing, and the extent and limitation of scientific work done so far and suggests some recommendations to limit the problem in Ethiopia. Ethiopia is a country rich in cultural diversity. The variety of foods and beverages, processed and consumed among the various ethnic groups are indicators of this diversity. Ethiopia is one of the countries where a wide variety of traditional fermented foods and beverages are prepared and consumed. Fermented products prepared from plant and animal sources play an important role in human food due to changes in the physical, chemical, and nutritional properties by microorganisms .Fermented beverages constitute a major part of the diet of traditional African homes serving as from inebriating drinks to weaning foods; in addition, these beverages also play a major role in social functions in which they are consumed in different occasions such as marriage, naming, and rain making ceremonies , at festTraditional fermented condiments are among the various traditional fermented foods, which are produced by microbial fermentations under highly variable conditions in different parts of the world . Such foinjera,” “ergo,” “ititu,” “ayib,” “qibe,” “arrera,” “kocho,” “tella,” “siljo,” “datta,” “awaze,” “borde,” “tej,” “areki,” “cheka,” “azo,” “keribo,”and “korefe.” Of these “tella,” “cheka,” “tej,” “borde,” “areki,” “keribo,”and “korefe” are among the varieties of fermented beverages consumed in Ethiopia, while “awaze,” “datta,” “siljo,” and “azo” are among the varieties of traditionally fermented condiments known in Ethiopia. Therefore, the aim of this paper was to review microbiological studies made by various researchers on fermentation, other processing methods, and microbial safety of traditional Ethiopian beverages and condiments.Ethiopian local fermented foods and beverages are products of acid-alcohol type of fermentation. These include “Tella” is a popular Ethiopian traditional beverage, which is made from diverse ingredients. “Tella” has various vernaculars in the various regions and is based on substrates such as barley, wheat, maize, millet, sorghum, “teff,” or other cereals [Vernonia amygdalina) and water several times. Then, the cleaned container is inverted over smoking wood fragments of “weyra” (Olea europaea) for about 10–15 minutes. This will remove microorganisms sensitive to wood smoke and adds the desired flavor to product. “Bikil” , the source of amylase from corn or barley or wheat grain is moistened in a container and left to germinate for about three days and finally sun-dried. The gesho plant (Rhamnus prinoides), local hops, which is different from hop (Humulus lupulus) is widely cultivated in Ethiopia and is available in the dried form in the local market. Kita is broken into small pieces. Barley flour is separately toasted on a metal pan sprinkling water on it during toasting until it turns dark brown. This is called enkuro. The color of “tella,” which may vary from light yellow to dark brown, is determined by the extent of baking kita or toasting enkuro. This mixture of enkuro, the rest of the germinated grains (bikil), some gesho, and water are added to the container. The mixture is kept covered overnight, after which more water is added, and the container is kept sealed for 5 to 7 days, until the beverage is ready. “Tella” can be kept for 10 to 12 days [Saccharomyces spp., (mostly S. cerevisiae) and Lactobacillus spp., (mostly Lactobacillus pastorianumi)  .Areki” is a distilled, colorless, clear, traditional alcoholic beverage in which fermented products are prepared in almost the same way as “tella” except that the fermentation mass in this case is more concentrated [Areki” fermentation product is known as Yereki-tinsis which is prepared by mixing powdered gesho leaves and powdered bikil (1 : 2 ratios) with water to give a mixture of free flowing consistency and will be put aside to ferment for about five days. Traditionally areki is classified as terra-areki and dagim-areki. The term dagim in Amharic refers to “second time” and designates that it is distilled second time, whereas the term terra in Amharic refers to “ordinary” [“entrated , 19. “Arrdinary” . The alcrdinary”  and varirdinary” . Dagim-ardinary” . It was rdinary” . Since trdinary” .Cheka” is a cereal and vegetable-based fermented beverage which is consumed in Southwestern parts of Ethiopia [Cheka is mainly prepared from cereals such as sorghum (Sorghum bicolor), maize (Zea mays), and vegetables such as leaf cabbage (Brassica spp.), moringa (Moringa stenoptella), and decne (Leptadenia hastata). In some localities, few households also use dried edible leftovers of injera, kitta, or kurkufa. The processes of cheka preparation are very complex and vary among households, villages, and localities. Three types of cheka are produced in the study districts such as hiba (parshota), chaqa (fasha), and menna (poh-kedha or madhot). The duration of cheka fermentation varies from 12 hours for menna to months for parshota [“Ethiopia . Cheka iparshota   and put back to fermenting [tej, relies on the microorganisms (lactic acid bacteria and yeast) present in the substrates, fermentation vats, and equipment. Their metabolic products contribute to acidity and also add distinctive flavor and aroma to the fermenting material. Major yeast species in tej were Saccharomyces cerevisiae, Kluyveromyces bulgaricus, Debaromyces phaffi, and Kluyveromyces veronae. The lactic flora consisted of Lactobacillus, Streptococcus, Leuconostoc, and Pediococcus species [“rmenting . Honey,  species , 23 , barley (Hordeum vulgare), wheat (Triticum sativum), finger millet (Eleusine coracana), sorghum (Sorghum bicolor), and tef (Eragrostis tef)) traditionally fermented beverage and is widely consumed in the southern and western parts of Ethiopia. It is produced by spontaneous fermentation using a simple equipment. Borde is a whitish-grey-to-brown-coloured beverage, with a bushy consistency and a sweet-sour taste [Borde is considered to be a low-alcoholic beverage even though the duration of its fermentation is long enough (4 days) to result in a considerable accumulation of ethanol [“ur taste , 25. Bor ethanol  . The spices added to it include garlic (Allium sativum), ginger , sweet basil (Ocimum sanctum), rue , cinnamon (Cinamommum zylanicum), clove (Eugenia caryophyla), Ethiopian caraway (Trachyspermum copticum), Ethiopian cardamom (Aframomum anguistifolium), and salt. Awaze fermentation starts by whipping a portion of the ground pepper-spice ingredient with warm water until it attains a thick consistency and left to ferment at ambient temperatures. The most dominant microorganisms in fermentation of awaze are aerobic mesophilic microflora such as Bacillus spp. and Lactic acid bacteria (LAB) [It is known that, fermented food, beverage, and condiment products are commonly produced throughout the world. Different countries of Africa's protein-rich food ingredients are often fermented to make condiments which result from the microbial fermentations of vegetable-spice mixtures . Awaze iia (LAB) , 34.awaze mainly in the southern part of Ethiopia and is consumed with other items on the basis of their desirable aromas and flavors resulted from the microbial fermentations of vegetable-spice mixtures. The major substrate in making of datta is the small chili pepper (Capsicum frutescence) at its green stage. Garlic and ginger, small amounts of fresh sweet basil and seeds of rue, were peeled, washed, and cut into small pieces and mixed together [Datta  is a condiment of similar use as that of together , 35.The mixed ingredients were manually wet-milled on a flat smooth traditional stone-mill into a greenish paste and transferred into a 500 ml screw-cap bottle to ferment at 20 to 25°C. Lactic acid bacteria (LAB) initiated fermentation, and later the homolactic LAB started and dominated the fermentation for the first 2 days and the heterolactic LAB took over thereafter .datta fermentation with Salmonella typhimurium [E. coli O157: H7 [E. coli O157: H7, although the pathogen survived for more than seven days at refrigeration storage [Challenge studies on himurium  and E. cO157: H7  showed t storage .siljo fermentation. Siljo is one of the traditionally fermented condiments of Ethiopia made up of safflower (Carthamus tinctorius) extract and faba bean (Vicia faba) flour. It is a popular condiment during the long fasting period before Easter [Lactobacillus acidophilus, L. plantarum, and L. delbrueckii and the yeasts Saccharomyces cerevisiae, Rhodotorula glutinis, Yarrowia lipolytica, and Saccharomyces rouxii. The fermentation was, but, initiated and later dominated by L. plantarum and L. acidophilus. The fermented product has protein and fat contents of 28 and 25%, respectively [A typical example of legume fermentation practiced in Ethiopia is e Easter . The blaAzo is a traditionally fermented semisolid condiment prepared from cereal flour and leaves of endod in northwest Ethiopia Tigray regional state. Of course, the way of processing and ingredients added for the preparation of this condiment differ from one community to other. Cereal flours and fresh leaves of endod are primary ingredients used for Azo preparation along with different variety spices, namely rue, garlic, ginger, korerima, black cumin, black mustards, and as minor ingredients for flavor enhancement. Azo-fermented product is consumed during Easter fasting season. Fermented condiments are used as taste enhancers in many traditional dishes [azo fermentation is a few scientific work undertaken so far (l dishes . Regardin so far .Because many fermented foods are produced using microorganisms, the risk of toxin contamination is high. During natural fermentations, food-poisoning flora and coliforms may also grow with the fermented food product. These microorganisms need to be eliminated to make fermented foods safe for consumption . SeveralIn conclusion, Asia is well-known for its exotic traditionally fermented food and beverage products produced using a wide range of raw materials, microorganisms, and fermentation processes. The indigenous methods of fermentation were aimed to preserve and balance the availability of food sources. Furthermore, many scientific research studies have exhibited promising and sustainable opportunities related to these traditionally fermented food products. The nutritional values of fermented foods are related to a unique group of microflora that may enhance health benefits directly through the interaction with the host or indirectly through metabolites synthesized during fermentation. The bioactive compounds and other interactions within fermented food can add novel flavors to food and impart potential health benefits. However, future studies need to be conducted in order to explore various aspects of fermented food products, such as the determination of biomarkers for fermented food health benefits, safety concerns related to these products, and the bioaccessibility of microbial metabolites.


