Staphylococcus xylosus and Lactobacillus sakei ssp. carnosus were investigated. The changes in aroma-active compounds were determined by application of aroma extract dilution analysis in combination with gas chromatography-mass spectrometry/olfactometry for identification, and by stable isotope dilution assays for quantification. A total of 30 aroma-active compounds for non-fermented and fermented samples were identified. The aroma profile of LPI fermented with Lactobacillus sakei ssp. carnosus was characterized as roasty and popcorn-like. Staphylococcus xylosus generated cheesy impressions, being in line with the fact that the main aroma compounds acetic acid, butanoic acid, and 2/3-methylbutanoic acid could be identified. Quantification of butanoic acid further confirmed these findings with the highest concentration of 140 mg/kg for LPI fermented with Staphylococcus xylosus. Our study provides insights into how fermentation utilizing different fermentative microbial strains, namely Staphylococcus xylosus and Lactobacillus sakei ssp. carnosus alters the aroma profile of lupin protein isolates. This demonstrates the potential of shaping fermented protein-based foods via targeted microbiological refinement.Aroma-active compounds of lupin protein isolate and lupin protein isolate fermented with  In recent years, an increasing trend of plant-based food products can be observed in the global market. More and more people are avoiding animal products in their diet for ethical, ecological, health and animal welfare reasons. This also increases the demand for plant-based raw materials, especially proteins, in order to continue to supply the body with sufficient amounts of the essential nutrient. Legumes, such as lupins, are promising alternatives to animal protein due to their high protein content of 39% up to 55%  and a baThe use of legumes in some food is still limited due to their functional properties, such as low solubility in the acidic range. In addition, legume proteins often have a characteristic overall aroma, which is described with green, grassy and beany aroma impressions ,5,6,7. IFermentation is a promising approach for modifying the aroma impressions of legumes considerably. For example, lactic acid fermentation has already been shown to decrease the green smelling n-hexanal content of pea protein extracts . Other sStaphylococcus xylosus and Lactobacillus sakei ssp. carnosus, respectively, to investigate the effect of fermentation on the functional and sensory properties of lupin protein isolates [Staphylococcus xylosus and Lactobacillus sakei ssp. carnosus. In particular, a sweaty/cheesy and a roasty/popcorn-like impression clearly emerged. Since the changes in the sensory properties of the other microorganisms used in the study were less pronounced, it was obvious to take a closer look at the aroma profile of the lupin protein isolate fermented with Staphylococcus xylosus and Lactobacillus sakei ssp. carnosus. Commonly, both microorganisms are commonly used as starter cultures in the fermentation of meat products [In a previous study, lupin protein isolates were fermented with isolates . Schlegeisolates  observedproducts ,16,17,18Staphylococcus xylosus and Lactobacillus sakei ssp. carnosus. Therefore, an aroma extract dilution analysis in combination with gas chromatography-mass spectrometry/olfactometry was carried out for the identification of the flavor compounds. Stable isotope dilution assays were used for their quantification.The aim of this study was to investigate the changes in the main aroma compounds of lupin protein isolates after their fermentation with Staphylococcus xylosus  and Lactobacillus sakei ssp. carnosus  for 24 h were prepared as previously described by Schlegel, Leidigkeit, Eisner and Schweiggert-Weisz [S. xylosus and L. sakei ssp. carnosus contained of 80.1% and 80.6%, respectively [Lupin protein isolate (LPI) preparation, and fermentation of the isolate with rt-Weisz . The proectively .−4 mbar, with a water bath temperature of 50 °C and an apparatus temperature of 55 °C. The obtained distillate was dried over anhydrous sodium sulfate, filtered and concentrated to ~3 mL at 50 °C by means of Vigreux distillation (50 cm × 1 cm i.d.), and finally to a total volume of ~100 µL by microdistillation according to Bemelmans [A total 20 g (±0.3 g) of each sample were extracted using dichloromethane  by stirring vigorously for 30 min at room temperature in a closed vessel. After filtration, volatiles were separated from the non-volatiles using the solvent-assisted flavor evaporation (SAFE) technique according to Engel, et al. . For a gThe sample distillates were analyzed by GC-O using a Trace Ultra gas chromatograph  using a DB-FFAP  capillary column. Aliquots (2 µL) of the distillates were manually injected by the cold on-column technique at 40 °C. After 2 min, the temperature was raised at 8 °C/min to 240 °C and held for 5 min. The