Leptospirosis is the world’s most common zoonotic disease. Mitigation and control rely on pathogen identification and understanding the roles of potential reservoirs in cycling and transmission. Underreporting and misdiagnosis obscure the magnitude of the problem and confound efforts to understand key epidemiological components. Difficulties in culturing hamper the use of serological diagnostics and delay the development of DNA detection methods. As a result, especially in complex ecosystems, we know very little about the importance of different mammalian host species in cycling and transmission to humans.Leptospira, and to identify the circulating species. Microscopic Agglutination Tests with a panel of 22 different serovars showed anti-leptospira antibodies in 36 sampled dogs (75%), and 10 serotypes were detected. Two DNA-based detection assays revealed pathogenic Leptospira DNA in 18 of 19 dog urine samples (94.7%). Amplicon sequencing and phylogenetic analysis of 16s rDNA and SecY genes from 15 urine samples revealed genetic diversity within two of three different Leptospira species: noguchii (n=7), santarosai (n=7), and interrogans (n=1).We sampled five indigenous Kichwa communities living in the Yasuní National Park in the Ecuadorian Amazon basin. Blood and urine samples from domestic dogs were collected to assess the exposure of these animals to Leptospira DNA provides strong evidence for high rates of past and current infections. Such high prevalence has not been previously reported for dogs. These dogs live in the peridomestic environment in close contact with humans, yet they are free-ranging animals that interact with wildlife. This complex web of interactions may explain the diverse types of pathogenic Leptospira observed in this study. Our results suggest that domestic dogs are likely to play an important role in the cycling and transmission of Leptospira. Future studies in areas with complex ecoepidemiology will enable better parsing of the significance of genotypic, environmental, and host characteristics.The high prevalence of antibodies and  Leptospira. Our results suggest that a high percentage of dogs are shedding the pathogen and we identified three different Leptospira species. Our serological analysis suggests the presence of ten serovars in dogs and their high exposure to Leptospira. These findings provide important insights into the epidemiology of leptospirosis in this ecosystem, suggesting that dogs are likely to play a critical role in the transmission of the disease.People around the world interact with a wide range of animals, but one of the closest is the domestic dog. Dogs can be reservoirs of several zoonotic infectious diseases, including leptospirosis. The frequent ecological interactions between people, dogs and wildlife in indigenous communities living in the Amazon basin might increase the complexity of leptospirosis transmission, in comparison with what has been described for other settings. In the Amazon basin, wild animals and domestic animals may act as reservoirs of the pathogen, excreting the bacteria through their urine. In this work we analyzed serum and urine samples from dogs living with Kichwa communities from the Yasuní National Park in Ecuador. Serum samples were analyzed with MAT and urine samples with a qPCR that detect the presence of pathogenic  Leptospira spp. bacteria by eating contaminated meat, direct contact with urine, or contact with urine-contaminated environments [Leptospirosis is a zoonosis that continues to be an important, albeit neglected infectious disease affecting humans and animals worldwide . Leptospronments –8. Mild ronments ,10. Unforonments ,11,12.Leptospira in their urine [Leptospira [A diverse array of domestic and wild animals likely serve as reservoirs by excreting ir urine –15. Howeir urine , and repir urine –27. Thesptospira . For effHumans across the world interact with a variety of animals, but one of the closest is the domestic dog. The exposure of dogs to pathogenic leptospires may depend on: 1) interactions with, or proximity to other domestic and wild animals, and 2) attributes of the local environment in which the animals live, such as hygiene and local weather conditions –31. DogsLeptospira, increases the likelihood of exposure and transmission of pathogenic Leptospira species to humans and wildlife. Thus, the proximity of dogs to people [Kichwa people, one of the indigenous ethnic groups that live in the Ecuadorian Amazon basin, own domestic dogs. However, unlike dogs in most urban settings, these animals are not confined indoors or the immediate vicinity of the homes; they drink water from the river or stagnant pools, roam freely in the forest, and hunt small wild animals ,44. ThisIn this study, we investigated whether domestic dogs from five Kichwa communities living on the riverbanks of the Napo River in the Ecuadorian Amazon were exposed to leptospirosis. In addition, we aimed to determine if these dogs were shedding leptospires by detecting DNA of the pathogen in their urine and thus exposing their owners to leptospirosis. Our results suggest that domestic dogs likely play an important role in the local epidemiology of leptospirosis.terra firme forest mixed with smaller extensions of palm swamps. There are still large expanses of continuous undisturbed vegetation in the eastern and southern portions of the park, but its northern and western boundaries are surrounded by a growing matrix of pastures, agricultural lands, and secondary vegetation [This study was carried out in the northern part of Yasuní National Park, in eastern Ecuador . This protected area contains parts of the territory of five Kichwa indigenous communities (approximately 88 000 ha). Yasuní National Park is located on the western Amazon and has been recognized as a major biodiversity hotspot . Encompagetation .With a population of more than 100,000 people, the Kichwa constitutes Amazonian Ecuador’s largest indigenous group . This stDuring June and November 2018, a dog census was performed by the Wildlife Conservation Society – Ecuador (WCS – Ecuador). The aim of this census was to serve as a baseline for population trends and a health surveillance program in the five Kichwa communities in northern Yasuní National Park . It was carried out by a local biologist to assess the number of dogs owned by each household. The percentage of households with dogs in the census varied between communities . The total domestic dog population was 550 dogs, and approximately 10% of the dogs (n=51) were sampled to test for various infectious diseases.® . Serum and urine samples were stored and transported on ice to the Institute of Microbiology at Universidad San Francisco de Quito and thereafter maintained at −20°C until DNA extraction.Ethical approval for sample collection was issued by CEIDA-USFQ (2017–010). Urine and blood samples were collected in August 2019 from domestic dogs living in the communities as follows. Blood samples we collected from the cephalic vein for all domestic dogs except for three, resulting in a total sample size of 48 dogs: Pompeya (n=7), Sani Isla (n=12), San Roque (n=12), Indillama (n=5), and Nueva Providencia (n=12). We collected urine samples from 19 male dogs by transurethral catheterization. It was not possible to get this type of sample from all dogs due to the absence of urine in the bladder of some animals. Transurethral catheterization is difficult in female dogs and thus urine samples from female dogs were not collected. To prevent DNA degradation, 4 ml of urine was added to 4 ml of 2X DNA/RNA ShieldThe 48 dog serum samples were analyzed by the National Reference Laboratory for Animal Diagnostics (AGROCALIDAD) using Microscopic Agglutination Test (MAT) performed with a panel of 22 different available serovars . Reactive samples with titers ≥ 1:100 were considered positive for detecting anti-leptospiral antibodies. MAT results were visualized by dark field microscopy, and the final titer was assigned as the serum dilution that promotes 50% of agglutination. The serovar with the highest titer was recorded in samples that reacted with multiple serovars. If a sample reacted with more than one serovar with the same titers and without showing a unique highest titer, samples were labeled as “cross-reactive”. Characterized local strains are not available in Ecuador, therefore the serology is performed with non-local strains and cross-reaction results are common.Leptospira spp. was performed on the 19 dog urine samples. Samples were thawed on ice and centrifuged at 4500 × g for 20 min at 4°C. The supernatant was discarded, and 200 μL of the pellet material was used for DNA extraction using DNeasy Blood and Tissue kit . DNA was stored at −20°C. Two previously described Taqman assays were used for molecular detection of pathogenic Leptospira: one assay targets the lipL32 gene [16S rDNA gene of pathogenic Leptospira [lipL32 or SNP 111) detected leptospiral DNA. The redundancy of two independent assays reduces the likelihood of false negatives that, in our experience, is relatively common and due to the large genetic diversity of pathogenic Leptospira. Using 2 assays also minimizes false negatives by mitigating against the inherent stochasticity of capturing a PCR target in low quantity samples.Molecular detection of pathogenic L32 gene  and the ptospira . A sampl16s rDNA and SecY genes from positive samples. Amplicons were sequenced using Oxford Nanopore Technologies. PCR amplicons were obtained using the Q5 High-Fidelity Master Mix , 0.4 μM each primer, and 2.5 μL of DNA template in a final reaction volume of 25 μL. PCR protocol consisted of an initial step at 98°C for 30 seconds, followed by 30 cycles of 10 s at 98°C, 30 s at 58°C or 54°C for rrs2 and SecYIV, respectively, and 30 s at 72°C, followed by final extension step for 2 minutes at 72°C. Amplicons were purified using AMPure XP magnetic beads  following manufacturer instructions and then quantified in a Qubit 3.0 fluorometer (Thermo Fisher Scientific) using the Qubit™ 1X dsDNA, high sensitivity kit . The quantified samples were normalized to a concentration of 3,0 ng/uL and sequenced following the Oxford Nanopore Library preparation protocol of the Ligation sequencing kit (SQK-LSK109) . Finally, 5,72 ng of the library was loaded into a MinION flow cell (FLO-MIN 106). Most reads were obtained during the first 12 hours of the run. Reads were basecalled and demultiplexed using the Guppy software (version 3.4.5)  [https://github.com/rrwick/Porechop) respectively.Primers rrs2 and SecYIV, described by Ahmed et al. ,53, wereLeptospira sequences were initially screened using BLASTn command line software (version 2.9.0–2) [Leptospira reads obtained during sequencing. Then, sequences were aligned with minimap2 (version 2.22) [L. interrogans serovar Copenhageni FioCruz L1–130 chromosome 1 (NC_005823.1) was used as a reference genome for the alignment. All the reads that were mapped to the corresponding genes (16s rDNA and SecY) were filtered to a new file and aligned in MEGA-X (version 10.1.8) [https://www.ebi.ac.uk/Tools/msa/emboss_cons/ [Leptospira obtained from GenBank. A phylogenetic tree was built in MEGA-X using the Neighbor-Joining method [2.9.0–2) . This ston 2.22)  and visuon 2.22) . L. intee 1 NC_0023.1 was ss_cons/ . Sequencg method , with thg method  and 500 g method .All raw sequence reads were deposited in the NCBI’s Sequence Read Archive (SRA) under Bioproject Number PRJNA758395, and SRA accession numbers SRX12007895 -SRX12007909.Anti-leptospira antibodies were registered in 36 of the 48 dogs (75% - CI: 60.4– 86.4) with titers ≥1:100. Tarassovi serovar was the most predominant (n=5) followed by Australis (n=3), Pyrogenes (n=3), Saxkoebing (n=2), Wolffi (n=2), Canicola (n=2), Hardjo (n=1), Grippotyphosa (n=1), Sejroe (n=1) and Shermani (n=1). 