Barkam center, and the changes in allele diversity, heterozygosity and inbreeding coefficient with increasing generations. The results show that the population has high genetic diversity, but the genetic structure of different generations in the population shows that the extent of inbreeding is slowly increasing with increasing number of generations. This suggests that the BMS-RM is effective for maintaining genetic structure diversity of population, but captive forest musk deer in Barkam center still face the risk of inbreeding increasing. So, it is necessary to optimize the BMS-RM of captive forest musk deer or introduce male forest musk deer from an unrelated population. This study revealed the status of genetic diversity of captive forest musk deer in Barkam center and the current risks of inbreeding for providing an important understanding to the application of BMS-RM.Forest musk deer is an endangered species. Musk produced by male musk deer is precious natural flavor and an important ingredient of traditional Chinese medicines. Forest musk deer is a territorial animal, and it is very difficult for captive forest musk deer to record the accurate pedigree using the “Breeding Management System of Rotated Mating” (BMS-RM). In the study, we examined the genetic structure of captive forest musk deer population in Moschus berezovskii) in Barkam Musk Deer Breeding Centre using twelve microsatellite markers, and then analyzed the change in genetic structure of successive generation groups from the population. The data provide a new understanding for the evaluation and usage of the breeding management system. Microsatellite marker analysis detected 141 alleles with an average of 11.75 alleles for each marker. The average expected heterozygosity (HE) was 0.731. Performing an F-statistical analysis on the data showed that the genetic diversity of population decreased, and the inbreeding coefficient significant increased with the increase of generation, and FIS of the 1st generation is significantly lower than that of the second to fifth generation (p < 0.01). The result suggested that the captive population was facing the pressure of inbreeding (FIS = 0.115) and the subsequent loss of genetic diversity. Therefore, it is necessary to improve the breeding management system of the captive population by preventing close relatives from mating or inducing new individuals from the exotic population.We investigated the genetic diversity of the population of captive forest musk deer ( Moschus berezovskii) is an Asian ungulate that is listed as an endangered species by the International Union for Conservation of Nature (IUCN) [The forest musk deer (e (IUCN) . It is ie (IUCN) . The fore (IUCN) . Musk pre (IUCN) ,5.Barkam, Miyaluo, Dujianyan, Fengxian, Yaan, have been established to relieve the severe hunting pressure to wild populations and provide markets with legal musk obtained from captive animals for traditional Chinese medicine [Barkam center was the first to be established [Barkam center are of great importance for maintaining genetic diversity, expanding the population, and assessing the breeding management system of the captive forest musk deer. Forest musk deer is a solitary, territorial species that is easily frightened [The breeding of captive forest musk deer originated in 1958 in China. The early breeding technique was mainly to expand the population, explore the method of musk collection and improve the husbandry conditions of timid forestry musk deer . After tmedicine . Howeverablished , and conightened . If maleightened . HoweverMicrosatellite DNA, also known as simple sequence repeats (SSRs) or short tandem repeats (STRs), consists of 1–6 core sequences repeated in series. With good stability, wide distribution, dominant inheritance, and many other advantages, SSRs provide excellent markers for measuring genetic diversity ,11 and lBaisha of Sichuan Province and Jinfeng Mountains by Zhao et al. [Sichuan and Shaanxi Provinces, China, by comparing their mitochondrial DNA-loop region’s sequence structure. Since then, more microsatellite loci have been found in forest musk deer [Miyaluo, Jinfeng Mountain and Barkam by using eleven microsatellite loci. The results revealed 142 different alleles in 121 individuals. The expected heterozygosity (HE) in the three populations was 0.552, 0.899, and 0.894, respectively, which indicated the three populations had high genetic diversity. The genetic diversity of two musk deer populations in Baisha and Jinfeng Mountain of Sichuan Province was calculated using 15 microsatellites and 22 AFLP primers [Miyaluo using seven microsatellite markers, and the genetic diversity of the younger musk deer was higher than that of the adult group [Other molecular genetic methods have been applied to the study of genetic diversity in forest musk deer populations. The amplified fragment length polymorphism (AFLP) marker technique was used to compare the genetic diversity of two populations of captive forest musk deer in o et al. . Peng eto et al. ,23 and Fo et al.  analyzedusk deer ,25,26. Gusk deer  profiledlt group .Barkam and examine the change in genetic structure of successive generations based on the polymorphisms at twelve microsatellite loci. We then also compared the distribution of private alleles and inbreeding coefficients of different generations and provided a scientific reference for the inbreeding control and genetic diversity maintaining of BMS-RM. If there is a high genetic diversity in the closed population, this implies the present BMS-RM is effective in maintaining the stability of genetic structure. If the inbreeding coefficient does not significantly increase with the increasing number of generations, this impliesy the present BMS-RM is effective in avoiding inbreeding. Otherwise, the BMS-RM will need to be improved in the future.In this study, our objective was to evaluate genetic structure of the population at the musk deer farm of Sichuan Institute of Musk Deer Breeding in Songgang town, Barkam, Sichuan Province, and there are more than 300 forest musk deer in Barkam center for genetic protection and musk production. The population originated from 18 wild forest musk deer in 1958, and some wild individuals were added to the population until 1980. The generations of these individuals were recorded, and their blood samples were collected at the time of musk collection or vaccination. These blood samples have been collected continuously for nearly 20 years, and were kept at −80 °C. All operations strictly obeyed Wildlife Protection Acts and Regulations of China, and were also approved by the Wildlife Protection Committee of Musk Deer Research Institute (MDRI-2009-02), and the Ethics Committee of Use of Endangered Wild Animals in Research and Teaching (2018YSZH0019).The forest musk deer were housed and raised in the farm of captive forest musk in Barkam center is strictly adheres to the rules of BMS-RM, and all individuals avoid sib-pair and parent-offspring mating  in a breeding group are housed in the same shed, and the male and female forest musk deer mate freely. The two male forest musk deer are transferred to another shed for mating with another 15 females in the next breeding season. The offspring of 3-year-old male and 2.5-year-old female musk deer begin to be used for reproduction in the next generation [g mating .We chose 238 forest musk deer blood samples with generational record. All individuals  were from 5 successive generations . The blood samples were preserved at −80 °C after addition of citric acid-EDTA as anticoagulant. Genomic DNA was extracted using the phenol-chloroform method  and stor2+ (25 mmol/L), 1 μL dNTPs (2 mmol/L), 1 μL of each upstream and downstream primer (10 μmol/L), 1 μL of template DNA, 1 U of Taq DNA polymerase , and 7.5 μL ddH2O. PCR amplifications were performed using a Perkin Elmer 9700 thermocycler . The PCR amplification conditions were: 95 °C pre-denaturation for 5 min, 94 °C denaturation for 30 s, annealing at a specific temperature , are shown in perature  for 50 sO), expected heterozygosity (HE), and polymorphism information content (PIC) were determined using CEVUS 3.0 software [IS), and population differentiation coefficient (FST) of each microsatellite locus were calculated with the FSTAT 2.9.3 software package. FIS of all generation in 12 microsatellite loci were compared by F-test. HE of different generation was compared by the Wilcoxon nonparametric test using GENEPOP 3.4 [IS increment (ΔFISE1) of the population of random mating  was obtained using the formula: mN is the number of male individuals, while fN is the number of female individuals. The expected FIS increment (ΔFISE2) of the breeding group  was obtained by the formula: mN = 2 and fN = 15. The actual FIS increment (ΔFISA) in the breeding management system was obtained according the formula: i is generation and i = 1, 2, 3, 4 and 5 in the study [The genetic diversity parameters of the population, including the number of alleles (A), observed heterozygosity (Hsoftware . Linkagesoftware . The genEPOP 3.4 . The expE) was 0.731. The population had a large number of rare alleles. We identified 78 rare alleles at the 12 microsatellite loci with an average of 6.5 alleles per locus. In addition, the FIS value also demonstrated a moderate degree of inbreeding risk in the population, since the inbreeding coefficient was 0.115, which is greater than 0.05 [p < 0.01), while the remaining eight were in balance. After a correction using the Bonferroni method, the results of linkage disequilibrium analysis showed that the twelve microsatellite loci did not have any linkage between each other.Our captive musk deer population had a high degree of genetic diversity . 