flow rate of the helium carrier gas was 2 mL/min. At the end of the capillary column, the effluent was split in a 1:1 ratio into an odor detection port (ODP) and a flame ionization detector (FID). The flame ionization detector and the ODP were held at 300 °C and 250 °C, respectively. Linear retention indices (RIs) of the aroma-active compounds were calculated using a homologous series of n-alkanes  as described by Van Den Dool and Kratz . To avoiv/v). GC-O analyses were performed for the undiluted distillates (FD = 1), and all subsequent dilutions. The respective FD factor assigned correlated to the highest dilution in which the compound that was still perceivable at the ODP.To enable a comparison between the fermented and non-fermented isolates, the same amounts were extracted, subjected to SAFE distillation, concentrated to the same final volume, and, finally, the same volume was used for gas chromatography–olfactometry (GC-O). The flavor dilution (FD) factor of each aroma compound obtained from the distillates of LPI and fermented LPI were determined using AEDA by diluting the distillate stepwise with DCM  and a Trace DSQ mass spectrometer  using a DB-FFAP  capillary column. Aliquots of 2 µL were injected via the cold on-column technique at 40 °C using a multipurpose autosampler MPS 2 . After 2 min, the temperature was raised at 8 °C/min to 240 °C, and held for 5 min. The flow rate of the helium carrier gas was 2 mL/min. At the end of the capillary column, the effluent was split into an ODP and a mass spectrometer using two deactivated fused silica capillaries . Mass spectra in positive electron impact mode were generated at 70 eV ionization energy with a m/z range was 35–399.The 2D-GC-MS/O system consisted of two gas chromatography systems (Agilent Technologies Germany GmbH & Co. KG) housing capillary columns with different polarities  connected via a CTS 1 cryotrap system (Gerstel GmbH & Co. KG). A total 2 µL were injected automatically using the cold on-column technique at 40 °C by a Multipurpose Sampler MPS 2XL (Gerstel GmbH & Co. KG). At the end of the capillary column of the first GC system the effluent was split 1:1 by volume and led to an ODP (290 °C) and FID (240 °C). After 2 min, the temperature was raised at 8 °C/min to 230 °C, and held at the final temperature for 5 min in the first GC oven. The helium carrier gas flow was 8 mL/min. Afterwards, the aroma-active areas of interest were transferred to the cryogenic trap (−100 °C). After thermodesorption in the second GC system at 250 °C, the volatiles were transferred to the second column at a starting temperature of 40 °C. The temperature was raised at 8 °C/min to a final temperature of 250 °C and held for 1 min. At the end of the capillary of the second GC system, the effluent was split and passed in equal parts to both an ODP and a MS. Mass spectra were recorded in positive electron impact ionization mode at 70 eV ionization energy. The 2H)-furanone and 3-(methylthio)propanal in LPI and fermented LPI was carried out using stable isotope dilution assays. Different amounts of sample material  were used for quantitation of the aroma-active compounds. The isotopically labeled internal standards were dissolved and stored in dichloromethane.The quantification of the selected aroma compounds acetic acid, butanoic acid, 2-acetyl-1-pyrroline, 4-hydroxy-2,5-dimethyl-3 were stirred (300 rpm) for 30 min at room temperature in a closed vessel. Further workup was performed as described above for the isolation of the volatiles. The quantitation experiments were performed in duplicates. The quantitation was performed via gas chromatography−mass spectrometry (GC-MS) or two-dimensional high-resolution gas chromatography−mass spectrometry (2D-GC-MS) as described above.Determination of response factors: for each odorant, a response factor was calculated by analyzing binary mixtures of defined amounts of the unlabeled analyte and the labeled standard in five different mass ratios  under the same chromatography conditions used for the samples.For the calculation of odor activity values (OAVs), orthonasal odor thresholds (OTs) of selected aroma compounds were taken from Czerny, et al. .First, the volatiles were extracted from the protein isolates by DCM followed by high vacuum distillation using the SAFE technique. The distillates obtained of each kind of protein isolate exhibited the typical characteristic overall aroma, when a drop of the distillate was put on a strip of filter paper, proving the successful extraction of all key aroma compounds. Next, the distillates were subjected to AEDA as a screening method for differentiation between aroma-active VOC and the bulk of odorless VOC. Application of AEDA by GC-O revealed in total 30 aroma-active regions for all samples in an FD factor range between 1 and ≥6561 . The GC-S. xylosus and L. sakei ssp. carnosus resulted in the detection of 30 aroma-active regions