15 samples cross-reacted with more than one serovar. MAT titers for each sample are detailed in Leptospira DNA was detected in 18 of 19 dog urine samples (97.4% - CI: 73.9– 99.8). Some samples that were negative for lipL32 were positive for SNP111 and vice-versa  was present in at least one sample from each community except for Nueva Providencia, Leptospira santarosai (n=7) was detected in all communities except for Indillama, and Leptospira interrogans was detected in a single sample from Pompeya , which was not expected because elsewhere, these are the most common serovars in dogs [Leptospira from multiple sources. Previous studies on Amazonian wildlife have shown a high diversity of Leptospira serovars [Our serological results in 51 dogs from a population of ~550 individuals suggests that a high percentage of dogs have been exposed to pathogenic  in dogs ,70–73 an in dogs ,43,72,74 in dogs . The divserovars ,22. TherLeptospira isolates.It is important to note that none of the dogs in our study were vaccinated against leptospirosis, and the high diversity of serovars encountered suggests that even vaccination, as routinely performed in Ecuador and other Latin American countries, would have little effective in preventing canine infection or carriage, although it may reduce the prevalence of certain serovars. In Ecuador, the health authority, AGROCALIDAD, has registered and approved multiple vaccines for canine leptospirosis . Most ofLeptospira, the highest percentage reported so far. Three pathogenic Leptospira species were identified in dog urine, L. santarosai, L. noguchii, and L. interrogans. These species have been previously identified in South America [L. santarosai and L. noguchii have been reported in asymptomatic wildlife and dogs, and all three have caused severe disease in humans [L. interrogans is the most common and widely distributed pathogenic species and known to also infect rodents and small mammals [L. interrogans was found only in a community close to the small town of Pompeya, although the presence of this species in more remote communities cannot be excluded due to the small number of samples collected. L. noguchii and L. santarosai were found in remote communities. The relatively high genotype diversity is consistent with the high serotype diversity and given the close interaction of dogs with the environment and wildlife, is suggestive of varied sources such as might be encountered in the adjacent forest.Dogs infected with the pathogen will begin to excrete bacteria in their urine 7 to 10 days post-infection, and this excretion can continue for several weeks or even years ,81. Addi America –86; L. sn humans ,83,87–95 mammals –98. In oLeptospira DNA from urine is complicated due to pH and degradation of leptospiral DNA at ambient temperatures or after freezing and thawing as might occur during transit [Leptospira DNA detection by combining the results of two highly sensitive TaqMan assays capable of detecting 1×101 copies/µL [The methods used to preserve and transport samples, coupled with detection and sequencing methods, reduced the likelihood of false positives. Recovering  transit . In the opies/µL ,51. By uLeptospira and interactions with diverse host species [Marmosa spp., coati, nine-banded armadillo, opossum, porcupine, rodents, primates, bats, and wolves are exposed to the disease or excrete leptospires in their urine [Researching leptospirosis within the framework of the One Health concept  by consi species ,29,101. ir urine –25,102. Leptospira DNA in dogs from five indigenous Kichwa communities provides strong evidence that infection and carriage of leptospirosis is very common among dogs in the Ecuadorian Amazon basin. The high serotype and genetic diversity of samples, coupled with the lack of a single dominant type suggest that there may not be any serotype of genotype that is specifically adapted to dogs, and sources of transmission to these dogs are likely to be varied. The domestic dogs in these communities are free-roaming and often hunt wildlife and interact with the ecosystem of the adjacent forest, providing frequent opportunities for the transmission of Leptospira to and from wild animals. Importantly, frequent interactions with humans and presence in the peridomestic space is likely to result in transmission between humans and dogs, and the high prevalence in dogs suggests that human leptospirosis in this region is likely greatly underestimated. To more completely understand the cycling and transmission of Leptospira in this environment, high-resolution genotyping of longitudinal samples collected from dogs, humans, wildlife, soil, and water would be ideal. This work however establishes that domestic dogs are likely to play an important role in leptospirosis epidemiology in this region, with implications in other regions of the world where peridomestic animals interact with surrounding environments and wildlife.A high level of seropositivity and prevalence of pathogenic Supplement 1


This study presents the development and characterization of a novel buffalo Petit Suisse cheese, enhanced with symbiotic properties through an innovative whey retention method and incorporating inulin and xanthan gum. The research focused on assessing the cheese’s physicochemical properties, shelf life, lactic acid bacteria viability, syneresis behavior, and the impact of varying concentrations of functional ingredients. The addition of inulin and xanthan gum, following a design of experiments, significantly influenced the cheese’s texture and consistency. Higher inulin concentrations were associated with increased fermentation activity, as indicated by total titratable acidity, which showed an increase from 1.22% to 1.50% over a 28-day period, and pH levels that decreased from 3.33 to 2.96. The syneresis index varied across trials, with the highest reduction observed in trials with increased xanthan gum concentrations, effectively reducing syneresis to 0%. Lactic acid bacteria viability also showed notable variations, with the highest cell survival percentage reaching 107.89% in formulations with higher inulin and xanthan gum concentrations. These results underscore the importance of inulin and xanthan gum in enhancing the cheese’s microbial stability and textural quality. The study concludes that the strategic use of inulin and xanthan gum improves the nutritional profile of buffalo Petit Suisse cheese and optimizes its textural and sensory attributes. The contemporary food industry is witnessing a transformative era, characterized by a burgeoning consumer appetite for health-focused and environmentally sustainable products. This evolution is particularly impactful in the dairy sector, where there is a growing emphasis on nutrient-rich and ethically produced offerings . Dairy cWith its rich nutrient profile and unique physicochemical properties, Buffalo milk has emerged as a key player in this evolving dairy narrative . DespiteAmong various dairy products, Petit Suisse cheese, a creamy delicacy of French origin, is undergoing a transformative phase in its production process . TraditiInulin, a soluble dietary fiber, has been increasingly used in dairy products, offering a range of benefits health benefits, including improved mineral absorption and immune function . AccordiXanthan gum, a polysaccharide used as a food additive, has a significant impact on the properties of dairy products. Research by El-Sayed et al. (2002) found that xanthan gum increases the viscosity, curd tension, and sensory properties of dairy products, without markedly affecting pH, total solids, or lactic acid bacterial counts . This enGiven the potential of buffalo milk and the functional benefits of inulin and xanthan gum, this study aims to develop a novel buffalo Petit Suisse cheese with enhanced symbiotic properties. By incorporating an alternative whey retention method and exploring the synergistic effects of inulin and xanthan gum, the research focuses on a holistic analysis of the cheese’s physicochemical properties, shelf life, viability of lactic acid bacteria, syneresis behavior, and overall quality. This investigation is poised to offer valuable insights into sustainable dairy production while aligning with the growing trend of health-centric, functional foods. The study also seeks to address the gap in understanding the combined effects of these functional ingredients in buffalo milk-based dairy products, potentially setting a new standard in the dairy industry.Bubalus bubalis). Milk composition can be viewed in The milk was sourced from Murrah buffalo breed  was allowed to equilibrate to approximately 10 °C. Using a centrifuge operating at a maximum speed of 4000 rpm, the milk was centrifuged for approximately 20–30 min to ensure optimal cream separation. The duration was adjusted based on visual inspection of separation efficiency. The extracted cream was then collected into sterilized containers and stored at 4 °C until further use. Buffalo skimmed milk was fermented using kefir grains in a sterilized glass container. A proportion of 15 g of kefir grains was used for every 500 mL of milk. The mixture was allowed to ferment for 24 h at room temperature. The kefir grains were separated after fermentation, and the fermented milk was stored at 4 °C.Fresh milk from the Murrah buffalo breed  and sugar (63.7 g) were weighed and added to the buffalo milk , and then cooled to 35 °C; 5% fermented kefir milk was added, stirred for two minutes, and then rennet was added according to the manufacturer’s instructions; subsequently, the mixture was incubated at 35 °C in a bacteriological oven for a 24 h period; t the end of this period, the mixture was cooled, processed, and added with the remaining ingredients—xanthan gum, inulin, fruit pulp (126 g), aroma (2.8 g), and potassium sorbate (0.35 g)—one at a time under processing, until fully homogenized; finally, the cheeses were packed in sterile plastic containers and stored at 4 °C. The amounts of xanthan gum and inulin on the final formulation varied according to a full factorial experimental design 2eriments . The indThe water content, ash and protein content were determined according to the official methods of AOAC International (2010) . To measThe determination of total sugars was carried out according to Lane and Eynon 1934) . Briefly34 . Brie2 at a temperature of 22 °C. Measurements were performed in triplicate, with the probe being extensively washed first with distilled water. As a result of these experiments, force-time curves were built and analyzed to determine some mechanical parameters  [Texture was analyzed using the AACC method 74-09 2000) [00 [20], esivity) .During the storage period, samples were stored at 4 °C for 28 days. Analyses were carried out on the product on days 0, 7, 14, 21, and 28, regarding the parameters of pH, acidity, lactic bacteria count, and syneresis using the previously described methods.−1 dilution. Serial dilutions were then carried out, and 1 mL of the 10−3 and 10−4 dilutions were inoculated onto Petri dishes, and then MRS culture medium was added (deep plating). After complete agar solidification, the plates were inverted and incubated at 37 °C for 72 h in a microaerophilic atmosphere. At the end of the incubation period, the number of colony-forming units (CFU/mL) was calculated based on the number of confirmed colonies and the inoculated dilution. A Gram stain was then carried out, followed by the catalase test to confirm the lactic bacteria according to the methodology described by Silva, Junqueira, and Silveira (2001) [The procedure for enumerating lactic bacteria were performed according to the following methodology, where 25 g cheese samples were homogenized in 225 mL of peptone saline solution (0.85% NaCl and 0.1% peptone), achieving a 100 represents the bacterial count at the beginning of the storage (CFU/mL) and t is the storage time. The specific growth rate (μ) was calculated using Equation (2):0 and N are viable cell numbers measured within the exponential growth phase at times t0 and t, respectively. The percentage of cell survival was defined as shown in Equation (3).In this equation, N refers to the bacterial count at a particular storage period (CFU/mL), N0 denotes the initial viable cell count (CFU/mL) at the beginning.Here, N represents the number of viable cells count (CFU/mL) at a particular period and NSyneresis was determined according to the methodology proposed by , using Ep < 0.05) among the treatments. The regression model used is represented in Equation (5).For each obtained response, a multiway ANOVA was carried out through linear regression to verify the influence of the factors on the obtained values and to check if there were significant differences  and average comparison by Tukey’s test at 5% probability, using GraphPad Prism version 10.0.0 for MacOS GraphPad Software, Boston, MA, USA, www.graphpad.com.Here, y is the independent variable, βi is the model parameter estimators and xi is the coded factors (independent variables). In cases with a statistically significant difference, response surfaces were generated to visualize the optimization range for process, product, and cost improvements. ANOVA statistical analyses and modelling were performed using Python (version 3.12) [Python Software Foundation, Python Language Reference, available at The present work studied the inclusion of inulin and xanthan gum on petite Suisse cheese from buffalo milk, potentially creating a dairy product that is both prebiotic and probiotic. The results of the physicochemical properties are presented in Our experimental analysis revealed a notable correlation between the incorporation of inulin and the water content of cheese. Specifically, the