238 indhan 0.05 . Four miR = 6) in the 1st generation compared to other subsequent generations, and the number of private alleles continuously decreased from the 1st to the 4th generation because some male musk deer offspring didn’t participate in the breeding. The number of private alleles increased from the 4th to the 5th generation because rotation mating induced new male musk deer from other shed. The number of private alleles might vary up and down around a constant value, if more generations were examined because the breeding obeyed the BMS-RM in the closed population. At the same time, the number of rare alleles (less than 5% of the population) was highest in the 4th generation group (RA = 49). However, the risk of loss of private and rare allele was always present, as some male individuals were not involved in breeding.Female forest musk deer are seasonal breeding animals, and produce one generation about 3 or 4 years . The allO) and the expected heterozygosity (HE) gradually decreased from the 1st to the 5th generation. The HO and the HE were, respectively, 0.743 and 0.742 in the 1st generation, and 0.632 and 0.731 in the 5th generation. The Wilcoxon nonparametric test showed that there was no significant difference in HE between the 1st and the 5th generation (p > 0.05). The population inbreeding coefficient (FIS) showed a gradual increase from the 1st generation to the 5th generation. The minimum at 1st generation was 0.001, and the degree of inbreeding in the 2nd (FIS = 0.134), the 3rd (FIS = 0.112), the 4th (FIS = 0.137) and 5th (FIS = 0.139) generation extremely significantly increased compared with the 1st generation (p < 0.01). The expected FIS increment (ΔFISE1) of the population of random mating was 0.005, and the expected FIS increment (ΔFISE2) of the breeding group was 0.050, while actual FIS increment (ΔFISA) in the breeding group was 0.034. The result suggested the BMS-RM was effective to maintain diversity in a captive population. However, the inbreeding of close relatives must be prevented to control the increase of inbreeding coefficient.Five generations showed high heterozygosity . HoweverE), and polymorphic information content (PIC) are the important indicators of the level of diversity. Botstein et al. proposed a measure of PIC variability, with PIC < 0.25 being a low level, 0.25 < PIC < 0.5 moderate, and PIC > 0.5 a high level [Barkam center. Xia et al. studied genetic diversity in a population of 48 captive forest musk deer using six microsatellite loci and found A = 8.67, HE = 0.842 and PIC = 0.811 [Dujiangyan center of Sichuan Province and found A = 9.4, HE = 0.820, and PIC = 0.778. Guan et al. [Miyaluo center, Jinfeng Mountains center and Barkam center, and found A = 12.91, HE = 0.899, and PIC = 0.884. Huang et al. [Barkam center, and found A = 24, HE = 0.854, and PIC = 0.837. Our results  were similar to theirs, confirming the high genetic diversity of captive musk deer in China. Other wild deer populations have lower diversity: red deer [Cervus nippon pseudaxis, A = 5.70; HE = 0.600) [Odocoileus virginianus, A = 5.90–9.20; HE = 0.670–0.730) [Microsatellites are molecular markers with a high degree of polymorphism, good stability and wide availability in mammals. They are useful molecular markers in the field of population genetic analysis and have been widely used in genetic protection of wildlife ,31,32,33gh level . Zhou etgh level . In our n et al.  used 11 g et al.  used 7 m8–0.689) ; sika de= 0.600) , and whiBaisha of Sichuan and at a reserve in Jinfeng Mountains, and both populations had high genetic diversity [Jinfeng Mountain was higher than that in Baisha [Sichuan Province [Shaanxi Province, China [Shaanxi Province showed considerable variation [Barkam center was maintained a high level under closed breeding condition, and it implied that the BMS-RM was effective in inbreeding controlling.The AFLP was first applied to measure genetic diversity of two populations of captive musk deer populations in iversity ,27, but n Baisha . Peng etProvince , and Fene, China . They foariation ,24. The E = 0.742), and Wright’s inbreeding coefficient at the 1st generation was the lowest (FIS = 0.001) among the other generations. Consistent with our findings, Huang et al. reported that the genetic diversity of adult captive population was higher than that of younger population [Barkam center is at the risk of genetic diversity loss judged form the increase of inbreeding coefficient from the 1st generation to the 5th generation. On the contrary, our result also showed that the BMS-RM also maintained genetic diversity, even improved the genetic structure of the captive musk deer population judged from the average number of alleles and the allele richness: the average number of alleles increased from 8.25 at the 1st generation to 9.25 at the 5th generation, and the allele richness increased from 6.177 at the 1st generation to 6.325 at the 5th generation. New private alleles appeared at the Mb102C  and Mb34  locus. Conversely, a private allele at Mb116H locus was detected in the 1st generation, but was not detected in the 4th, 3rd and 2nd generation. The loss of private allele on Mb116H locus may be due to that some male offspring individuals didn’t involve in mating. Huang et al. came to a similar conclusion in their study [Protecting the gene pool of a population requires preserving its genetic diversity. DNA replication, non-random mating, selection and drift, and other factors all tend to lead to the loss of diversity . In our pulation . Captiveir study . Maybe, R = 5) and a higher number of alleles overall (A = 49). These rare alleles only were found in a few individuals, and they commonly disappeared from the gene pool. One founder of wild forest musk deer should be sired a sufficient number of offspring in order to prevent the pedigree bottleneck effect. Ballou’s [It is worth noting that the newly added genes in generation groups were mostly rare alleles with less than 5% of gene frequency. Our study showed that the 5th generation group had more private alleles , the appropriate number of individuals within breeding group (BGN), the number of breeding groups within the large population (mN) and the exchange of male musk deer across different generations were considered as important factors. It’s necessary to further clarify the proper size of N, BGN, and mN in order to achieve the minimum inbreeding increment using the BMS-RM under closed breeding conditions, and we advocate the exchange of individuals among different breeding centers, and to occasionally induce wild forest musk deer as the complementary strategy of BMS-RM in China.Judging from the inbreeding coefficient increment (ΔFIS), the BMS-RM decreased the risk of inbreeding compared with the mating system of random mating within the breeding group, and with the non-rotation mating system of male musk deer across different generations, but it also increased the degree of inbreeding compared with the random mating system in a large population. It is difficult to randomly mate in a large population of captive individuals. Indeed, random mating of large population is easy to cause the repeated mating and missed mating of female musk deer . TherefoBarkam center maintained a high level. The breeding management system of rotated mating (BMS-RM) is effective in maintaining high genetic diversity, but the genetic diversity of population slowly decreased and inbreeding coefficient slowly increased with increasing number of generations, which implied the captive population is facing the pressure of inbreeding and subsequent loss of genetic diversity. Therefore, it is necessary to improve the breeding management system or develop new breeding management system of the captive population in the future.In summary, the finding of our present study is that the genetic diversity of captive forest musk deer in


Trypoxylus dichotomus is one of the largest beetle species in the world and is commonly used in traditional Chinese medicine. Ten subspecies of T. dichotomus and a related Trypoxylus species (T. kanamorii) have been described throughout Asia, but their taxonomic delimitations remain problematic. To clarify issues such as taxonomy, and the degree of genetic differentiation of Trypoxylus populations, we investigated the genetic structure, genetic variability, and phylogeography of 53 specimens of Trypoxylus species from 44 locations in five Asian countries . Using specific‐locus amplified fragment sequencing (SLAF‐seq) techniques, we developed 330,799 SLAFs over 114.16M reads, in turn yielding 46,939 high‐resolution single nucleotide polymorphisms (SNPs) for genotyping. Phylogenetic analysis of SNPs indicated the presence of three distinct genetic groups, suggesting that the various subspecies could be treated as three groups of populations. PCA and ADMIXTURE analysis also identified three genetic clusters , which corresponded to their locations, suggesting that geographic factors were important in maintaining within population homogeneity and between population divergence. Analyses of SNP data confirmed the monophyly of certain subspecies on islands, while other subspecies  were found to be polyphyletic and nested in more than one lineage. AMOVA demonstrated high level of differentiation among populations/groups. Also, pairwise FST values revealed high differentiation, particularly between South and West, as well as between North and South. Despite the differentiation, measurable gene flow was inferred between genetic clusters but at varying rates and directions. Our study demonstrated that SLAF‐seq derived markers outperformed 16S and COII sequences and provided improved resolution of the genetic differentiation of rhinoceros beetle populations from a large part of the species’ range.The Japanese rhinoceros beetle  We estimate the evolution of morphological diversification as well as genetic differentiation, gene flow, and population genetic structure of Japanese rhinoceros beetle populations. Horn shapes and sizes diverge greatly even among closely related species and sometimes between populations of beetles, suggesting differential selective pressures on horns among different lineages ; T. d. xizangensis . With random amplified polymorphic DNA (RAPD) and mitochondrial DNA COII sequence analysis, homogeneity between small brown planthoppers from different geographic regions could be inferred  and 16S ribosomal RNA (rRNA) genes were employed in reconstructing the phylogeny of 22.1Trypoxylus individuals were caught by light trap or collected on sap exudate (food source for beetles) of broad‐leaved trees. A total of 128 individual beetles from 44 locations in five different regions of Asia  were obtained in this study as well as outgroup taxa included two beetles of Xylotrupes mniszechi from Tibet (China) and Xyloscaptes davidis from Vietnam. 