in each of the respective undiluted distillates (FD 1). Three aroma-active regions  in LPI fermented with S. xylosus were detected within the highest FD factor of ≥6561. For LPI fermented with L. sakei ssp. carnosus, five regions  were perceived within the highest FD factor of 729. For unequivocal identification of the most potent aroma-active compounds, the respective odor quality and intensity perceived at the odor detection port, the retention indices on two capillary columns of different polarities, and mass spectra in EI mode recorded by GC-MS/O and 2D-GC-MS/O (if trace odorants coeluted with other volatiles present in higher amounts) were compared with the data of reference compounds.Analysis of the distillates obtained from the LPI fermented with E)-non-2-enal (no. 10), (Z)-non-2-enal (no. 12), -nona-2,6-dienal (no. 14) and -nona-2,4-dienal (no. 17), whereas a metallic note was elicited by trans-4,5-epoxy-(E)-dec-2-enal (no. 15). The grassy, green and soapy smelling regions derived from hexanal (no. 1) and nonanal (no. 5). In addition, coconut-like and peach-like aroma-active regions derived from lactones such as γ-nonalactone (no. 20), δ-nonalactone (no. 23), γ–decalactone (no. 24), δ–decalactone (no. 26) and γ–dodecalactone (no. 28). Pea-like, green bell pepper-like aroma-active regions belonged to the class of pyrazines, such as 3-isopropyl-2-methoxypyrazine (no. 7), 3-sec-butyl-2-methoxypyrazine (no. 9) and 3-isobutyl-2-methoxypyrazine (no. 11). Cheesy and vinegar-like aroma impressions were identified as methylpropanoic acid (no. 13), butanoic acid (no. 15), 2-/3-methylbutanoic acid (no. 16 a/b), and acetic acid (no. 6). Apart from that, mushroom-like and geranium-like impressions were traced back to 1-octen-3-one (no. 2) and (Z)-1,5-octadien-3-one (no. 4). Moreover, 2-acetyl-1-pyrroline , 3-(methylthio)propanal , 3-hydroxy-2-methyl-pyran-4-one , octanoic acid , and 4-hydroxy-2,5-dimethyl-3(2H)-furanone  were identified. In addition, nonanoic acid , 3-hydroxy-4,5-dimethylfuran-2(5H)-one , 4-hydroxy-3-methoxy-benzaldehyde , and phenylacetic acid  were successfully identified in the lupin protein isolates.As a result, in all samples fatty smelling aroma-active VOC were identified as the aldehydes , 14.0 µg/kg (S. xylosus) and 14.0 µg/kg (L. sakei ssp. carnosus) and 3-(methylthio)propanal with 2.8 µg/kg (S. xylosus), 2.7 µg/kg (LPI), and 2.4 µg/kg (L. sakei ssp. carnosus). The 4-Hydroxy-2,5-dimethyl-3(2H)-furanone was quantified in the fermented samples S. xylosus and L. sakei ssp. carnosus with amounts of 45.0 µg/kg and 50.0 µg/kg, respectively, but were found to be below the limit of quantification (LOQ) for non-fermented LPI.Quantification revealed the highest concentration for acetic acid in all samples with the highest amount of 2500.0 mg/kg for  xylosus . For LPIOAVs are defined as the ratio of the concentration of an aroma substance in a sample to its odor threshold and are indicative of the relative importance of an individual aroma substance to the overall aroma. OAVs were calculated for the five quantified aroma compounds  using odS. xylosus and L. sakei ssp. carnosus fermented samples. Further, butanoic acid was found with an OAV of 18 for the sample fermented with S. xylosus, and was, therefore, potentially of higher sensory impact than the samples fermented with L. sakei ssp. carnosus (OAV 2) and LPI (OAV < 1). In addition, for acetic acid, an OAV > 1 was only achieved for S. xylosus (OAV 14), whereas an OAV of 2 was calculated for the remaining samples. The 4-Hydroxy-2,5-dimethyl-3(2H)-furanone did not exceed an OAV > 1 in any of the investigated samples.The highest OAV was calculated for 2-acetyl-1-pyrroline for all samples with values of 124 for LPI and 113 for S. xylosus and L. sakei ssp. carnosus as previously reported by Schlegel, Leidigkeit, Eisner and Schweiggert-Weisz [S. xylosus exhibited an intense cheesy aroma impression followed by a moderate intensity of oatmeal-like/fatty impressions. LPI fermented with L. sakei ssp. carnosus was described with popcorn-like/roasty as main aroma characteristic, followed by oatmeal-like/fatty with a moderate intensity.The comparison of the AEDA results obtained in this study with the results from the comparative aroma profile analyses of non-fermented LPI and LPI fermented with rt-Weisz  are in vrt-Weisz  by the rE)-non-2-enal , (Z)-non-2-enal  and -nona-2,4-dienal  in LPI are likely to be responsible for the fatty aroma impression obtained during aroma profiling. Similar impressions and components were also detected in the samples fermented with S. xylosus and L. sakei ssp. carnosus. The aroma impression cooked potato-like and roasty/popcorn-like can be attributed to the presence of the components 3-(methylthio)propanal  and 2-acetyl-1-pyrroline , respectively.The aroma attributes during aroma profiling pea-like/green bell pepper-like for LPI could be related to the presence of 