data indicated that higher levels of inulin, as observed in Trials 2 and 4, were associated with an increased water content, resulting in a reduction of total solids. This phenomenon is likely attributable to the hydrophilic characteristics of inulin, which can bind and retain water within the cheese matrix . On the Variations in ash content indicate mineral content was relatively minor across the different trials. The most substantial ash content was recorded in Trial 4, which also contained the highest concentration of xanthan gum. This observation suggests a potential relationship between xanthan gum levels and mineral or ash retention within the cheese. However, the relationship did not exhibit linearity, as variations in xanthan gum content did not consistently correlate with ash content, implying the influence of additional factors on the ash content of the cheese. Similar lack of correlation results were found on white cheese .The study found that the alterations in inulin and xanthan gum levels had minimal impact on the fat and protein content of the cheese, which remained relatively consistent across all trials. This stability indicates that the nutritional profile of the cheese, in terms of fat and protein, is not significantly affected by these additives . Such cop < 0.05. Equation (6) presents the first-order model for total sugars.The content of total sugars exhibited more variability, with the highest sugar content aligning with the trials that had increased amounts of inulin . Given that inulin is a form of carbohydrate, this result was anticipated. The trials with a higher inulin ratio, specifically 16 g/100 g, showed a substantial rise in sugar content, whereas Trials 1 and 3, with only 6 g/100 g of inulin, presented the lowest sugar levels. This apparent correlation indicates that adjusting the inulin content can effectively modulate the sweetness and possibly the caloric value of the cheese, which can be tailored to meet consumer preferences and nutritional objectives. After modeling, the multiway ANOVA revealed that the total sugar model was significant for p-value of 0.0144. The coefficients indicated a negative influence of inulin (β1 = −0.0003) and a positive influence of xanthan gum (β2 = 0.9067) on firmness, along with a significant interaction term (β3 = 0.1267). The model’s R2 value was 0.9580, suggesting a high explanatory power. The positive coefficient for xanthan gum suggests its crucial role in increasing the firmness of the cheese, likely due to its thickening properties. This xanthan gum feature is correlated with its ability for form secondary structures due to this polysaccharide side-chains [The results of the texture-dependent mechanical properties are presented in  β3 = 0.17. The moe-chains . The intp-value of 0.0152. The coefficients showed a strong positive effect of xanthan gum (β2 = 16.7733) on consistency, while inulin had a moderate positive influence (β1 = 0.524). The R2 value was 0.9565, indicating the model’s high explanatory power. Xanthan gum’s substantial positive impact on consistency aligns with its role in improving the structural integrity of the cheese. This finding reveals that xanthan gum impacts the stabilization of the whey protein gel network [p-value = 0.008934) with an R2 value of 0.969559. Xanthan gum had a significant positive effect (β2 = 4.426667), while inulin had a negative impact (β1 = −0.202000). Xanthan gum’s positive influence confirms its role in increasing viscosity, a desirable property for spreadable cheeses. The negative effect of inulin might suggest its role in reducing viscosity, possibly due to its interaction with the water content in the cheese. Similar result was found for Petit Suisse cheese [2 value indicates that the model effectively captures the relationship between the ingredients and the viscosity index. Equations (8) and (9) presents the first order model for consistency and viscosity index, respectively.The consistency model yielded a significant  network . Inulin’e cheese . The higp-value = 0.035189) with an R2 of 0.923346. Both inulin and xanthan gum showed negative coefficients , but their interaction had a positive effect (β3 = 0.2300). The negative coefficients for inulin and xanthan gum suggest that higher concentrations of these ingredients independently may reduce cohesivity. However, the positive interaction term indicates they might improve cohesivity when used together, possibly due to a balancing effect between moisture retention (by inulin) and thickening (by xanthan gum). Equation (10) presents the model for cohesivity.The cohesivity model was significant , a specific range of firmness (3–6 N), optimal viscosity index (13), and higher cohesivity (1.29). The optimization process identified that an inulin level of approximately 7.29 g/100 g and a xanthan gum level of around 1.06 g/100 g would yield the most desirable product attributes. This result was obtained by maximizing the desirability functions corresponding to each product property, namely total sugars, firmness, viscosity, and cohesivity. The overall desirability score at these optimal levels was approximately 0.64 on a scale from 0 (completely undesirable) to 1 (fully desirable). This score reflects a balanced compromise among the various attributes, considering their individual importance and interplay. The desirability functions were designed to prioritize lower sugar content and a texture profile that balances firmness with cohesivity and viscosity, catering to the dual demands of health and sensory appeal.In terms of pH, there were significant changes over the storage period. Initially, the pH values were generally higher, but as the storage time increased, a gradual decrease in pH was observed in most trials, aligning with the increase in acidity levels due to lactic acid production. For example, in Trial 1, the pH decreased from 3.82 on Day 7 to 2.96 on Day 21. Similarly, in Trial 4, the pH decreased from 3.42 on Day 7 to 3.10 on Day 28. These pH changes are indicative of the ongoing microbial activity and the production of lactic acid as the primary fermentation product.The changes in pH and acidity in the cheese over time are critical for understanding the maturation process and flavor and texture development. The variations across different trials underscore the impact of inulin and xanthan gum concentrations on these processes. Inulin, particularly at higher levels, appears to accelerate the fermentation process, enhancing the growth and activity of lactic acid bacteria . XanthanThe consistency of results, shown by the low coefficient of variation percentages, suggests a reliable influence of formulation changes on cheese acidity and pH. This study demonstrates the significant roles of inulin and xanthan gum in fermentation, highlighting their potential in optimizing cheese formulations for desired sensory qualities, textural characteristics, and probiotic benefits. Further research could explore how these changes in acidity and pH impact the cheese’s overall flavor profile, texture, and shelf-life, contributing to the development of high-quality, health-beneficial dairy products .In the context of the viability of lactic acid bacteria(LAB), also presented in 6 to 1.16 × 106. This consistency provided a standardized baseline for assessing the influence of inulin and xanthan gum on bacterial growth. Over the 28 days, a general trend of increasing bacterial counts was observed in all trials, with Trials 2 and 4 showing higher levels (16 g/100 g), showing a more pronounced increase. By day 28, these trials reached bacterial counts of 1.20 × 106 and 1.23 × 106 CFU/mL, respectively. This trend suggests that increased inulin concentrations may foster a more conducive environment for the growth of lactic acid bacteria, potentially due to its prebiotic properties.The initial counts of viable lactic acid bacteria (CFU/mL) at the outset of the storage were relatively uniform across all trials, ranging from 1.10 × 10−1). This indicates that this specific formulation might be more effective in maintaining cell viability. Correspondingly, the specific growth rates varied across the trials, with Trial 4 exhibiting the highest growth rate (0.00426 day−1), aligning with the higher bacterial counts observed. This implies that the combination of higher inulin and xanthan gum concentrations supports an environment favorable for bacterial growth [The specific rates of cell death, indicated by negative values across all trials, reflected a decline in the rate of cell death over time. Particularly notable was Trial 4, which combined high levels of inulin (16 g/100 g) and xanthan gum (0.8 g/100 g) and showed the most significant decrease in cell death , reinforced the notion that higher concentrations of inulin and xanthan gum are advantageous for bacterial growth and survival.These findings suggest that the formulation of buffalo Petit Suisse cheese can be strategically optimized by adjusting the concentrations of inulin and xanthan gum to enhance the growth and survival of lactic acid bacteria. This has implications for the cheese’s probiotic potential and its overall quality and shelf life. The results provide a valuable foundation for further research into developing functional dairy products that are both nutritious and have an extended shelf life, aligning with the evolving consumer preferences for health-oriented and sustainable food products.The effect of varying concentrations of inulin and xanthan gum on the syneresis of buffalo Petit Suisse cheese across several trials over a 28-day period is presented in Remarkably, Trials 3 and 4, with 6% and 16% inulin combined with a higher xanthan gum concentration (0.8%), exhibited no syneresis over the 28 days. This underscores the effectiveness of xanthan gum at higher concentrations in stabilizing the cheese matrix and preventing the expulsion of liquid.The control group  with an intermediate concentration of inulin (11%) and xanthan gum (0.65%) showed no syneresis for the first 14 days. However, a minimal syneresis was recorded thereafter, peaking on day 14 5.636%) and then gradually decreasing. This pattern indicates that these intermediate concentrations provide a balance, minimizing syneresis while maintaining the quality of the cheese. When retained in the cheese matrix, whey proteins can interact with inulin and xanthan gum, potentially forming a more cohesive and less syneresis-prone structure . This in% and theThe observed patterns of syneresis in the study reflect these macromolecular interactions. In formulations with higher inulin levels but lower xanthan gum , the initial high syneresis rate that gradually decreases over time could be attributed to the restructuring of the inulin-protein network, influenced by the presence of whey proteins and lactose. This result suggests that weak gels are initially formed and become stronger, reducing the tendency for syneresis . In contIn summary, our research contributes to the field of dairy product development by demonstrating the successful creation of a buffalo Petit Suisse cheese with enhanced symbiotic properties, achieved through an innovative whey retention approach and the incorporation of inulin and xanthan gum. The pivotal findings of this study are the significant roles played by these additives in modifying the cheese’s texture, consistency, and microbial environment. One of the key insights is the positive effect of higher inulin levels on the fermentation process. Our results showed that increased inulin not only enhanced the growth of lactic acid bacteria but also contributed to the overall probiotic quality of the cheese. This suggests a promising avenue for further exploration into the use of inulin in dairy products, particularly for enhancing their probiotic attributes.Additionally, the role of xanthan gum in reducing syneresis and maintaining the creamy texture of the cheese underscores the importance of texture-modifying agents in dairy product formulation. The interaction between xanthan gum and inulin, along with the whey retention strategy, was found to be crucial in balancing the texture and microbial stability of the cheese. These findings highlight the potential of combining different functional ingredients to optimize the textural and microbiological properties of dairy products. For future research, we recommend a deeper exploration into the synergistic effects of various functional ingredients in dairy formulations. There is also a need to investigate the long-term stability and sensory attributes of such products, which are crucial for consumer acceptance. Furthermore, extending this research to other types of dairy products could provide broader insights into the applicability of these methods in the dairy industry.