53 specimens of Trypoxylus belonging to two species (T. kanamorii and T. dichotomus) and nine subspecies from five regions of Asia were selected for SLAF‐seq analysis and phylogenetic analysis based on 16S and COII sequences .During the season of peak activity (from May to mid‐August) in the years 2017 to 2018, live 2.2Xylotrupes was treated as an outgroup in our comparisons and thought be a more distantly related to Trypoxylus than Xyloscaptes  based on Dutrillaux et al.  for 1 min, and 72°C for 1 min; and a final extension step of 72°C for 10 min. The PCR products were purified and visualized by gel electrophoresis on 1.5% agarose gel to confirm amplification. Sanger sequencing was performed at TSINGKE. All sequences are available from GenBank under accession numbers MT103602–MT103655 for COII and MT080110–MT080164 for 16S rRNA.Genomic DNA was extracted from thoracic muscle tissue using the QIAGEN DNeasy Tissue Kit in accordance with manufacturer's instructions. For sequence‐based phylogenetic analysis, amplicons from cytochrome c oxidase II (COII) and 16S ribosomal RNA (16S rRNA) were used as they are common loci in barcoding. The 16S rRNA region was amplified by two primer sets (16sF/16sR and 16SB/16SA) , , which were employed because they successfully amplified species in 2.416S and COII sequences were edited using Seqman v. 7.1.0 in the DNASTAR Lasergene core suite software , and aligned using MAFFT v.6  analysis of concatenated 16S and COII alignment was performed in PAUP v.4.0b10  were performed using MrBayes v.3.1.2 with Markov chain Monte Carlo (MCMC) and Bayesian posterior probabilities  was performed by RAxMl v. 1.3  was used to predict enzyme digestion. DNA fragments of 264–364 bp were selected as SLAFs and prepared for paired‐end sequencing on the Illumina High‐Seq 2,500 sequencing platform  at Biomarker Technologies Corporation.For SLAF‐seq analysis, DNA concentration was quantified using a NanoDrop‐2000 spectrophotometer, and all DNA samples were diluted to 50 ng/μl. SLAF library construction was carried out following Sun et al.  with min2.6Raw pair‐end reads were clustered based on sequence similarity. Sequences with over 90% identity were grouped in one SLAF tag, SLAFs with low‐depth coverage were filtered out  were constructed using the neighbor joining (NJ) method employed in MEGA X with p‐distance and pairwise deletion option parameters  and expected heterozygosity (He) were defined as the probability that two randomly chosen alleles from the population are different  were used to measure the level of genetic differentiation among T. dichotomus populations based on genetic polymorphism in the SNP data  as applied in ARLEQUIN v. 3.5.1.2, which is a method of quantifying at what level  genetic variation is greatest  were inferred using MIGRATE‐N v. 3.6.4 , Tibet (China), Sagaing (Myanmar); 24 T. d. dichotomus from Southern China; 11 T. d. septentrionalis from Northeastern China, Korean Peninsula, Jeju Island (Korea), Hokkaido (Japan), Hyogo (Japan), and Fukushima (Japan); three T. d. politus from Yunnan (China) and Wiang Papau (Thailand); two T. d. shizuae from Yakushima (Japan); three T. d. tsunobosonis from Taiwan; two T. d. takarai from Okinawa (Japan); and two T. d. tsuchiyai from Kuchinoerabu‐jima (Japan).External morphology of all ed Table . MorpholTrypoxylus taxonomy) was individually proved to be unambiguously species‐ or subspecies‐specific. As such, additional morphological characters should be used in the further taxonomic study of Trypoxylus. For example, male genitalia have been successfully employed to delimit numerous Coleopteran species.The coloration of cuticle ranges from black to red; body length, including horn, ranges from 3 cm to over 9 cm, and all other external morphological characters had similarly broad and sometimes overlapping values across regions. Similarly, none of the analyzed morphological characters  = 274; consistency index (CI) = 0.818; retention index (RI) = 0.839; and rescaled consistency index (RC) = 0.686. ML and BI trees were similar in topology to the MP tree.To assess the monophyly of the t Figure . The aliTrypoxylus subspecies  and T. d. inchachina  in Japan, as well as a clade of T. kanamorii and T. d. politus from Myanmar and China.The phylogenetic tree based on 16S and COII sequence dataset did not clearly resolve the relationships among the s Figure , as repr3.3D. ponderosae (Coleoptera: Scolytidae) as a reference, the restriction fragment length of RsaI‐HaeIII, ranging from 264 to 364 bp, was used for defining SLAF tags in our study taxa. Polymorphism analysis of the SLAF markers was summarized in Table Using the genome of mountain pine beetles Trypoxylus taxa.SNP markers were developed based on high depth mapped to the reference genome in each SLAF tag. We identified 2,127,917 SNPs, with an integrity score between 30.16% and 53.93%. To avoid bias in the estimation of the baseline differentiation and to reduce sequencing and PCR error from the SNPs dataset, SNPs with a minor allele frequency (MAF > 0.05) on average across sampling locations were retained and used for analyses. It has been shown that very low‐frequency SNPs (MAF < 0.05) created biases in quantifying genetic connectivity and should therefore be excluded when inferring demographic processes  and Yunnan (China) were clustered together with T. d. politus and T. d. dichotomus from Thailand, Yunnan (China), Hainan (China). These samples also clustered with T. d. dichotomus from Sichuan and Guangxi in China. The rest of samples from Eastern China formed a paraphyletic clade. Two T. d. septentrionalis  from Korea, and one T. d. dichotomus (T‐38) did not cluster with these three genetic clusters and may represent hybrid or relictual lineages.Within group North, PCA also separated specimens into three major clusters Figure , which wK were assessed across a range from 1 to 10, with the optimum cluster value of 3 (K = 3) determined using the cross‐validation error rate , followed by genetic cluster North (He = 0.356) and lowest in the West genetic cluster (He = 0.29). However, observed heterozygosity  was lower than expected in all genetic clusters suggesting past genetic bottlenecks or some other demographic or selective process is common in Trypoxylus beetles. The mean PIC also revealed that group South was highly polymorphic (0.281), whereas group West exhibited the lowest PIC (0.240).The genetic diversity of the three genetic clusters inferred by genome‐wide SNP data is presented in Table 3.6Analysis of molecular variance (AMOVA) indicated that 35.89% of the total variation attributed to variation among populations, while 28.29% and 35.82% were attributed to among individuals within populations and within individual, respectively Table .FST revealed differing levels of divergence between North, West, and South genetic clusters. The highest differentiation was between genetic clusters North and South (FST = 0.345), while the least differentiation was between West and North (FST = 0.094) (Table Pairwise 4) Table , which wNm) between North and West genetic clusters was 3.70 and 2.13 between South and West genetic clusters. Gene flow was inferred to be unidirectional from the South to North genetic cluster at a rate of Nm 0.94 (<1), and the genetic differentiation  between these genetic clusters was also inferred to be the highest among the clusters we compared. As such, a strong barrier to gene flow was inferred between the two population groups  from the southern part of West genetic cluster form a well‐supported clade within the West genetic cluster suggesting a recent radiation with subsequent isolation. In general, the partitioning of individuals into North and South genetic clusters follows previous observations for Trypoxylus populations).The genetic partitioning of individuals is clearly related to geographic distribution. For instance, the South genetic cluster is made up of individuals found on the islands of Taiwan, Okinawa, and Kumijima. The phylogenetic branching pattern within this genetic cluster is consistent with the pattern expected for an island radiation wherein individuals from the same island cluster together and the distance of islands from one another is reflected in branching order   and Gyungsang num‐do (T‐20) did not cluster with beetle populations from Northeastern China and Japan. They could represent different independent lineages as a result of historic separation or recent geographic isolation.Despite the geographic isolation of various subspecies/population, some individuals were inferred to be of admixed origins Figures  and 4. Gd Nagai, . Throughd Nagai,  and shou4.2T. dichotomus and closely allied species T. kanamorii have been described from different parts of Asia, including the countries China, Japan, Korea, Myanmar, Thailand, Laos, and India , and/or biotic factors seen in other species  had significantly longer horns for a given body size, than those specimens in Taiwan and Yakushima (largely corresponding to group South) in this study.Using broadly distributed SNP markers, subspecies were resolved in three well‐supported genetic clusters that generally corresponded to geographic origins. Although T. d. tsuchiyai and T. d. shizuae from Japan that were resolved as monophyletic with high branch support (PP = 1). Both subspecies were similar to T. d. septentrionalis. However, T. d. tsuchiyai was smaller and wider in both sexes, with pronotum and middle frontal areas of elytra clothed with pubescence, and the female individuals had shorter and coarser pubescence on pygidium comparing to T. d. septentrionalis. T. d. shizuae lacked an obvious presence absence morphological trait but was different in body size ratio to T. d. septentrionalis among individuals examined for this study. Based on our results, T. d. tsuchiyai and T. d. shizuae should be retained as subspecies and considered for elevation to species following further study including genitalic dissections. T. d. septentrionalis is paraphyletic with the inclusion of T. d. tsuchiyai and T. d. shizuae. However, the resolution of several well‐supported clades within the North genetic cluster suggests that T. d. septentrionalis should be reexamined and possible refined to account for these well‐supported clades.Several subspecies circumscribed by morphological features were supported by the molecular data in this study. The best example was T. d. inchachina and T. d. takarai have been reported from the Okinawan Islands (Japan), and together, they formed a monophyletic clade with small genetic distance. T. d. takarai has been separated from T. d. inchachina as being a smaller and black bodied beetle, with shorter pronotal horns on male individuals. However, these two subspecies were found to be very similar to each other in morphological traits  that result in the creation of basal paraphyletic grades does not adhere to the goal of species delineations recognizing monophyletic groupings.The West genetic cluster comprised of 23 specimens of a Nagai, . The cla5Trypoxylus beetles. The current study provides an important dataset, analyses, and method for genotyping (SLAF‐seq) that can be used and built upon to answer unresolved questions of systematics, character evolution, chemical and immunological chemistry, and phylogeography in Trypoxylus and related taxa.Our study demonstrated that SLAF‐seq derived markers outperformed 16S and COII sequences in beetle taxonomy and provided improved resolution of the genetic differentiation of rhinoceros beetle populations from a large part of the species’ range. Phylogenetic analysis of SNPs indicated the presence of three distinct genetic groups, suggesting that the various subspecies fall into three distinct and well‐supported lineages. PCA and ADMIXTURE analysis also identified three genetic clusters , which corresponded to their origin, suggesting that geographic factors were important in maintaining within population homogeneity and between population divergence. Further study including more detailed morphological work, increased taxon sampling, and taxonomic circumscription is needed to improve the current systematics of All authors declare that they have no competing interests.Huan Yang: Data curation ; Formal analysis ; Writing‐original draft . Chongjuan You: Data curation ; Funding acquisition (lead); Methodology ; Writing‐original draft . Clement Tsui: Investigation ; Methodology ; Writing‐review & editing . Luke Tembrock: Writing‐review & editing . Zhiqiang Wu: Writing‐review & editing . Depo Yang: Supervision .