3-isopropyl-2-methoxypyrazine , 3-sec-butyl-2-methoxypyrazine , and 3-isobutyl-2-methoxypyrazine . The identified components , butanoic acid  and 2-/3-methylbutanoic acid . The high FD factor of >6561 for butanoic acid and of 729 for 2-/3-methylbutanoic acid provide further evidence for a high relevance of the two components for the overall aroma profile of fermented LPI with S. xylosus, corresponding with the pronounced intensity of the sweaty-cheesy aroma impression in the sensory evaluation. The main aroma impression roasty/popcorn-like of LPI fermented with L. sakei ssp. carnosus in sensory analysis can likewise come from 2-acetyl-1-pyrroline .For all the mentioned aroma-active VOC for LPI, an FD factor of 9 was determined. Although the FD factor of the individual components was not particularly high, roasty/popcorn-like and cooked potato-like impressions seem to have a high relevance for the overall aroma profile of LPI. Possibly, the presence of odorants with similar aroma impressions added up to yield the intensity of the overall sensory impression ,24. AddiS. xylosus) and roasty/popcorn-like (L. sakei ssp. carnosus), respectively, corresponding with the observation that the AEDA showed higher FD factors for these odorants in the fermented samples compared to non-fermented LPI.In summary, the sensory data strongly corresponded with the odorants identified here, as well as the AEDA results in so far as the fermented samples showed stronger sensory impressions of sweaty/cheesy  with an FD 9 for LPI was determined with a higher FD in the sample fermented with S. xylosus (FD 729) and with L. sakei ssp. carnosus (FD 27), which was supported by the quantitative data. LPI had a concentration of 13.0 mg/kg, compared to 83.0 mg/kg for fermented LPI with L. sakei ssp. carnosus and a concentration of 2500.0 mg/kg acetic acid for the one fermented with S. xylosus. Correspondingly, with an OAV of 14 in samples fermented with S. xylosus, this compound was more pronounced in the same sample than in samples fermented with L. sakei ssp. carnosus and non-fermented LPI (both OAV < 1). Moreover, 2-and 3-methylbutanoic acid (no. 16 a/b) were characterized with an FD factor of 729 in the fermented LPI, being higher than in non-fermented LPI with an FD of 9. Butanoic acid (no. 15) had an FD factor of ≥6561 in LPI fermented with S. xylosus, being likewise considerably higher than in the sample fermented with L. sakei ssp. carnosus (FD 243). Butanoic acid and methylpropanoic acid (no. 13) were not detectable in the undiluted distillate of non-fermented LPI using GC-O. Quantitative analyses of butanoic acid confirmed the higher FD factor for LPI fermented with S. xylosus compared to L. sakei ssp. carnosus with concentrations of 140.0 mg/kg to 15.0 mg/kg, respectively, whereas for LPI, a concentration of 570.0 µg/kg was determined. In addition, results of the OAV calculation showed values of 18 for samples fermented with S. xylosus, and 2 for L. sakei ssp. carnosus. In this case, the OAV for LPI was <1. The higher concentration of butanoic acid in the S. xylosus fermented sample was also consistent with sensory profiling, where the aroma impression cheesy was the main impression of the S. xylosus fermented sample. Nevertheless, LPI fermented with L. sakei ssp. carnosus also contained butanoic acid and 2/3-methylbutanoic acid (same FD as S. xylosus). However, these components do not appear to be of major relevance to the overall aroma profile of the L. sakei ssp. carnosus fermented sample. In addition, higher FD levels of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (no. 22) were determined in LPI fermented with S. xylosus (FD 81) and L. sakei ssp. carnosus (FD 729), than in LPI (FD 3). In this respect, the results of the quantitative analysis were in good agreement and showed concentrations of 50.0 µg/kg for the L. sakei ssp. carnosus fermented sample and 45.0 µg/kg for the S. xylosus fermented sample, whereas levels < LOQ (19.7 µg/kg) were detected in non-fermented LPI. Accordingly, the calculated OAV was for all samples <1 and therefore most likely not of relevance for the overall aroma, although 4-hydroxy-2,5-dimethyl-3(2H)-furanone was detected during AEDA. This is due to the fact, that in AEDA the influence of the matrix on the aroma release is not effective.When comparing the identified components and respective FD factors of LPI and LPI fermented with L. sakei ssp. carnosus showed roasty/popcorn-like as the main aroma impression, only differences in one FD range for 2-acetyl-1-pyrroline (no. 3) was detected between LPI fermented with L. sakei ssp. carnosus (FD 9), fermented with S. xylosus (FD 3) and non-fermented LPI (FD 3). This was also confirmed by the quantitative analysis of 2-acetyl-1-pyrroline by SIDA, where LPI fermented with L. sakei ssp. carnosus and S. xylosus showed a concentration of 14.0 µg/kg each. The highest concentration of 2-acetyl-1-pyrroline was 15.0 