New findings demonstrate two phases of population expansion occurring 22,000–26,000 and 2500–3800 years ago, coinciding with the Last Glacial Maximum, and Neolithic demographic transition, respectively. This research significantly enhances our understanding of the maternal genetic history of Thailand and MSEA, emphasizing the influence of South Asian admixture. Moreover, it underscores the critical role of prior information, such as mutation rates, within the Bayesian framework for accurate estimation of coalescence times and inferring demographic history.The evolutionary dynamics of mitochondrial DNA within the Thai population were comprehensively explored with a specific focus on the influence of South Asian admixture. A total of 166 samples were collected through randomized sampling, ensuring a diverse representation. Our findings unveil substantial genetic and haplogroup diversity within the Thai population. We have identified 164 haplotypes categorized into 97 haplogroups, with a notable inclusion of 20 novel haplogroups. The distribution of haplogroups exhibited variations across different populations and countries. The central Thai population displayed a high diversity of haplogroups from both the M and N clades. Maternal lineage affinities were discerned between several Mainland Southeast Asia (MSEA) and South Asian populations, implying ancestral genetic connections and a substantial influence of South Asian women in establishing these relationships.  Numerous archaeological sites, dating back to 10,000–45,000 years ago, have been well documented throughout the area4. During the late Pleistocene to Holocene periods, the Hoabinhian cultural system was widespread among hunter-gatherer groups spanning from southern China to Sumatra5. However, only two ancient DNA samples have been recovered from Pha Faen, Laos (7888 ± 40 years before the present (YBP)) and Gua Cha, Malaysia (4319 ± 64 YBP)6 and modeled as a deeply diverged East Eurasian lineage. Furthermore, the genetic data revealed a relationship with modern Andamanese hunter-gatherers such as Önge and MSEA Negritos, including Jehai7. The Neolithic population exhibits an admixed genetic profile between a deeply diverged east Eurasian lineage of hunter-gatherers and East Asian of southern Chinese agriculturalists, which is like modern-day Austroasiatic-speaking populations such as Mlabri7. The Bronze Age in MSEA emerged in the late second millennium BCE, with the earliest evidence of bronze metallurgy dating to around 1100 BCE, reflecting continued cultural interaction with southern China8. The genetic study of Bronze Age individuals from Northern Vietnam found substantial genetic turnover suggesting an additional wave of migration from Southern China during the Bronze and Iron Ages7. In the late first millennium BCE, the region was incorporated into the Maritime Silk Road through connections between regional networks linking Southeast Asia, China, South Asia, and beyond. The presence of walled urban settlements which are associated with centralized policies and can be classified as incipient states such as Angkor Borei, Oc Eo, and Khao Sam Kaeo, dating back to the fourth century BCE, indicates the existence of well-established and dynamic local and regional networks that facilitated integration9. The cultural interaction between South Asian and local MSEA populations created synergies that influenced early state formation in MSEA9.Mainland Southeast Asia (MSEA) has a rich and complex history. Archaeological evidence indicates that anatomically modern humans inhabited the region for at least 50,000 years10. Kutanan et al.11 initially analyzed 1234 complete mtDNA sequences from 51 Kra-Dai and Austroasiatic groups in Thailand and Laos, revealing high genetic heterogeneity across the region with 212 haplogroups. Significant genetic differentiation existed among samples from the same ethnolinguistic group. Notably, Kra-Dai groups exhibited more genetic homogeneity than Austroasiatic groups, which displayed a higher proportion of basal mtDNA lineages. In a subsequent study, Kutanan et al.12 analyzed an additional 560 complete mtDNA sequences from 22 distinct populations, leading to the identification of 62 new haplogroups. Researchers also found West Eurasian-associated haplogroups in some populations in MSEA, which could be a signal of South Asian admixture12. Previous genome-wide studies had documented the presence of South Asian genetic admixture in MSEA populations particularly those with strong cultural influences from India including the Bamar, Cham, Khmer, Malay, Mon, and Thai16. However, it is absent in present-day hunter-gatherer groups or relatively isolated groups from the highlands of northern Thailand13.Due to prehistoric and continuous human habitation, the region exhibits significant ethnolinguistic and genetic diversity. Specifically, Thailand, located in the center of MSEA, is home to approximately 68.61 million people who speak 73 recognized languages from five linguistic families: Kra-Dai, Austroasiatic, Sino-Tibetan, Austronesian, and Hmong-MienRecent archaeological discoveries and investigations into South Asian admixture in MSEA populations have highlighted the present-day Thai population’s complex mtDNA evolutionary history and diversity. This study aims to expand our knowledge of Thailand’s mtDNA landscape by comprehensively analyzing an additional 166 whole mtDNA sequences from the randomized samples of self-identified Thais, regardless of their ethnic backgrounds. This approach will minimize potential biases associated with targeting specific groups, providing a more comprehensive overview of the genetic diversity of the present-day Thai population. By examining the data in conjunction with previous studies and considering South Asian admixture, this study will achieve a more comprehensive understanding of the complex evolutionary history of mtDNA in Thailand  while the previous studies were 5.82 (1234 samples/212 haplogroups)11, and 3.22 (560 samples/174 haplogroups)12 respectively. A higher ratio implies that a larger number of samples is used to identify a smaller number of haplogroups, indicating lower genetic diversity or greater relatedness among the samples. The haplogroups observed belong to macrohaplogroup M , R9 , B , and N , respectively. The most frequent haplogroups are B5a1a , F1f. , and F1a1a1 . We employed HaploGrep2 to generate DOT graphs represented in Fig. 18. The phylogenetic tree generated by Bayesian Evolutionary Analysis Sampling Trees (BEAST) reveals the distinctions among individual samples in B5a1, F1a, and F1f. clades . It was noted that all samples collected from the northern region of Thailand were assigned to the R clade, while most of the samples from the southern region fell under the M clade  were grouped by Thai government administrative region. Samples are from Bangkok and Central , Northeast , South , East , North , and West    and 0.17% (P > 0.01), respectively12. These results suggest that the language family may be a better indicator of the genetic structure of Thai/Lao populations than geography. However, we caution that language family and geographic classification may be poor indicators of genetic structure due to the high levels of variation within populations and the low levels of variation between populations. Furthermore, a Mantel test for correlations between genetic and geographic distances indicated no correlation  supporting the limited impact of geography on the genetic structure of populations in Thailand.Analysis of Molecular Variance (AMOVA) revealed that only 0.4% of the total genetic variation was attributable to differences between populations classified by geographic regions. However, the observed genetic differentiation  was not statistically significant at the 0.05 significance level  was performed, and the result is shown in Fig. To examine the relationship of our data to other populations, we initially generated a nonmetric multidimensional scaling (NMDS) plot at the global population level Fig. a. Africa20. Thailand has been found to show a notable prevalence of haplogroup D in all studies  over time. In Fig. Ne underwent a substantial and remarkable increase. In particular, the Ne value exhibited a more than 20-fold surge, highlighting a significant population expansion event. After the initial expansion, Ne remained stable until approximately 3800 YBP, after which another growth phase was observed. This subsequent growth phase persisted from approximately 3800 YBP to 2500 YBP, during which Ne experienced a notable fourfold increase. Interestingly, from 2000 YBP to the present, the Ne exhibited a stable trend. These findings suggest a complex demographic history characterized by different population dynamics. The initial population expansion, followed by a period of stability, and the subsequent phase of growth indicate intricate patterns of population growth and contraction. The stable population size observed in recent history suggests a relative equilibrium in the population dynamics.The Bayesian skyline plot (BSP) is a valuable tool for visualizing the demographic history of populations based on changes in effective population size  approximately 7.2% using f-statistic-based Admixtool2 with autosomal SNPs and estimated the admixture date around 4 CE during the Angkor period using haplotype-based Globetrotter. However, a subsequent study found significant South Asian admixture (ca. 40–50%) in a protohistoric individual from the Vat Komnou cemetery at the Angkor Borei site in Cambodia. Radiocarbon dating places this individual in the early period of Funan (1 CE), suggesting the possibility of South Asian migration to MSEA and intermarriage with local populations before or during the early stage of state formation14. This study provides additional evidence from mtDNA that contemporary Cambodian populations carry genetic contributions from South Asian ancestry. Moreover, the result suggests a