Bariatric surgery can effectively treat morbid obesity; however, micronutrient deficiencies are common despite recommendations for high-dose supplements. Genetic predisposition to deficiencies underscores necessary identification of high-risk candidates. Personalized nutrition (PN) can be a tool to manage these deficiencies.Medline, PubMed, and Google Scholar were searched. Articles involving genetic testing, micronutrient metabolism, and bariatric surgery were included.Studies show associations between genetic variants and micronutrient metabolism. Research demonstrates genetic testing to be a predictor for outcomes among obesity and bariatric surgery populations. There is limited research in bariatric surgery and micronutrient genetic variants.Genotype-based PN is becoming feasible to provide an effective treatment of micronutrient deficiencies associated with bariatric surgery. The role of genomic technology in micronutrient recommendations needs further investigation. SNPs arVitamin D is essential for many functions of the body. Deficiency of vitamin D is associated with many cancers, autoimmune disorders, and cardiovascular disease as well as significantly affects musculoskeletal function –41. ObesGC) ; 7-dehydrocholesterol reductase (DHCR7) and CYP2R1 (involved in 25-hydroxylase production) are associated with fasting plasma 25(OH) D concentrations [The heritability of vitamin D status is estimated to be 30% and common variants group-specific component  D levels [CYP2R1, 24-hydroxylase (CYP24A1), and vitamin D receptor (VDR) genes [One randomized controlled trial looked at older Australians randomly assigned to monthly doses of 30,000 IU or 60,000 IU vitamin DD levels . AnotherR) genes . There iR) genes . HoweverVitamin B12 is a coenzyme, cofactor, and essential component in vitamin B complex. It is essential for cardiac health  and cognFUT2 gene) that codes for an enzyme in the vitamin B12 pathway are associated with B12 levels [Genetic variants may impact the proteins involved in vitamin B12 absorption, cellular uptake, and intracellular metabolism –63. Gene2 levels . Transco2 levels . In a st2 levels . This de2 levels .Vitamin B12 along with folate influences one-carbon metabolism. Cubulin (CUBN) is the intestinal (IF) and polymorphisms of this gene have been associated with chronic diseases in individuals with low B12 status . A studyFolate plays a role in one-carbon metabolism, methylation and DNA synthesis, and methionine regeneration –72. FolaSeveral studies have shown an association between SNPs related to folate metabolism, folate deficiency, and elevated homocysteine . A commoThiamine is essential for glucose, amino acid, and energy metabolisms –92. DefiMutations in thiamine transporter genes, SLC19A2 and SLC19A3, have been observed in cases of thiamine deficiency due to decreased absorption of thiamine that leads to neurological dysfunction . SLC35F3Iron is essential for metabolic processes like oxygen transport, deoxyribonucleic acid (DNA) synthesis, electron transport, as well as cellular functions can affect one’s well-being . In indiConsidering the results of several GWAS, there is strong evidence of genetic regulation of iron metabolism, and mutations in transmembrane serine protease 6 (TMPRSS6) gene that encodes for an enzyme that regulates hepcidin involved in iron homeostasis, iron carrier transferrin (TF), and transferrin receptor-2 (TFR2) genes have been associated with iron deficiency . A GWAS TMPRSS6 mutations have been associated with refractoriness to oral iron and studies confirm the role of TMPRSS6 in predicting oral iron response , 115. OnThe vitamin and mineral supplementation studies that focus on treating genetic disorders are mainly case studies. Supplementation studies for vitamin D-related genetic variants have been conducted in populations that are overweight and have obesity. Limited data is available on micronutrient supplementation according to genetic variants in bariatric surgery populations. Table Genetic expression patterns can be a predictive tool for responsiveness to nutritional treatments. Some studies have indicated that surgery-induced weight loss was associated with remodeling of the epigenome that helps regulate metabolic gene expression , 126. OnFrom the review, it is evident that the deficiencies of the studied micronutrients are influenced by genetic mutations. Postbariatric surgery, patients frequently have these deficiencies and knowledge of these mutations may have bearing on its management. Additional research is needed to establish this association. This review confirmed the scarcity of research that has been conducted in the area of bariatric surgery and micronutrient genetic variants, with only two articles being found in this search. This limitation should be considered when interpreting the findings in this discussion. Furthermore, the treatment regimen for those who have micronutrient genetic variants and undergoing bariatric surgery should be an area of future research.Personalized dietary and supplement advice derived from genetic testing should be based on appropriately designed studies. Utility of data from GWAS in providing dietary advice is limited because it is not known what diet and supplement intakes are required to prevent and treat the deficiencies that might be caused by micronutrient genetic variants. Identifying how a genetic variant modifies the response to supplementation on the micronutrient status and possibly identify responders and non-responders will be required to understand this population and area of research. Genotype along with micronutrient blood levels would be the initial step in applying PN among bariatric surgery patients. Genetic marker is only one factor that influences improvements related to micronutrient status .Future work should focus on genotyping for multiple variants in the micronutrient metabolic pathways and their additive and interactive effects to get a complete understanding of the influence of genetic factors on micronutrient metabolism. Then, utilizing genomic technology to understand this influence on the responses to micronutrient supplementation is also important. This would involve micronutrient status, genetic variations, and genetic interactions within metabolic pathways involving the micronutrient, its molecular targets, and environmental stressors .Furthermore, studies should focus to understand the role of the gut microbiome and its influence on metabolism and physiology. The human gut microbiota (which has its own genome) can modulate signaling pathways and regulate gene expression . Diet, lTechniques used for genetic testing will determine the cost. Methods being used in healthcare and research to identify genetic variations are known as next-generation sequencing (NGS) . SequencConcerns and limitations involved in genetic testing include  the rapiPN is the delivery of dietary advice at an individual level and future work should verify if this targeted nutrition can change behaviors and have an impact on health outcomes. Dietitians currently provide PN advice based on diet and phenotype; however, genotype-based PN advice is not so readily available. One review, involving a variety of populations such as patients with a history of weight loss failures, people with obesity, as well as healthy men and women of various ages, examined the evidence for genotype-based personalized information on motivating behavioral change, and factors which may affect the impact of genotype-based personalized advice . The res