µg/kg for LPI. The odor threshold for 2-acetyl-1-pyrroline is 0.12 µg/L [S. xylosus and L. sakei ssp. carnosus, an overall contribution in all samples. Thereby, it can be assumed that the roasty/popcorn-like impression was more prominent in the fermented sample with L. sakei ssp. carnosus may be due to additive or suppressive effects of other matrix ingredients. Likewise, the aroma profile of LPI was described with a moderate intensity of a cooked potato-like note but only weak intensities of this aroma impression for fermented samples with S. xylosus and L. sakei ssp. carnosus. However, comparison of the FD factors and concentrations of 3-(methylthio)propanal (no. 8) revealed, only minor differences between all samples. The odor threshold for 3-(methylthio)propanal is 1.4 µg/L [While the results of the sensory analysis for LPI fermented with .12 µg/L , resulti1.4 µg/L , and theS. xylosus and L. sakei ssp. carnosus are widely used as starter cultures in meat and sausage fermentation. Their metabolic pathways and products as well as the formation of aroma-active VOC have already been described in numerous studies. Acetic acid is a typical aroma compound of dry fermented sausages [S. xylosus und L. sakei ssp. carnosus and sausage products [S. xylosus and L. sakei ssp. carnosus [Lupinus angustifolius L. cultivar Boregine with a content of about 7% and isoleucine and valine with contents of about 4% and 3%, respectively [δ- and γ-nonalactone, -decalactone and dodecalactone are important aroma compounds in some fermented fruit, dairy, and meat products [δ- and γ-hydroxy acids or triglycerides [γ-nonalactone, γ-decalactone, and γ-dodecalactone in fermented LPI with S. xylosus is due to an intensified formation of free fatty acids during fermentation. However, such postulated formation pathways would require more detailed investigation of the formation/degradation of free fatty acids. Finally, 2-acetyl-l-pyrroline and 4-hydroxy-2,5-dimethyl-3(2H)-furanone have been described as thermally induced volatiles from proline [sausages ,25,26,27sausages ,26. Anotsausages . Butanoiproducts ,13,25. Icarnosus ,25,29,30carnosus ,16,31,32products . They caproducts , or theyycerides . Possibl proline ,36 and s proline , respect proline  and were proline ,36,37. TS. xylosus and L. sakei ssp. carnosus affects the aroma profile of lupin protein isolate. Identification of aroma-active VOC by GC-O in fermented lupin protein isolate showed similarities to aroma compounds formed during fermentation of meat and sausage products . Nevertheless, the aroma profile was characterized as cheesy after fermentation with S. xylosus and as roasty, popcorn-like with L. sakei ssp. carnosus. Lupin protein isolate fermented with S. xylosus could therefore be a promising approach for the production of plant-based cheese alternatives without the addition of flavorings. L. sakei ssp. carnosus could be suitable for its use in, e.g., meat alternatives or baked goods due to its roasty aroma profile.Fermentation with This study shows that fermentation could be a promising approach for the development of protein ingredients with tailor-made sensory properties. As the effect of fermentation strongly depends on the starter culture and the raw material used, it is worth investigating even more microorganisms and fermenting even more raw materials. This could expand the range of plant proteins and thus serve the ever-growing market for protein ingredients with new fermented products. These ingredients can then be used for the development of alternatives to animal products without the addition of a long ingredients list.


Picea abies) belonging to three elevational classes; namely, low- (acuminata), medium- (europaea), and high-elevation (obovata) form, each represented by 150 trees. After rigorous filtering, we used 1916 Genotyping-by-Sequencing generated SNPs for analysis. Outputs from three multivariate analysis methods  indicated the presence of a distinct genetic cluster representing the high-elevation ecotypic group. Our findings bring a vital message to forestry practice affirming that artificial transfer of forest reproductive material, especially for stands under harsh climate conditions, should be considered with caution.We investigated the genetic structure of three phenotypically distinct ecotypic groups of Norway spruce ( Picea abies (L.) Karst, has been the subject of numerous genetic diversity studies utilizing both biochemical and molecular markers8. Overall, most studies were conducted on the population level and aimed to identify the species extent of genetic differentiation12.As a species of substantial economic and ecological importance, Norway spruce 3, through more informative microsatellite DNA markers16, to those relying on next-generation sequencing methods which made it possible to address more specific questions such as the impact of evolutionary factors on the genetic makeup of the