substantial contribution of South Asian women to the genetic makeup of present-day Cambodian populations.The primary objective of this study was to enhance our understanding of the evolutionary patterns of mtDNA in the Thai population and its correlations with neighboring countries, while also taking into consideration the impact of South Asian admixture. We observed a significant cluster that includes the haplogroups B5a1, F1a, f1f., and M7b1a1 Fig. . Notablyons Fig. b suggestons Fig. b. The dions Fig. b and hapons Fig. b highlig22. Machold et al.23 found that the Y-haplogroups R1a-M420 and R2-M479, associated with west Eurasia, occurred at low frequencies in the Ede population  and in the Giarai population (3.7% and 3.7%). Changmai et al.13 using qpAdm, qpGraph, and SOURCEFIND methods, consistently inferred the presence of South Asian ancestry in the Ede and Giarai populations . The comprehensive genetic evidence derived from Y-haplogroups, and autosomal DNA admixture strongly supports the relationship between the Ede, Giarai, and South Asian populations23. The NMDS plot  positions Ede and Giarai relatively closer to South Asian populations compared to other Vietnamese groups. However, the gap between them remains wider than seen in Cambodian and Myanmar populations. Therefore, our findings suggest that while Ede and Giarai show some maternal lineage affinity with South Asians, mtDNA evidence alone is insufficient to decisively establish a conclusion about the contribution of South Asian women to their genetic makeup.The Ede and Giarai are two distinct populations residing in Vietnam, all of which have Austronesian language roots due to cultural diffusion11, we have taken an additional step by visualizing and representing the genetic association between Mon and South Asian populations in a phylogenetic tree. Interestingly, Mon and Punjabi individuals in Lahore, Pakistan, share several haplogroups in the M clade, including M, M30, M45, M4a, and M5a, suggesting a common ancestral population. Mon also shares some haplogroups in the M clade, such as M40a1, M45a, and M5c1, with the Indian Telugu population in the UK. The Telugu-speaking people primarily reside in the states of Andhra Pradesh and Telangana in South India, while Punjabi-speaking people are predominantly found in the Punjab region spanning across India and Pakistan. It is surprising to note that Mon harbors haplogroups from both the South and North Indian subcontinents. The qpAdm analysis infers approximately 12% South Asian ancestry within the Mon population13. Additionally, the Mon group exhibits minor occurrences of West Eurasia-associated haplogroups, including J (5%) and R (16%)24. Furthermore, we observed that Mon shares specific haplogroups within the D clade , with the Wanchoo people , as well as with the Gallong people 25. This observation aligns with the findings of Changmai et al.13 and Kutanan et al.15, who reported the presence of additional ancestry from a Tibetan-related source in the Mon population. They hypothesized that this Tibetan-related ancestry in Mon might have been acquired through interactions with Sino-Tibetan speaking populations in Myanmar. Based on the aforementioned observations, we have performed additional analysis to further investigate the relationship between the Mon population and Sino-Tibetan speaking populations applying f4-statistics in the form of f4 and f4. The result of f4-statistics , which indicates that the Tibeto-Burman groups share more genetic drift with Mons than with other Thailand groups. In this study, we also found that Sino-Tibetan shared 12.5% mtDNA haplogroups  with Mon. This proportion is similar to the autosomal SNPs that Mon and Sino-Tibetan showed an admixture proportion of around 10% when inferred using SOURCEFIND13.Building upon the data obtained from the study conducted by Kutanan et al.11. The discrepancy in timing can be attributed to the variation in mitochondrial mutation rates and mtDNA partition schemes applied in the respective studies. The mutation rate of mtDNA is not constant but fluctuates over time, influenced by factors such as population size and dynamics. In growing populations, there is a higher number of lineages, leading to an increased accumulation of mutations per lineage. These lineages also persist for longer periods, resulting in an accelerated evolutionary rate that surpasses the mutation rate. Conversely, in declining populations, the evolutionary rate decreases.Our study identified two phases of population expansion in Thailand: an initial phase of around 22,000–26,000 YBP and a second phase of around 3800 YBP. The initial phase contrasts with a previous study that proposed an expansion of around 55,000 YBP while the second phase has not been reported in the previous study−8 substitutions per site per year based on a recent study26. However, there is no consensus on the best partitioning scheme and mutation rates to use in coalescent dating and BSP analyses. To assess the sensitivity of our results to the choice of partitioning scheme and mutation rate, we performed two additional analyses. The first analysis used a single model with a single mutation rate of 2.285 × 10−8 site−1 year−1, which was previously applied by Maier et al.27. This analysis showed that the timeframes shifted to around 48,000 YBP for the initial expansion and 7500 YBP for the second expansion , the flooding of the Sunda shelf (marine transgression)33 likely caused a contraction of inhabitable areas towards inland regions, stabilizing population expansion35. During LGM, several new haplogroups emerged, providing additional evidence for population expansion. Notable among them are the Southeast Asian lineage M91 , the Northeast Indian haplogroup M49e , M49e1 , and Indochina R9b 37. We also observed a second phase of population expansion occurring between 3800 and 2500 YBP, which had not been previously documented in studies on mtDNA in the Thai population. Moreover, the sudden increases in effective population size might indicate the arrival of new groups or gene flow from neighboring regions. Gignoux et al.38 demonstrated a global Neolithic expansion through mitochondrial lineage analysis. They also indicated that the population size of Southeast Asia began to expand around 4700 years ago (CI 3000–5700 years ago), which aligns with our findings. This period corresponds to the Neolithic demographic transition, characterized by the adoption of agriculture by prehistoric societies, resulting in increased food production and decreased mobility compared to foraging practices. In Southeast Asia, this transition took place between 2500 and 1500 BCE and brought about significant changes in the biological, linguistic, and cultural evolution of the region39. Around 6000–4000 BCE, rice (Oryza sativa japonica) was fully domesticated in the mid-lower Yangtze River valley of central China40. Subsequently, rice and millet farmers from the Yangtze and Yellow River regions migrated southward through different routes, reaching Baiyangcun in Yunnan around 2650 BCE41. By 2200–2000 BCE, they had reached coastal Vietnam and Thailand, and by 1700–1500 BCE, they had expanded to the interior Khorat Plateau, as reported by Higham et al.42. Archaeological excavations have yielded artifacts such as domestic rice, pottery styles, and tools, demonstrating a connection to southern China. Notable examples include Khok Phanom Di (ca. 2000–1500 BCE), Ban Chiang, and Non Nok (ca. 1100–1000 BCE) as well as Tha Kae and Khok Charoen (ca. 2000/1800–1100 BCE)43. In addition to archaeological findings, the studies on autosomal DNA have provided supporting evidence for a subsequent increase in the effective population size. Neolithic populations in MSEA were a mixture of deeply diverged East Eurasian lineages and East Asian agriculturalists who migrated from South China approximately 4000 years ago7 and the genetic structure of Bronze Age individuals from Northern Vietnam (ca. 2000 years ago) suggests another migration wave from southern China to MSEA7.The initial population expansion phase Fig.  correspo48 while the maximum genetic diversity theory is an alternative framework. Therefore, we approach our conclusions with caution.Our study provides insights into the mtDNA) landscape of the present-day Thai population and its connections with neighboring regions. However, some findings are based on the neutral theory of molecular evolution such as BSP, which has been challenged by many authorsf4-statistics indicate a Tibeto-Burman genetic element in Thailand's Mon population. Our findings indicate two population expansion phases, aligning with the LGM and Neolithic transition. Importantly, this work enhances our understanding of the mtDNA landscape of Thailand. Moreover, it also emphasizes the critical role of prior knowledge in the Bayesian framework for accurate inference of demographic history.In summary, our study investigated mtDNA evolution within the Thai population, focusing on South Asian admixture. We identified 20 novel haplogroups from 166 randomly selected individuals. Haplogroup distribution was found to vary across populations and countries. Maternal lineage affinities connect MSEA and South Asian populations, highlighting ancestral genetic association and the significant influence of South Asian women. Notably, This study was conducted to investigate the mtDNA landscape of the Thai population in strict adherence to ethical guidelines and principles. All experimental protocols received approval from the Institutional Review Board (IRB) of the Faculty of Medicine, Chulalongkorn University (IRB No. 817/64). The need for informed consent was waived by the IRB of the Faculty of Medicine, Chulalongkorn University under 45CFR46.101(b), as all 166 blood samples were taken from medicolegal autopsies authorized by the Faculty of Medicine, Chulalongkorn University (Certificate of Exemption No. 049/2021). The Thai ethnicity of the deceased individuals was