Plasmodium falciparum malaria. Present study explores the possible evolutionary drivers shaping the genetic organization of Pfrhs in Indian parasite population. It simultaneously evaluates a putative gain-of-function variant of PfRH5, a keystone member of PfRH family.Reticulocyte binding protein-like homologs (RHs) are currently being evaluated as anti-erythrocytic stage vaccine targets against Pfrh1, Pfrh2a/b, Pfrh4 and whole Pfrh5 were amplified using blood samples of P. falciparum malaria patients from Chhattisgarh and West Bengal and sequenced. Assembled sequences were analysed using MEGA7 and DnaSPv6. Binding affinities of recombinant PfRH5 proteins with basigin (BSG) were compared using in silico (CHARMM and AUTODOCK) and in vitro  methods.Receptor binding regions of Pfrh1 (0.5), Pfrh2a/b (0.875), Pfrh4 (0.667) and Pfrh5 (0.778) sequence changes corresponded to low frequency (< 0.05) variants which resulted in an overall negative Tajima’s D. Since mismatch distribution of none of the Pfrh loci corroborated with the model of demographic expansion, a possible role of natural selection formulating Pfrh sequence diversity was investigated. Among the 5 members, Pfrh5 displayed very high dN/dS (5.7) ratio. Nevertheless, the model of selective sweep due to presence of any advantageous substitutions could not be invoked as polymorphic nonsynonymous sites (17/18) for Pfrh5 exceeded significantly over the divergent (62/86) ones (p = 0.0436). The majority of extant PfRH5 sequences (52/83) differed from the reference Pf3D7 allele by a single amino acid mismatch (C203Y). This non-conservative alteration was predicted to lower the total interaction energy of that PfRH5variant with BSG, compared to PfRH53D7. Biophysical evidences validated the proposition that PfRH5variant formed a more stable complex with BSG. Thermodynamic association constant for interaction of BSG with PfRH5variant was also found to be higher (Kavariant = 3.63E6 ± 2.02E6 M−1 and Ka3D7 = 1.31E6 ± 1.21E6 M−1).Pfrhs is principally shaped by purifying selection. The most abundant and ubiquitous PfRH5 variant harbouring 203Y, exhibits a greater affinity for BSG compared to PfRH53D7 possessing 203C allele. The study underscores the importance of selecting the functional allele that best represents circulating strains in natural parasite populations as vaccine targets.Together, the study indicates that the genetic architecture of  Plasmodium falciparum remains a global health concern. Intervention of malaria is typically based on two regimens those include (a) control of anopheles mosquito vector by the use of insecticidals and (b) treatment of patients using anti-malarial drugs. Drug resistant parasites arising due to wide and indiscriminate deployment of chloroquine, sulfadoxine-pyrimethamine and mefloquine based mono-therapies, is causing resurgence of disease in many parts of the world . Re. ReP. fa. Pf3D7) , 19. Thiity, CA) . Number Pfrh1: PF3D7_0402300; Pfrh2a: PF3D7_1335400; PfRH2b: PF3D7_1335300; Pfrh4: PF3D7_0424200; Pfrh5: PF3D7_0424100) .−1 in the wavenumber range 1000–4000 cm−1. 10 ul of solution containing 1ug/ul protein was utilized for each FTIR analysis.FTIR was carried out using PerkinElmer spectrum 100 FT-IR Spectrometer at a resolution of 0.5 cm3D7, PfRH5M, PfRH53D7–BSG and PfRH5M–BSG were observed in different Δλ at 25 °C 3D7 and r3D7–BSG and rPfRH5M–BSG complexes were subjected to FTIR spectroscopy  compared to rPfRH53D7–BSG (0.198)  in comparison to rPfRH5M –BSG   of rPfRH5M with was found to be 2.77 fold higher (Ka = 3.63E6 ± 2.02E6 M−1) than that between rPfRH53D7 and BSG (Ka = 1.31E6 ± 1.21E6 M−1). This validated a distinct functional impact of C203Y polymorphism in relation to ligand-receptor interaction  than rPfRH53D7–BSG (− 8340).Finally, isothermal titration calorimetry was employed to compare the thermodynamic aspects of complexes formed between rPfRH5s and BSG. ITC yielded negative heat deflection, indicating the binding between rPfRH5P. falciparum. For example, RTS,S which is close to obtaining licensure, displays a modest vaccine efficacy in terms of blocking infection [P. falciparum parasites, the level and pattern of genetic variability of Pfrh family of ligands, which take important part in host-parasite interaction during erythrocyte invasion by P. falciparum, are thoroughly investigated.More than 50 years of continued research and control initiatives have caused a commendable reduction of malaria incidence and mortality globally in the last 15 years . In keepnfection . In addinfection , 49, 50.P. falciparum and PfRH5 antibodies have been shown to exhibit strain-transcending invasion inhibition [Not all members of PfRH family are essential for parasite propagation, as they can be knocked out without affecting parasite growth and few members of EBA and PfRH families even perform overlapping function , 52. PfRhibition , 53. ThuPfrh1, Pfrh2a/b, Pfrh4 and Pfrh5 sequences reveals majority of the polymorphic sites  are rare (frequency < 0.05) among the parasite isolates of Chhattisgarh and West Bengal. For each of these loci, the estimate of nucleotide diversity, π is lower than Watterson’s θ, resulting in a negative Tajima’s D, which typically denotes that evolutionary drivers such as recent population expansion and/or purifying selection/selective sweep may reconstruct the genetic landscape of Pfrh genes in P. falciparum parasites of these study regions. The observed pattern of mismatch distribution rules out possibility of any past demographic processes. Despite the abundance of low frequency variants, a very high dN/dS of 5.7 is observed for Pfrh5, which suggests a possibility of genetic hitchhiking effect. Even so, a model for selective sweep due to any advantageous amino acid substitutions cannot be invoked, as the locus harbours significantly high ratio of within-species non-synonymous to synonymous amino acid variations compared to between-species divergence (p = 0.0436). It is thus surmized that genetic architecture of Pfrh family members has been shaped predominantly by purifying selection. To compensate for the limited sample size, additional sequences for each of Pfrh genes retrieved from NCBI were analysed. Despite an overall abundance of low frequency polymorphisms, the structures of the parasite population from different geographical regions showed remarkable genetic differentiation possibly due to factors such as local adaptation and/or random genetic drift. Nevertheless, frequency of C203Y was found to be high in Kenya and Mali, as well [Analysis of receptor binding regions of e p = 0.06. It is  as well .Pfrh5 variants results in amino acid replacements, an attempt has been undertaken to weigh their possible functional values. Since only C203Y is predominantly present among Indian P. falciparum strains, the present study evaluates any functional advantages associated with this replacement in relation to the PfRH5′s interaction with BSG. Analyses of circular dichroism data of purified PfRH5 alone or in complex with BSG show significant differences in the composition of alpha-helices, beta-sheets, and random coils due to C203Y replacement. Pattern of intrinsic fluorescence and anisotropy measures for rPfRH5M–BSG complex indicate that alteration in secondary structure of rPfRH5M has further affected the three-dimensional folding of ligand-receptor complex. Estimation of interaction energy and thermodynamic association constant rPfRH5M –BSG further establish a favourable biochemical impact of C203Y polymorphism on the affinity between rPfRH5M and BSG. This data however contrasts a recent study conducted in Mali which fails to find any correlations between amino acid substitutions on PfRH5 with malaria risk [Since, 17  out of the 18 ria risk , 55. TakPfrhs is dominated by presence of rare variants due to possible action of purifying selection. C203Y polymorphism located on PfRH5 is present in overwhelming proportion in the study population. This C to Y replacement strengthens the interaction between PfRH5 and BSG. The implication of this genetic variant in the context of host-parasite conversation during merozoite invasion needs to be explored further. Nonetheless, present data is sufficiently informative while designing a rational multivalent malaria vaccine with PfRH5 being one of the components.The data presented here clearly demonstrate that genetic architecture of Additional file 1: Table S1. Details of oligonucleotide primers used for amplification and sequencing.Additional file 2: Table S2. Comparison of genetic diversity of Pfrh loci in publicly available global dataAdditional file 3: Fig. S1. a Neighbor joining network of Pfrh5 haplotypes (excluding the singletons) observed in India, Kenya and Mali. Size of a circle was proportional to relative frequency of respective haplotype and each brunch of the network represented a mutational step. b Frequency of the haplotypes in respective regions.Additional file 4: Fig. S2. Synchronous fluorescence data of recombinant target proteins and their complexes with BSG at different Δλs.