species22. Historically, isoenzyme markers showed a low level of inter-population diversity24. Microsatellite markers are frequently used for population-level studies. However, due to their neutral nature, they are not affected by evolutionary forces such as selection and adaptation processes26.As genetic markers evolved, the answers on examined hypotheses improved in their precision, starting with allozyme27, wood formation28, disease resistance29, morphological characteristics30 or local adaptations31.At present, the availability of genomic-based genotyping platforms offers vast opportunities to address specific Norway spruce research questions, such as identifying candidate genes associated with drought response32 such as cold hardiness and bud set33, growth cessation34, or latitudinal gradient for red/far-red light ratio in connection to local adaptation for shade tolerance35. Also, a provenance effect on grown parameters and phenology was shown36. Only a few studies have paid attention to epigenetic factors in Norway spruce38 even that they may play an essential role in adaptative responses40.Despite extensive gene flow and limited genetic differentiation among populations detected with neutral markers, Norway spruce populations possess strong local adaptation in phenological and climate-related traits along latitudinal gradients8. A more recent study showed that the current distribution of Norway spruce genetic diversity is more admixtured due to repeated evolutionary events such as ancient splits, hybridization, and bottlenecks20. Norway spruce is a wind-pollinated species characterized by the high gene flow41. Along with the species outcrossing nature, other factors enhance gene flow level; namely, artificial spreading of spruce outside the natural grown area42 and man-influenced forest reproductive material transfer establishing non-autochthonous forests from non-local gene sources43.The demographic history of Norway spruce has been widely studied, suggested current distribution originated from two main refugia44. Despite a relatively continuous variance in crown parameters within stands, three prevalent morphotypes-ecotypic groups, are distinguished46 along an altitudinal gradient47. The low‐elevation ecotype, acuminata, is characterized by broad crowns, toothed cone scales, and comb‐like branches. In central Europe, this form occurs predominantly up to 500 m.a.s.l. In contrast, the high-elevation ecotype, obovata, typically has a narrow crown, round cone scales, and flat branches (500–1000 m.a.s.l) and the medium‐elevation ecotype, europaea, with rhombic toothed cone scales and a mostly conical crown with medium width and brush-type branches that are usually shorter, denser, and down hanging (1000 m.a.s.l up to tree line). It is essential to emphasize that these forms are not exclusively limited to one elevation or latitudinal zone, although any given Norway spruce ecotypic form is usually prevalent within particular stands44. Crown shape has been studied with association to altitude30; however, with the exception for our previous study utilizing microsatellite markers16, there is a paucity of information addressing the underlying genetic causes for the observed architectural differences among these various ecotypes.Norway spruce possess noticeable variability in crown morphology, especially across substantial altitudinal gradients48 present in conifers49. By choosing appropriate REs, the repetitive regions of genomes can be avoided, and lower copy regions are targeted with higher efficiency50.Here, we aimed to elucidate the level of genetic differentiation among three phenotypically identified Norway spruce ecotypes determined by their crown morphology and the altitude of their stands using Genotyping-by-Sequencing (GBS) generated SNP data. GBS utilizes restriction enzyme digestion (RE) to focus on sequences in low-copy genomic regions while minimizing reads in the abundant repetitive sequences51), a method that effectively detects even weak, multiloci molecular signatures to identify SNPs with genotype-environment association signals.Using rigorously filtered data, we applied three widely used multivariate methods for genetic structure analysis. Results provided consistent outputs identifying a group of high-elevation individuals as a genetically distinct cluster. Additionally, we utilized the Redundancy Analysis  within the Czech Republic. Per each ecotype, 150 trees were sampled (N = 450). Stands representing the high- (HE), medium- (ME), and low-elevation (LE) ecotypic forms are located in the Giant Mountains National Park, the Jizera Mountains protected area, and the Bohemian Switzerland National Park, respectively . HE individuals were sampled across two subplots . We targeted mature trees (DBH ≥ 25 cm) of the characteristic crown morphology to their respective ecotypic form  according to the manufacturer's instructions. DNA concentration