recorded according to their identity cards, which were provided by their relatives.The sampling frame for this study was all deceased individuals who underwent a medicolegal autopsy at the Faculty of Medicine, Chulalongkorn University in 2019 and 2020. We employed a randomized sampling approach to minimize bias. The samples were randomly selected from the available medicolegal autopsy cases. This approach allowed us to collect samples without any specific bias toward gender, geographic location, or ethnolinguistic background. By doing so, we aimed to minimize the associated bias and the risk of selecting samples that might not accurately represent the overall population. We were able to collect 166 samples from the sampling frame. The sample consisted of 143 males and 23 females, with an age range of 3–87 years . We acknowledged that gender imbalance in our sample could influence the representation of certain maternal lineages in our study.DNA extraction using the QIAamp DNA Blood Mini Kit (Qiagen). All dsDNA was quantified using NanoDrop™ 2000 spectrophotometers (Thermo Scientific) and diluted to 0.1 ng/µl. A long-length amplification was performed with two sets of specific primer pairs  using the LA PCR Kit (TaKaRa) following the manufacturer’s protocol. The PCR products were qualified and quantified by Agarose gel electrophoresis and NanoDrop™ 2000 spectrophotometers, respectively. These PCR products for each sample were diluted to 0.2 ng/µl and pooled in 1 well with 10 µl for each PCR product.DNA libraries were prepared using Nextera XT DNA Library Preparation Kit for the first 90 samples and Nextera DNA Flex Library Preparation Kit (Illumina) for another 78 samples with 1 ng of DNA input. Library preparations were performed following the manufacturer’s protocols with a bead-based normalization method using 1% spiked-in PhiX control v3 (Illumina). Pool DNA libraries were sequenced by MiseqFGx (Illumina) using Miseq Reagent Nano Kit v2 (300 cycles) with a 2 × 150 bps read length.50 with HaploGrep251 following the established criteria of EMPOP.The sequencing data were aligned with the revised Cambridge Reference Sequence , mapping and determining variants using mtDNA Variant Processor and mtDNA Variant Analyzer v1.0.0 (Illumina). MtDNA haplotypes were assigned to haplogroups based on PhyloTree Build 1752, including geodist, vegan53, Ape54, Adegenet55, and Ade456. The results were visualized using the ggplot257 package. We acknowledge that our results may be influenced by ethnolinguistic background, gender ratio imbalance, and sample size bias. We are aware that some regions have few samples, and our interpretations of findings from these regions should be approached with caution, as they may not accurately represent the regional population. Findings from these regions should be considered preliminary and ideally validated through larger-scale or targeted group research to ensure the reliability and validity of the results.To investigate the mtDNA landscape of the Thai population, we grouped samples into six administrative regions and plotted their geographic locations to determine data distribution Fig. . We obse18, and data from previous studies59, which all detail can be found in Supplementary Table dist.genpop” from the Adegenet55 package and then applying the “metaMDS” function from the Vegan53 package in R. The results were visualized using the ggplot2 package. The application of NMDS allowed the identification of patterns and differences among these populations, further contributing to our understanding of the genetic diversity in this region.To provide a comprehensive population representation, a dataset of 6533 complete mtDNA sequences was compiled, incorporating 166 newly generated data from our study along with 2504 samples from the 1000 Genome Project Phase 3 as provided by Weissensteinersmartpca60 version 18,140 from the EIGENSOFT package (https://github.com/DReichLab/EIG). The computed principal components (PC) for populations from East Asia, Europe, South Asia, and Thailand were analyzed and visualized using the ggplot2 package in R.PCA was performed to observe a cluster of samples following to mitochondrial sequencing variant using HaploGrep2 based on PhyloTree Build 17. The “lineage” function generated a DOT file that was visualized with Graphviz and adapted in Inkscape. We complemented the HaploGrep2 tree with Bayesian inference using BEAST61 to ensure consistent topologies. Multiple sequence alignment of sequences to rCRS using Geneious Prime. All data conversion, alignment, and file formatting steps were performed in Geneious Prime. Software jModel test 2.1.762 was used to select the most suitable model during the creation of the input file for BEAST using BEAUTi v1.863. We applied the HKY + Γ model to our data. Exponential priors were employed to specify the prior distributions for the gamma shape parameters in the analysis. Additionally, lognormal priors were utilized to define prior distributions for the kappa parameters of the HKY model. These choices were made based on the previous study27 to incorporate prior information and assumptions regarding the parameter values. The strict clock model was tested in our analysis, utilizing a lognormal prior distribution for the mutation rate (μ). Numerous mutation rates have been published  mutations site−1 year−1 for the oldest period  to 4.33 × 10–8 (95% CI 3.90–4.82 × 10–8) for the most recent period26. We reasoned that this mutation rate is appropriate for our study because we study present-day populations. We also performed two additional analyses to assess the sensitivity of our results to the choice of partitioning scheme and mutation rate. The first analysis used a single model with a single mutation rate of 2.285 × 10−8 per site per year, which is an average of mtDNA mutation rates reported in the literature72 and applied by Maier et al.27. The second analysis partitioned mtDNA into coding and non-coding regions and applied mutation rates of 1.708 × 10−8 and 9.883 × 10−8 respectively. It is the same approach applied by Kutanan et al.12 in the studies of populations in Thailand. The results of these additional analyses are presented in Supplementary Table The phylogenetic tree was generated directly with 66. Therefore, to achieve the calibration of the phylogenetic tree, 20 ancient samples with associated tip ages measured in calibrated years before the present  were assigned based on dates available from previous studies79. By incorporating such reliable temporal information, we were able to enhance the accuracy and reliability of the inferred evolutionary relationships within the studied mtDNA lineages. We performed 4 × 107 MCMC steps by sampling every 4 × 103 steps. Tracer80 was used to visualize and diagnose MCMC output and to assess a stationary distribution of posterior samples, within and between MCMC chains. We used several tools from the BEAST package to analyze and visualize the posterior tree samples. First, we used LogCombiner to combine the posterior tree samples generated from the MCMC analysis. This step allowed us to merge the individual trees into a single file for further analysis. Next, we summarized the combined tree samples using TreeAnnotator, another component of the BEAST package. TreeAnnotator enabled us to derive the maximum clade credibility (MCC) tree, which represents a summary tree that captures the most credible evolutionary relationships among the sampled sequences. To visualize and interpret the results, we utilized iTOL; therefore, we were able to depict and analyze the MCC tree, facilitating the interpretation of the evolutionary relationships and providing a visual representation of the inferred phylogenetic patterns.In BEAST, tip date calibration refers to the process of assigning dates or age estimates to the tips  of the phylogenetic tree based on known or estimated dates of those samples. BEAST used Bayesian inference to estimate the posterior distribution of parameters, including divergence times, substitution rates, and tree topology, given the input data and prior knowledge Tip date calibration has been reporting no significant influence on the reconstructed topology but has a notable effect on substitution rate and divergence time estimatesNe over time based on the number of coalescent events. It is important to note that the inferred population demography obtained from the BSP analysis primarily reflects the dynamics of the samples included in the analysis. Therefore, to focus specifically on the population dynamics of Thailand, the 20 ancient samples that originated from regions outside of Thailand were excluded from the analysis. To ensure an accurate estimation, the BSP was conducted using a substantial number of MCMC chains, totaling 40,000,000. A burn-in period of 4,000,000 generations was implemented and excluded from the analysis to allow the chains to reach a state of convergence. By reconstructing the population's demographic history through time, we can gain valuable insights into the fluctuations in Ne and better understand the underlying processes that have influenced the population dynamics in Thailand.In this study, we employed a Bayesian Skyline analysis, which utilizes a Markov Chain Monte Carlo (MCMC) approach, to estimate F4-statistics analysis was performed using the Diploid genome-wide SNP. The dataset was derived from the Affymetrix Human Origins SNP "array and merged with previously published ancient and present-day population data. Detailed information regarding the SNP data can be found in the original article13. To calculate the f4-statistics, we employed the “qpdstat” function from the R package “ADMIXTOOLS2”81 with default settings (https://uqrmaie1.github.io/admixtools/index.html). We focused on two specific population combinations for our analysis: f4 and f4. To visualize the results, we generated plots in Supplementary Figs. ggplot2 package in R.Supplementary Information.Supplementary Tables.