ST between virtual and DNA pools, a phylogenetic tree, principal component analysis (PCA) and evaluation of functional SNP classes were conducted. An F-test was performed to reveal significantly differentiated allele frequencies between colonies. Variation within a colony was quantified as expected heterozygosity.Göttingen Minipigs (GMP) is the smallest commercially available minipig breed under a controlled breeding scheme and is globally bred in five isolated colonies. The genetic isolation harbors the risk of stratification which might compromise the identity of the breed and its usability as an animal model for biomedical and human disease. We conducted whole genome re-sequencing of two DNA-pools per colony to assess genomic differentiation within and between colonies. We added publicly available samples from 13 various pig breeds and discovered overall about 32 M loci, ~ 16 M. thereof variable in GMPs. Individual samples were virtually pooled breed-wise. FPhylogeny and PCA showed that the GMP is easily discriminable from all other breads, but that there is also differentiation between the GMP colonies. Dependent on the contrast between GMP colonies, 4 to 8% of all loci had significantly different allele frequencies. Functional annotation revealed that functionally non-neutral loci are less prone to differentiation. Annotation of highly differentiated loci revealed a couple of deleterious mutations in genes with putative effects in the GMPs .Differentiation and annotation results suggest that the underlying mechanisms are rather drift events than directed selection and limited to neutral genome regions. Animal exchange seems not yet necessary. The Relliehausen colony appears to be the genetically most unique GMP sub-population and could be a valuable resource if animal exchange is required to maintain uniformity of the GMP. The Göttingen Minipig (GMP) is an animal model with growing importance . CreatedManaging the GMP in independent colonies in closed barriers is beneficial from a safety point of view. Additionally, a production unit close to the main sales market minimizes negative effects on animal welfare through long transports and prevents import complications and unnecessary bureaucracy. On the other hand, splitting a population reduces the effective population size of each sub-population, which increases the risk of genetic drift or the manifestation of recessive disorders . Two conIn this study we try to assess whether the genetic management was able to maintain the uniformity of the GMP breed, or if the isolated production units are already genetically diversified such that an exchange of breeders is inevitable. This was done by re-sequencing two representative DNA pools from each unit: candidates were sampled for low average relationship within a pool, but elevated relationship towards the remaining colony, allowing an assessment of the diversity within and between units.a) compared to the mean relationship of the candidate set with the remaining colony mates (b). Both, the absolute level of relationship and the difference between a and b were lowest for Relliehausen (RE) and highest for North Rose (NR), while Dalmose barrier 2 (DA2) and 3 and Nisshin (NI) were at the same level and exhibited similar difference between a and b  total non-monomorphic SNPs, thereof 4′121’427 (incl. 263′498 on chr. X) novel SNPs not documented in dbSNP were retained. Discarding loci, monomorphic in the minipigs and X-chromosomal loci for the analysis within minipigs left a data set containing 16′498’773 autosomal SNPs.Principal component analysis (PCA) on the reference allele frequencies shows clear separation of the GMPs, Asian and European breeds, with the Mini-LEWE clustering close to the Asian breeds, by the first component Fig. . The secST and the Reynolds genetic distance (DR) were estimated. When applied to the variable set of large breeds and minipig pools, both measures provided a similar picture of three strongly differentiated groups  the second lowest and the Asian the highest  (Table R (0.36 vs 0.37) and FST (0.26 vs. 0.27) to the GMP than within the group of Asian breeds.As measures of differentiation and genetic distance between the different breeds, Fps Table . In prinnce Fig. , but proST values calculated with genome wide SNP data shows a clear clustering of the GMPs from the other breeds. The next level clusters contain (in that order) Xiang, Meishan, South Chinese wild boars, the Mini-LEWE and the North Chinese wild boars. The European breeds form their own cluster. Throughout 100 resamplings, the GMP cluster, the Mini-LEWE cluster and European cluster are rediscovered in every iteration, while the nodes connected to the Asian breeds seem unstable with resampling probability between 18 and 87%, with the exception of the Japanese wild boar, that behaves like an outgroup sample. Even though, the European and the GMP clusters are distinct, the order within the clusters is variable. The node support within the European cluster spans from 56 to 72%, and between 18 to 86% in the GMP cluster. The most stable structure with 86% contains the RE pools and the least stable (18%) contains the DA and NI herds.The UPGMA tree Fig.  producedST values between colonies, differences in the average expected heterozygosity within pools by a variation based approach employing an F-test statistic. Resulting loci detected by the aforementioned statistics were functionally annotated and imbalances between the various classes were checked for potential biases towards differentiated loci.The genetic differences within the GMP were determined by comparing pools in terms of allele frequency differences, such as oppositely fixed alleles, extreme FST, add probabilistic evidence on differentiation between pools. On average, the NI colony had the lowest proportion of significantly  differentiated loci, overall 4.7%, followed by DA3 and 2 with 5.3 and 5.4%, respectively, and RE with 5.7%. With 6.9%, NR had the highest proportion of significantly differentiated loci , Arrestin Beta 1 , Autoimmune Regulator  and Calcium/Calmodulin Dependent Protein Kinase II Beta . RVIS scores for those genes were not extreme, with − 0.84 (among 11.18% most intolerant genes) ARRB1, − 0.62 (17.16%) for CAMK2B, − 0.57 (19.01%) for AIRE and 0.27 (70.58%) for CHST12.In the highest FST annotations  or phenotypic differences , while New Zealand and American Texel appeared indistinguishable (FST = 0.025). Studies comparing clearly distinct pig and cattle breeds, respectively, found FST values between 0.06 and 0.40 [ST of ~ 0.1 was found between relatively similar breeds, for example Large White and Landrace, while values higher 0.3 indicated major differentiation, such as between Nellore and Holstein cattle or Asian and European pig breeds. These values matched our findings between the European, Asian and GMP groups. Within the GMP, even two randomly composed pools from the same unit had a minimum differentiation of about 0.05. Between the aforementioned clusters FST was about 0.06 to 0.8 and therefore between the differentiation observed between the sheep breeds from separate origins and clearly distinct breeds. We explain this by genetic drift and slight differences in the actual breeding management, since the three clusters are confounded with the three partners in GMP breeding, even though all follow the same general breeding goal. We also assume that ascertainment bias affecting the variant discovery procedure might have an elevating effect on differentiation since incorporation of various pig breeds in the discovery sample might allow for calling more variants and especially more heterogeneous variants than calling in a GMP only sample [While it seems particularly easy to distinguish the GMP from other pig breeds, it is more difficult, but relevant from a breeders’ point of view, to determine, if genetic isolation of the five breeding colonies has led to differentiated subpopulations. Applying PCA on the 10 GMP pools, we were able to see a trend to three subgroups consisting of NR, RE, and a cluster comprising NI and DA, respectively. The presence of a certain level of stratification is expected and has been observed beforehand, i.e. in studies in dogs  or sheepand 0.40 , 17. FSTy sample .ST levels found in the aforementioned studies implies that our colonies are at the edge of splitting into sub-populations, and we did not expect all genomic regions between all pairwise combinations of the five units being similarly differentiated, when focusing on individual loci. The F-test  and between the animals in the set and all remaining animals in the colony (b) were calculated. Both values were combined in an index I = 0.8*a − 0.2*b, to minimize relationship within the samples while maximizing relationship with the sample and the remaining colony. This sampling was repeated up to 25′000 times and restarted every time a new index value went below the previously recorded one. The procedure was stopped after 25′000 rounds without improvement.A joint pedigree was created from the pedigrees of all colonies in the five separated barrier facilities (Research Farm Relliehausen: Relliehausen (RE); Ellegaard Göttingen Minipigs A/S: Dalmose barrier 2 and 3 ; Marshall BioResources: North Rose (NR); Oriental Yeast Co., Ltd.: Nisshin (NI)). Numerator relationship matrices were constructed with Wrights coefficient of relationship  for eachDNA of two times ten animals per colony, randomly chosen from the available samples from the previously selected 30 candidates, was pooled using equimolar amounts of the individual DNA. 150 bp paired-end sequencing was done on an Illumina HiSeq 4000 with an aim coverage of 30X and an insertsize of about 420 bp. Publicly available data from 13 various pig breeds –31 as usX, which contained samples in rows and genetic loci in columns. Elements of X were the respective reference allele frequencies. Every locus was centered and scaled beforehand. Due to the nature of data from short-read sequencing, matrix X contained numerous missing values. To account for these, missing information was recoded in matrix N with the same dimensions as X, in which records and missing positions were recoded as 1 or 0, respectively. The adjusted covariance structure was subsequently modeled as V and eigenvalues λ were achieved bei eigenvalue decomposition of E. Loadings L where the corrected eigenvalues where negative values were set to zero. Principal components were calculated by multiplying the eigenvectors with a diagonal-matrix containing square roots of L.PCA was constructed from matrix ST) and Reynolds distance (DR) were estimated between breed pools. Therefore read information of individuals was virtually pooled by breed-wise summation of reads supporting the reference and the alternative allele, respectively.Fixation index . GenetiAdditional file 1 Supplementary Table 1. Genome wide FST (upper triangle) and Reynolds distance (lower triangle).Additional file 2 Supplementary Table 2. Number of SNPs significant at 5% Bonferroni corrected significance level in F-Test.Additional file 3 Supplementary Table 3. Functional annotation of SNPs oppositely fixed between one unit and a distant pool from another unit.Additional file 4 Supplementary Table 4. Functional annotation of SNPs dependent on the FST value between two colonies.