was measured using a NanoDropTM 2000 spectrophotometer . Both intact and HindIII  digested DNA were run on 0.8% agarose gel to assure DNA quality.Undiluted aliquots of 40 µl DNA  were placed into 96-well PCR plates (95 samples + one negative control) and shipped under dry ice for analysis.From each sample, approximately 50 mg of cambial and adjacent layers of wood tissue were scratched with a scalpel and frozen in liquid nitrogen, homogenized using a mixer mill MM400  for 3 min at 30 Hz. The genomic DNA was extracted by the DNeasy56. Reads occurring at a minimal count of three were stored in a FASTQ file format.DNA samples were digested with the 6-base cutter restriction enzyme EcoT22I. Library and sequencing preparation were done in 96-plex, using 64 bp tag sequences. Sequencing ran on NextSeq 2000  platform at the Cornell University sequencing facilities  according to GBS protocolPicea abies reference genome v1.0., ftp://plantgenie.org/Data/ConGenIE/Picea_abies/v1.0/58 with Burrows-Wheeler Aligner . Aligned sequence tags were stored in SAM format file and SNP calling was performed using TASSEL-GBS Pipeline (version 3.0.174)61.FASTQ file was aligned against the 62.Low-quality SNPs were filtered out by minimum minor allele frequency (MAF) > 0.01 and missing data < 90% and the data were finally converted into Variant Call Format (VCF) file62 as follow: (1) only biallelic and non-indel SNPs were kept, (2) only loci with no-missing data were kept, (3) MAF was set > 0.05, (4) the Quality value threshold for loci was set ≥ 20, (5) only loci with mean depth values between 10 and 60 were retained, and (6) SNPs within 1000 bp distance were thinned.VCF file (raw data) was subjected to a more stringent filtering process before analysis. All filtration steps were performed by VCFtools software (version 0.1.16)http://www.worldclim.org/bioclim) in 0.5 arc-minute resolution (approx. 1 × 1 km). We utilized getData function call from the raster package and assigned each tree stand with given climatic variables. After removing highly correlated variables, seven out of the original 19 climatic variables, were retained for further analysis .Primary data analysis was run with VCF tools. For Principal Component Analysis (PCA), as well as Discriminant Analysis of Principal Components (DAPC), we utilized functions implemented in R package adegenet64, the input file was generated with PGDSpider65 by VCF file conversion. To detect the most likely number of clusters, we generated K from 1 to 10  using Evanno's approach66.Bayesian clustering was done in STRUCTURE v2.3.4.GST67, Jost's D68 and Hedrick's GST69 as measures of genetic differentiation among populations. We created genind objects for each pairwise comparison with the R package adegenet63 and performed 1000 bootstrap samples, with each subpopulation resampled according to its size, with the function chao_bootstrap of the mmod R package70. Then, for each set of the permutated datasets, we obtained the observed genetic distance value and its normalized 95% confidence intervals (CI)  with the function summarise_bootstrap in mmod package. We considered the genetic differentiation index to be statistically significant if their lower bound of the CI was greater than zero.We used Nei's provesti.dist from poppr package71 and geographical and altitudinal distance matrices. The test uses Pearson's regression coefficient between geographic and genetic distance matrices. The statistics and the test's p value correspond to the proportion of times the randomized regression coefficient is equal or greater than the observed one72.We employed the Mantel test from the ade4 package with 100,000 randomizations to estimate the correlation between the genetic distance matrix  with 1000 permutations. Then, we identified candidate loci based on locus scores  that were ± 3 SD from the mean loading on the first three constrained ordination axes. We recognized the environmental variables exhibiting the strongest associations with each candidate adaptive locus using Pearson's correlation coefficient (r). We utilized a permutation test with 100,000 randomizations to estimate p value of such correlation and we adjusted α-level by Bonferroni correction (20 candidate SNPs and 7 climate variables) to value 0.00035.Redundancy analysis (RDA) implemented in vegan packagehttps://doi.org/10.6084/m9.figshare.16845445.v1).The formal identification of the plant material used in our study was done by Jiri Korecky and confirmed by Jan Stejskal. We declare that the sample collection permissions were obtained from the responsible authorities. LE individuals were sampled in the Bohemian Switzerland National Park under the permission nr. SNPCS 0573/2015, HE individuals were sampled in the Giant Mountains National Park under the permission nr. OSML 38-11/2015, and ME individuals were collected in the Jizera Mountains protected area under the permission nr. LČR 8/2016. Norway spruce is not considered a species at risk of extinction or endangered species. Collection of plant material complied with institutional, national, and international guidelines and legislation. Trees in our study are categorized based on crown morphology , most likely due to the low DNA quality. Among the tested DNA restriction enzymes , EcoT22I, a rare cutter with the potential of good read depth for heterozygotes, was deemed the most appropriate enzyme based on fragment size optimization. There were 96 samples sequenced in one lane turning in approx. 