SPEVs) suggests their involvement on fertility via transmitting information between the original cells and recipient cells. SPEVs-coupled miRNAs have been shown to affect sperm motility, maturation, and capacitation in mammals, but rarely in poultry species. The present study aims to reveal the profile of SPEVs miRNAs and their potential effect on sperm storage and function in poultry. The SPEVs was successfully isolated from 4 different chicken breeds by ultracentrifugation and verified. Deep sequencing of SPEVs small RNA library of each breed identified 1077 miRNAs in total and 563 shared ones. The top 10 abundant miRNAs  accounted for around 60% of total SPEVs miRNA reads and are highly conserved across species, predisposing their functional significance. Target genes prediction and functional enrichment analysis indicated that the most abundantly expressed miRNAs may regulate pathways like ubiquitin-mediated proteolysis, endocytosis, mitophagy, glycosphingolipid biosynthesis, fatty acid metabolism, and fatty acid elongation. The high abundant SPEVs-coupled miRNAs were found to target 107 and 64 functionally important mRNAs in the potential recipient cells, sperm and sperm storage tubules (SST) cells, respectively. The pathways that enriched by target mRNAs revealed that the SPEVs-coupled miRNA may rule the fertility by affecting the sperm maturation and regulating the female's immune response and lipid metabolism. In summary, this study presents the distinctive repertoire of SPEVs-coupled miRNAs, and extends our understanding about their potential roles in sperm maturation, capacitation, storage, and fertility, and may help to develop new therapeutic strategies for male infertility and sperm storage.The presence of EVs in seminal plasma ( Extracellular vesicles are bowl-shaped vesicles with a bilayer structure secreted into the extracellular system of cells. Almost all cell types especially liver cells, tumor cells, red blood cells, and epithelial cells, etc. are able to produce extracellular vesicles . ExtraceSPEVs) has been recently demonstrated in chickens and actively involved in sperm viability and motility regulations  (miRNAs) are a class of endogenous ncRNAs of 20-25 nucleotides long with regulatory functions found in eukaryotes. Extracellular vesicles miRNAs have been shown to be associated with immune function and endothelial cell proliferation  . MicroRNferation . The miRferation . The expferation .The miR-210-3p plays a protective role under hypoxic stress through regulating cell survival, proliferation, inflammation, and metabolism . The hypSST) cells of uterovaginal junction during the sperm storage in females.The transportation of sperm through vas deferens and sperm storage within the female reproductive tract is important for gaining and maintaining fertility . ExtraceHowever, quite few studies have been focusing on this topic and limit the understanding of extracellular vesicle-coupled miRNA profiles of chicken seminal plasma. Therefore, in order to further study the effect of SPEVs miRNAs on sperm function and reproductive breeding in poultry, 4 breeds of chickens were first selected for SPEVs extraction and verification, followed by the construction of miRNA profiles of SPEVs, and the potential interaction of high-abundance miRNAs with sperm and SST mRNA was explored.All animal experiments were approved by the Animal Care and Use Committee at the IAS-CAAS  and conducted at the institute. All of the experiments followed relevant guidelines and regulations set by the Ministry of Agriculture and Rural Affairs of the People's Republic of China.8/mL) were identified after the estimation using the computer-aided semen analysis (CASA) system .Chicken of 4 breeds, including the dual-purpose Beijing-You chicken , Dwarf chicken , and Recessive White chicken , and an elite layer breed White Leghorn  from the experimental farm of Institute of Animal Science, Chinese Academy of Agriculture Sciences (IAS-CAAS), were used in this study. Staring from 19 wk of age, they were reared in individual cages  with controlled temperature and light. Feed and water were provided ad libitum. At 50 wk of age, each 40 roosters per breed of normal semen quality (sperm motility > 0.7 and sperm concentration > 1.0 × 10g at 4°C for 10 min. The precipitation was discarded and the supernatant was collected and stored on ice. Sperm pellet was gently resuspended with 500 µL phosphate buffer solution (PBS) with protease inhibitor and centrifuged as at 1,000× g at 4°C for 10 min. The precipitation was resuspended with PBS and centrifuged again, and this procedure was repeated 3 times. The supernatant collected by 3 centrifugations was mixed evenly and centrifuged at 20,000× g at 4°C for 15 min. The precipitation was discarded and the supernatant was collected after centrifugation at 100,000× g at 4°C for 90 min. The supernatant was discarded, and the circular white precipitate was attached to the tube wall. The precipitate was resuspended in 200 µL PBS and stored at −80°C until further manipulation.Semen for extracellular vesicles isolation were collected form the 40 chickens from each breed using the torso-abdominal massage method. The necessary care was taken to avoid any contamination with transparent fluid and other cloacal products. Semen for extracellular vesicles isolation were collected form the 40 chickens from each breed using the torso-abdominal massage method. The necessary care was taken to avoid any contamination with transparent fluid and other cloacal products. The collected semen (around 20 mL per breed) was pooled and centrifuged at 1,000× SPEVs samples were isolated from the seminal fluid, fixed in 2% paraformaldehyde, dropped onto copper grids, and incubated without movement for 5 min. SPEVs adhered to copper grids were washed with PBS. The excess liquid was absorbed from the edge of the copper grids with filter paper, phosphotungstic acid negative staining solution was added, and the grids were again incubated without movement for 5 to 10 min. The excess staining solution was absorbed, after which the grids were allowed to dry naturally. The copper grids were placed in the sample seat for further observation with a H-7500 transmission electron microscope .NTA) at VivaCell Biosceinces with ZetaView PMX 110  and the corresponding software ZetaView 8.04.02. Isolated exosome samples were appropriately diluted using 1 × PBS buffer  before the measurement. NTA measurement was recorded and analyzed at 11 positions. The ZetaView system was calibrated using 110 nm polystyrene particles. Temperature was maintained around 26°C.We measured the SPEVs particle size and concentration using nanoparticle tracking analysis (ALIX), heat shock protein 70 (HSP70), and tumor susceptibility 101 (TSG101). After reaction on ice for 30 min with 100 uL RIPA lysate  added to the sample, the supernatant was collected for detection of protein concentration using the BCA protein Detection Kit  after centrifugation at 20,000× g at 4°C for 10 min. After the adding of 5× protein loading buffer , the samples were boiled at 95°C for 10 min. After gelation, 20 µg of proteins were loaded on 15% SDS-PAGE  at 90 V for 90 min, after which the proteins were transferred to polyvinylidene fluoride (PVDF) membrane at 300 mA for 60 min. The PVDF membrane was blocked with 5% skim milk powder  for 1 h. After cleaning, the primary antibodies were incubated at 4°C overnight. The 3 primary antibodies used were ALIX , HSP70 , and TSG101 . After washing, the membranes were incubated with secondary antibody  for 1 h at room temperature. The image of protein band signals was captured using the imaging system  after ECL luminescence solution processing.Immunoblotting was performed on 3 extracellular vesicles specific marker proteins including ALG-2 interacting protein X (PAGE). Then the 3’adapters were added and the 36 to 44 nt RNAs were enriched. The 5’adapters were then ligated to the RNAs as well. The ligation products were reverse transcribed by PCR amplification and the 140 to 160 bp size PCR products were enriched to generate a cDNA library and sequenced using Illumina HiSeq 2500 by Gene Denovo Biotechnology Co. .After total RNA was extracted by the Trizol reagent kit , the RNA molecules in a size range of 18 to 30 nt were enriched by polyacrylamide gel electrophoresis (rRNA), small cytoplasmic RNA (scRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), and transfer RNA (tRNA). Meanwhile all of the clean tags were aligned with small RNAs in Rfam database (Release 11.0) to identify and remove rRNA, scRNA, snoRNA, snRNA, and tRNA. All of the clean tags were also aligned with the reference genome (Ensembl_release106). Those tags that mapped to exons or introns might be fragments from mRNA degradation, and were therefore removed. The tags mapped to repeat sequences were also removed. All of the clean tags were then searched against miRBase database (Release 22) to identify known chicken miRNAs. So far, the miRNA sequences of some species were still not included in miRBase database. For those species the miRNAs alignment with other species was a dependable way to identify the known miRNAs. All of the unannotated tags were aligned with reference genome. According to their genome positions and hairpin structures predicted by software MiReap_v0.2, the novel miRNA candidates were identified. The miRNA profile was also compared with the reported SPEVs miRNA of human, bovine and porcine  to identify and remove ribosomal RNA  and in the SST cells . The regulatory network analysis of miRNA-mRNA was performed using the Cytoscape (v3.9.1).The profile of sperm mRNA and SST mRNA of potential importance was exacted from the previous publication and database (SST RNA-sequencing data was uploaded to NCBI and is not yet available) , i.e. thGO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed on 1) the target genes of miRNAs shared by the 4 breeds selected in order to comprehensively figure out their biological functions; 2) the target genes of the miRNAs of top 10 abundance; 3) the shared genes between targets of the SPEVs miRNA of top 10 abundance and the key sperm mRNA; and 4) the shared genes between targets of the SPEVs miRNA of top 10 abundance and the key SST mRNA. Gene ontology analysis were categorized into 3 categories, biological process, cellular component and molecular function. The GO and KEGG enrichment analysis was performed using the OmicShare tools (http://omicshare.com/tools).Gene ontology (TEM) and NTA. Transmission electron microscopy showed that SPEVs had an obvious teacup structure  were shared by all breeds, and 633 miRNAs were shared by at least 2 breeds. For the known miRNA cluster, 458 (42.5%) miRNAs were present in at least 2 breeds. For the novel miRNAs, 105 miRNAs were common to at least 2 breeds. In addition, 26 miRNAs were shared by all breeds with count >10,000 . All miRFor further investigating the potential function of SPEVs miRNAs, GO and KEGG pathway analyses was carried out for mRNA targets of the 563 common miRNAs from 4 breeds, and for mRNA targets of the 26 common miRNAs with count >10,000, respectively.Salmonella infection, peroxisome, apoptosis-multiple species, cysteine and methionine metabolism, fatty acid degradation, glutathione metabolism, and apoptosis-fly  ways. Besides the function during the spermatogenesis, SPEVs may internalize their cargo in spermatozoa during the transport through the vas deferens and in female tract epithelia after the insemination to regulate their functional activity . This maThe mRNA cluster in the sperm cells may play substantial roles in fertilization, embryo development, and even intergenerational epigenetics influence . The higIn the present study, the high abundant SPEVs-coupled miRNAs were found to target 64 functionally important mRNAs in chicken SST cells. During the natural mating or artificial insemination, the semen plasma also enters the females’ oviduct. The SPEVs may regulate the gene expression of SST epithelial cells via transporting the miRNA cargos. KEGG analysis of those 64 selected target genes was mainly enriched to lipid metabolism . It is reported that the superabundance of fatty acids secreted by SST cells had detrimental effects on sperm storage in the female reproductive tract . In addiIn conclusion, chicken SPEVs-coupled miRNA was firstly identified with a total of 563 common ones among 4 chicken breeds. Some high expressed miRNAs were highly conserved in other species, suggesting their biological importance. Bioinformatics studies have shown that these miRNAs influence spermatogenesis and maturation. Taking sperm and SST cells as the potential target cells of SPEVs-coupled miRNAs, the combined analysis revealed that the miRNAs may rule the fertility by affecting the sperm maturation during the transport via the vas deferens and regulating the immune response and lipid metabolism in the females SST cells. Our findings may extend the understanding about the roles of miRNAs of SPEVs in sperm maturation, storage, and fertilization, and may help to develop new therapeutic strategies for male infertility and sperm storage in chickens.