Landraces are a potential source of genetic diversity and provide useful genetic resources to cope with the current and future challenges in crop breeding. Afghanistan is located close to the centre of origin of hexaploid wheat. Therefore, understanding the population structure and genetic diversity of Afghan wheat landraces is of enormous importance in breeding programmes for the development of high-yielding cultivars as well as broadening the genetic base of bread wheat. Here, a panel of 363 bread wheat landraces collected from seven north and north-eastern provinces of Afghanistan were evaluated for population structure and genetic diversity using single nucleotide polymorphic markers (SNPs). The genotyping-by-sequencing of studied landraces after quality control provided 4897 high-quality SNPs distributed across the genomes A (33.75%), B (38.73%), and D (27.50%). The population structure analysis was carried out by two methods using model-based STRUCTURE analysis and cluster-based discriminant analysis of principal components (DAPC). The analysis of molecular variance showed a higher proportion of variation within the sub-populations compared with the variation observed as a whole between sub-populations. STRUCTURE and DAPC analysis grouped the majority of the landraces from Badakhshan and Takhar together in one cluster and the landraces from Baghlan and Kunduz in a second cluster, which is in accordance with the micro-climatic conditions prevalent within the north-eastern agro-ecological zone. Genetic distance analysis was also studied to identify differences among the Afghan regions; the strongest correlation was observed for the Badakhshan and Takhar (0.003), whereas Samangan and Konarha (0.399) showed the highest genetic distance. The population structure and genetic diversity analysis highlighted the complex genetic variation present in the landraces which were highly correlated to the geographic origin and micro-climatic conditions within the agro-climatic zones of the landraces. The higher proportions of admixture could be attributed to historical unsupervised exchanges of seeds between the farmers of the central and north-eastern provinces of Afghanistan. The results of this study will provide useful information for genetic improvement in wheat and is essential for association mapping and genomic prediction studies to identify novel sources for resistance to abiotic and biotic stresses. Triticum aestivum L.) is the primary source of calorie intake for more than one third of the global population and is ranked as the third most important crop in the world [Wheat , peas (Pisum sativum), rye , and chickpeas (Cicer arietinum) [Afghanistan lies close to the centre of origin of hexaploid wheat and enjoys rich topography, rich history, and a complex tribal system. It is considered the third largest centre of origin for many domesticated crops worldwide and has played crucial role in the domestication of many crops such as barley (ietinum) . During ietinum) ; in addiietinum) ,18,19,20The recent advances in the development of molecular markers and high-throughput systems have generated a wealth of information for a large number of plants ,22,23. SA total of 363 bread wheat landraces collected from seven provinces of Afghanistan were genotyped using Diversity Arrays technology (DArT). One of the landraces from Kabul did not correspond to the recorded geographic location and therefore has been either recorded incorrectly or is a mixture . A set oK). The results indicated that the population structure was optimum at K = 2. In the plot of K against ΔK, there was a steep slope at K = 2 following the flattening of the curve  and the results showed at least three sub-populations . Hence, The results of principal component analysis (PCA) is in accordance with the DAPC analysis and showed three clusters. The first, second, and third PCs explained 18.76%, 10.69%, and 4.34% of the total variation, respectively .It is to be noted that both the STRUCTURE and DAPC analysis clustered the population with high levels of admixture, which was evident from the neighbour-joining based clustering and minimum spanning network (MSN) . The neiThe analysis of molecular variance (AMOVA) based on the STRUCTURE results revealed that there was very small genetic variation (0.29%) between the two sub-populations. However, greater genetic variance was found within the two sub-populations (99.71%) . The samNa) and the number of effective alleles (Ne) of the two sub-populations identified by STRUCTURE were 1.999 and 1.591, respectively (I), the diversity index (He), and the unbiased diversity index (uHe) were 0.516, 0.345, and 0.346, respectively. Between the two sub-populations grouped by STRUCTURE, Sub-population 1  showed higher diversity than Sub-population 2 . Sub-population 2 clustered by the DAPC approach showed higher diversity  compared with Sub-population 1 and Sub-population 3. The overall mean values of the genetic indices for the populations derived from the DAPC approach were Na = 1.994, Ne = 1.579, I = 0.506, He = 0.338, and uHe = 0.341. When the landraces were grouped based on their geographic origin, the landraces from Badakhshan province showed the highest genetic diversity , whereas the landraces from Kabul showed the least genetic diversity . Since there was only one landrace accession from Konarha and Samangan, their genetic indices were not calculated and hence a comparison with other provinces was not possible.The mean values of different alleles  and Sub-population (3). The majority of the landraces from Takhar province were grouped into Sub-population 1 (77), the rest were grouped into Sub-population 3 (25) and Sub-population 2 (17). Six out of seven landraces collected from Kabul province were grouped into Sub-population 2, along with the two landraces from Konarha and Samangan province, whereas one landrace from Kabul province was grouped into Sub-population 3. A similar trend was observed when the whole panel was divided into two sub-populations using the STRUCTURE method, where the landraces from Badakhshan, Takhar, Kunduz, and Baghlan were assigned to both the sub-populations. However, the landraces from Kabul, Konarha, and Samangan were all grouped in Sub-population 2. In the overall trend, it was observed that the majority of the landraces from the neighbouring states were slightly favoured to group together than with the landraces from the provinces which are far away . HoweverThe potential use of wheat landraces for both abiotic and biotic stress resistance has been given significant importance in many breeding programmes ,29,30,31In this study, a set of approximately 5000 genotyping-by-sequencing (GBS) derived high-quality SNPs were used, obtained from 363 bread wheat landraces. This research shows the usefulness of GBS-derived markers for population structure and genetic diversity studies. In the current study, the B genome had the highest number of SNPs, followed by the A and D genomes, which is in agreement with many previous studies ,34. Genek = 2 value sometimes misrepresents the true structure in the population and could possibly mean that either there is no definite population structure, or the STRUCTURE program failed to successfully identify the underlying genetic structure of the collection [Population stratification analysis is useful for understanding genetic diversity and also facilitates GWAS analysis . In the llection . In addillection ,42,43. AGenetic differentiation due to population structure is measured by Fst. An Fst value ranges from 0 to 1 and a value of 0.15 is considered significant in distinguishing the populations . Thus, sThe allelic patterns and indices of genetic diversity shed light on the genetic diversity within sub-populations. Clustering based on STRUCTURE observed slightly higher genetic diversity in Sub-population 1 compared with Sub-population 2, which could be attributed to the size of Sub-population 1. In the case of three sub-populations, as grouped by DAPC analysis, Sub-population 2 showed the highest genetic diversity. Sub-population 2 comprised mainly landraces from Baghlan and Kunduz, identifying the highest genetic diversity amongst the landraces from these regions. The landraces from Baghlan also showed significant genetic diversity in an earlier study, thus confirming our results . Interesn = 66), Kabul (n = 7), Konarha (n = 1), Kunduz (n = 50), and Samangan (n = 1). Badakhshan, Baghlan, Takhar, and Kunduz belong to one agro-ecological zone , whereas Kabul, Samangan, and Konarha belong to three different agro-ecological zones, which are central, north and east, respectively. Details of the landraces used in the study are presented in the Three hundred and sixty-three Afghan bread wheat landrace accessions assembled from 7 north and north-eastern states of Afghanistan were used in the study. All landraces are conserved in the International Center for Agricultural Research in the Dry Areas (ICARDA) gene bank. The highest numbers of landraces were collected from Badakhshan and Takhar regions, with 119 accessions from each region. The rest of the accessions were collected from Baghlan (The landraces were genotyped by the high-throughput genotyping by sequencing (GBS) method using DArT technology  at the Gk = 2–10 under the admixture model of population structure and was assessed with a burn-in period of 50,000 and 50,000 Markov Chain Monte Carlo (MCMC) replications. To infer the optimal clusters/sub-populations, the best K value representing the optimum number of sub-populations was estimated as Delta K (ΔK) based on the change in the log probability of data between successive structure iterations using Structure Harvester [K value, membership coefficient matrices of three replicates from the STRUCTURE analysis were used in CLUMPP [In order to analyse the underlying population structure in the Afghan landraces, STRUCTURE software (v.2.3.4) was used, which implements model-based Bayesian cluster analysis . The proarvester . For then CLUMPP  to genern CLUMPP .K-means clustering of principal components to identify groups of individuals. It is run with different numbers of clusters, each of which gives rise to a statistical model and an associated likelihood. The best supported model, and therefore the number and nature of clusters, it is assessed using the Bayesian Information Criterion (BIC).The second approach to analyse the population structure was based on the discriminant analysis of principal components (DAPC), which was implemented using an R package, “adegenet” . DAPC usNa), the number of effective alleles (Ne), Shannon’s index (I), the diversity index (He), the unbiased diversity index (uHe), and the percentage of polymorphic loci (PPL) were measured using GenAlex v6.503 [In order to analyse the genetic variation amongst the 363 bread wheat landraces from Afghanistan, various diversity parameters like the number of different alleles (x v6.503  in R stux v6.503 . A neighx v6.503 .This study provided a detailed view of the population structure and genetic diversity of 363 bread wheat landraces from seven provinces of Afghanistan. In this study, SNPs obtained from high-throughput GBS technology were used to explore population structure and genetic diversity of bread wheat landraces and suggests that these markers with high genome coverage are useful for revealing the genetic diversity and population structure. The population structure analysis revealed that the landraces originating from different geographic regions of north and north-eastern parts of Afghanistan were best divided into three distinct groups based mainly on geographical background and micro-climatic conditions within the agro-ecological zones of the landraces. However, high proportions of admixture were also observed, revealing the possibility of historical free seed exchange among farmers within these provinces. Further, it was observed that the genetic diversity was higher within the landraces compared with between the sub-populations, which is unique to these particular landraces and has been previously reported as well. The study also found out that the number of markers on the D genome, interestingly, was nearly similar to those on the A and B genomes, which is also unique to these landraces, as previous studies on bread wheat reported that the D genome had 2–5 times fewer markers than the A and B genomes. This indicated high sequence diversity present in the D genome in the Afghan landraces. Thus, this suggests that this diversity can be utilized in the elite wheat breeding programmes to widen the genetic base of the overall breeding populations. This knowledge of the genetic diversity and population structure of landraces from central and north-eastern provinces of Afghanistan will be important for future studies in the region, conferring resistance to abiotic and biotic stresses from GWAS to identify and isolate marker–trait associations for important economical traits.