Wheat production and end-use quality are severely threatened by drought and heat stresses. This study evaluated stress impacts on phenotypic and gluten protein characteristics of eight spring wheat genotypes  grown to maturity under controlled conditions (Biotron) using RGB imaging and size-exclusion high-performance liquid chromatography (SE-HPLC). Among the stress treatments compared, combined heat–drought stress had the most severe negative impacts on biomass , grain yield, and thousand kernel weight. Conversely, it had a positive effect on most gluten parameters evaluated by SE-HPLC and resulted in a positive correlation between spike traits and gluten strength, expressed as unextractable gluten polymer (%UPP) and large monomeric protein (%LUMP). The best performing genotypes in terms of stability were Happy, Diskett, SW1, and SW2, which should be further explored as attractive breeding material for developing climate-resistant genotypes with improved bread-making quality. RGB imaging in combination with gluten protein screening by SE-HPLC could thus be a valuable approach for identifying climate stress–tolerant wheat genotypes. Wheat provides approximately 20% of global total human dietary calories and 21% of daily protein consumption  and wheat protein quality parameters are usually negatively related , the staThe aim of this study was to determine the effects of individual and combined heat–drought stresses on phenotypic plant growth characteristics, yield, and gluten protein quality parameters of spring wheat genotypes grown in highly controlled environments. Plant growth and development under heat, drought, and combined heat–drought stresses were monitored using RGB imaging, and the gluten protein quality characteristics of wheat grain were assessed by size-exclusion high-performance liquid chromatography (SE-HPLC). The stability of yield and of gluten protein quality parameters for different spring wheat genotypes grown under stress conditions was also evaluated.22.1Eight spring wheat genotypes  developed in the breeding program at Lantmännen Lantbruk, Svalöv, Sweden, were evaluated. The selected genotypes represented a range in gluten strength, as identified when grown in the field in our previous study . Diskett2.2The spring wheat plants were grown in a randomized complete block design under three stress environments , which were applied simultaneously. The plants were grown in plastic pots  in peat-based soil, with three plants per pot. Two weeks after emergence, a plant cone (61 × 25 cm) was inserted in each pot to support the growing plants. Each pot was considered a biological replicate, and four biological replicates were used per genotype.www.smhi.se). The daylight intensity of 400 μmol m-2s-1, produced with light-emitting diode (LED) lights, was provided during the growing period. Until the start of the stress treatments, all plants were watered every 2 days with approximately 500 ml water per pot. The drought, heat, and combined heat–drought stress treatments (information below) were introduced at the beginning of heading stage (Zadoks 50) , Alnarp, Sweden  Table S12.2.2Plants assigned to the drought treatment began heading (Zadoks 50) slightly earlier than the plants in the other treatments. Drought stress was thus applied to 51-day-old plants, by stopping all watering of the plants for 5 days. After 5 days, normal watering was resumed (500 ml water/pot every 2 days).2.2.3In this treatment, the 56-day-old plants received no water for 5 days and the temperature was maintained at 29°C during day and night Table S1Wheat plants still growing 8 days after stopping the stress treatments were considered recovered plants. The digital biomass of all plants was recorded at three time points  Table 1.2.3The biomass of the wheat plants was assessed digitally from the top and side through RGB imaging in a laboratory with LED light, using two Canon EOS 1300D digital single-lens reflex cameras with an 18–55 mm kit lens . The cam2.4The digital biomass of each plant was automatically extracted with EasyLeaf software . Since a2.5The height of all three plants in each pot was measured with a ruler as the distance from the soil surface to the tip of the spike, excluding the awns. To measure the spike length (mm), the tallest spike of each plant in the pot was selected and the length of the spikes was recorded from the base of the rachis to the tip of the terminal spikelet, excluding the awns. Spike width (mm) was measured on these same spikes, at a point halfway along the spike height. The weight of fresh biomass (g), including the weight of spikes (g), per pot was recorded. Thousand kernel weight (TKW) (g) was calculated as described by 2.6SE-HPLC was used to evaluate the gluten protein characteristics of the harvested grain. Seeds from the different genotypes grown at different stresses and control samples were milled into flour using a homogenizer  for 30 s at 30 Hz. The flours were freeze-dried  for 24 h in order to remove all moisture prior to SE-HPLC analysis. A two-step gluten protein extraction method was performed according to 2.7post hoc test (p< 0.05), to assess the effect of different treatments on the gluten protein parameters and phenotypic traits. Spearman correlation analysis (p< 0.05) (R package Corrplot) was applied for all gluten protein parameters and phenotypic traits in plants in each treatment. Genotype main effect plus genotype by environment interaction (GGE) biplots analysis (R package Metan) was performed to evaluate the stability of the studied genotypes in the different growing environments. The selected GGE tools were “mean vs. stability,” “which-won-where view of the GGE biplots,” and “ranking genotypes.” These tools facilitate the identification of the optimal genotypes based on performance and stability, identify the optimal genotype for each growing environment, and rank them according to suitability for a growing environment.Statistical analyses were performed using the software R . Princip33.1The digital wheat biomass assessed by RGB imaging at time points Tp1, Tp2, and Tp3 differed between the genotypes for the different stress conditions tested Figure 1Mean digital biomass measured at flowering (anthesis) was similar for all genotypes at Tp1 and decreased for all genotypes at both Tp2 and Tp3. The greatest impact of the stress treatments was observed in the drought and combined heat–drought treatments, followed by heat Figure 2Under control conditions, rather similar digital biomasses at Tp1–Tp3 were observed for all genotypes except SW1 and SW3 (3.2The impact of genotype (G) and stress environment (E) on the protein parameters was significantly dominated by the individual effect and not by G and E interaction (G × E) S3 in SIpost hoc test indicated a major impact of the combined heat–drought stress conditions on the gluten protein parameters, with significant effects on TOTE, TOTU, TPP, TMP, and %UPP in comparison with the other environmental conditions  explained 65.6% of the variation  Figure 4Yield, phenotypic characteristics, and gluten protein parameters explained 52.8% of the variation in PCA  Figure 5The Spearman’s rank correlation results indicated a significant impact of the treatments on certain gluten protein parameters, yield, and phenotypic traits Figure 6In all stress treatments, grain yield was significantly positively correlated with biomass (p< 0.001), while significant positive correlations were also found between grain yield and digital biomass under heat (p< 0.01) and under combined heat–drought stress (p< 0.05)  and most of the protein parameters studied   and %UPP  was observed  (Comparisons of the impact of the stress treatments on selected yield traits (grain yield and TKW) and gluten parameters  for individual genotypes revealed some variation (non-significant) between the genotypes Figure 7In terms of gluten protein characteristics, a clear increase in both %UPP and LPPs (gluten strength) was noted for SW2, which had the highest %UPP (52.5%) of all genotypes studied  plot for ‘mean vs. stability’ revealed the most promising genotypes  in the eight wheat genotypes studied. The drought treatment also significantly reduced the digital biomass, whereas the heat stress treatment had a relatively mild impact 2. A simower TKW . The magower TKW .In this study, wheat plants were exposed to the different 5-day stress treatments during anthesis, which is known to be one of the most critical growth stages, explaining the strong impact of the treatments involving drought. Applying heat stress alone had a mild impact on plant development characteristics and the biomass of individual genotypes; thus, it was not possible to evaluate plant response mechanisms to this stress. The chosen heat stress temperature (29°C) was based on findings in previous studies that a temperature of 27°C–30°C or higher prior to and during anthesis can substantially reduce grain size, numbers, and yield .As expected, drought and combined heat–drought stress decreased biomass accumulation Figure 3The digital tools used in this study to measure digital biomass showed good ability to evaluate wheat plants under severe drought and combined heat–drought stress conditions. The correlations observed between digital and measured biomass, e.g., under drought conditions (at Tp3) and combined heat–drought conditions (at Tp2), revealed the strong potential of RGB imaging to identify even small differences induced by stresses and to detect symptoms of the genotypes under the different environments. Under heat stress, the genotypes SW1 and SW3 were least affected (In the control (no stress) growing environment, most gluten parameters  showed a negative correlation with grain yield, TKW, biomass, plant height, and spike width . TKW andThis study examined the impact of stress factors on the most important gluten quality parameters, e.g., gluten strength, based on the concentrations of polymeric proteins. Under individual drought and heat stress, a negative correlation between %UPP and digital biomass (Tp2–Tp3) was observed. There was also a clear impact of stress on the %UPP and %LUPP fractions Table 3,Spike length is a strong indicator of yield  and is dA positive effect of abiotic stress on gluten protein polymerization has also been observed in previous studies performed in the field  and greeThe results obtained with the imaging tools employed to measure digital biomass at different stress time points  in this study were significantly and positively correlated with grain yield and with actual measured biomass, suggesting that RGB imaging could be a useful method for evaluating the impacts of plant stresses on phenotypic characteristics, such as grain yield.No significant effect was found for the interaction between the genotype and the environment (G × E) on gluten protein parameters (except for Mon/pol), contradicting findings in previous studies in the greenhouse  and in tAmong the eight genotypes compared, Happy was the most promising in terms of the stability of grain yield, TKW, %UPP, and TOTE in stressful growing environments. Happy also showed greater digital biomass in the combined heat–drought stress treatment. The breeding line SW2 showed not only lower digital biomass and yield under stress but also the highest stability and mean gluten strength (%UPP) in the control and in all three stress treatments, supporting previous findings . Genotyp5Assessing and controlling important phenotypic and grain quality–related traits in wheat is important for success in breeding programs seeking to produce desirable wheat material for use under future climate change. This study revealed significant impacts of combined heat–drought stress on plant phenotypic characteristics and gluten protein quality traits in eight spring wheat genotypes, while drought stress alone also had negative impacts. However, heat stress (29°C) had only mild effects on yield and phenotypic characteristics, although, in field conditions, the impact of heat stress could be much more severe.There were significant positive correlations between grain yield and digital biomass, and between digital biomass and actual measured biomass, in all stress treatments tested, indicating that RGB imaging can be a valuable tool in assessing stress in wheat plants.Individual drought and heat stresses significantly affected gluten strength (%UPP and %LUPP). There was a negative correlation between digital biomass and most gluten protein parameters analyzed, although the Mon/pol ratio was not affected by the experimental stresses studied.A surprising finding was that the number of spikes was significantly positively correlated with both %UPP and %LUMP under combined heat–drought stress, suggesting a correlation with not only polymeric glutenins (HMW and LMW) but also large monomeric proteins . The number of spikes is an indicator of yield and, together with gluten protein quality traits, could potentially be explored in screening for high yield and gluten protein quality in wheat under climate stress.The most promising genotypes in terms of performance and stability in the stress environments tested were SW3 and Happy for high yield, SW1 and Happy for high TKW, SW2 and Bumble for high %UPP, and Diskett and SW1 for high protein concentration (TOTE). In order to meet plant breeding targets for extreme climate resistance, these top-performing genotypes need to be further tested in field studies where their phenotypic and gluten protein characteristics are evaluated using the combination of tools tested in this study.The original contributions presented in the study are included in the article/RK and AC conceived the study. RK, AC, SL, and FL planned the greenhouse experiments. PV developed the breeding population set. SL and FL performed the greenhouse experiments, analyzed the data, and wrote the manuscript with inputs from RK and AC. RK acquired the funding. All authors contributed to data interpretation and approved the final version of the manuscript.