5 M reads per sample. The total sequencing coverage (for 20 Gb Norway spruce genome) has been estimated approximately 0.025×.The FASTQ file was aligned against the Norway spruce reference genome and from 52,203,003 read tags present, 51.6 and 30.8% were uniquely and multiply aligned, whereas 17.6% tags remain unaligned. Variant calling performed on uniquely aligned tags generated 9,190,592 raw SNPs. These SNPs were filtered by minimum minor allele frequency (MAF) > 0.01 and missing data < 90%, kept 6,970,550 SNPs that were stored in VCF file (mean heterozygosity 0.074 ± 0.012).only biallelic and non-indel SNPs were kept: 6,229,325 SNPs,only loci with no missing data were kept: 36,313 SNPs,MAF was set > 0.05: 16,982 SNPs,the Quality value threshold for loci was set ≥ 20: 16,982 SNPs,only loci with mean depth values between 10 and 60 were retained: 12,824 SNPs,SNPs within 1000 bp distance were thinned: 1,916 SNPs.The SNP data underwent a strict filtration in VCFtools software and was reduced from almost 7 million to 1,916 (0.03%) as follows:The mean observed heterozygosity of the final SNP dataset was 0.271 ± 0.125 (expected heterozygosity 0.237 ± 0.114).The PCA applied on the final dataset (1916 SNPs) separated the high-elevation from the medium and low-elevation ecotypes Fig. . Althoug74. As a trade-off between the power of discrimination and over-fitting, we used optim.a.score function and retained 37 PCs entering the analysis (n.pca = 37), n.da = 10. Similar to PCA analysis output, the DAPC differentiated the high-elevation ecotype from the low and medium elevation ecotypes. Again and similar to PCA analysis, individuals from both HE subplots overlapped and formed one cluster , likely due to shallow population structureAdditionally, clustering using STRUCTURE , respectively. While both correlations are low; they were significant, indicating that trees' genetic distance is correlated with altitudinal and geographical distances in an almost similar manner.The correlation of genetic distance with the geographical and altitudinal distances evaluated by the Mantel test were 0.062 and 0.068 (r) ranging from − 0.05 to 0.13, yet no correlation was statistically significant), 7 SNPs with bio6 , 2 SNPs having the highest correlation with bio12 , and 1 SNP with bio4 . See Supplementary Table We used the Redundancy Analysis (RDA) method to select candidate SNPs to depict traces of local adaptations to climatic conditions. The first RDA component separates the high-elevation from medium- and low-elevation populations. In contrast, the second RDA component tends to emphasize differences in low- and medium-elevation populations and the third RDA depicted the difference between HE1 and HE2 populations Fig. a,b. We s78. The only exception is minor allele frequency (MAF) as in similar studies it is conventionally set to 0.0581. However, settings to different thresholds (0.01 and 0.02) are also reported. Our study inspected a variation attributable to most individuals within the respective group (ecotypic variance), so we set MAF to a 0.05 threshold.Genotyping-by-sequencing is a cost-effective genotyping technique that provides large datasets but is frequently of variable quality. Therefore, initial data filtration is needed, and one should set up the filtration criteria depending on the research question and overall genotyping quality. However, optimal settings are not straightforward and vary widely among studies77, we thinned SNPs within a 1 kbp distance. Being aware of relatively low sequencing depth (mean depth for unfiltered 6.97 M SNPs data set is estimated 3.73), we set the minimum filtration depth for finally selected calling variants to 10, ensuring that only reliable SNPs are kept. We considered this to be the main reason for the low number of SNPs remained after filtering. Although we reduced the primary dataset significantly (preserving approximately 0.03% data out of the primary dataset), the remaining SNPs are robust and reliable.We excluded multiallelic SNPs and indels during the first filtration step, which reduced the dataset to 89.4% of its original size. Further significant reduction occurred when excluded missing data . Filtering for minor allele frequency additionally further reduced the data set by about one-half. To diminish the effect of linkage disequilibrium82, in our data, separation of HE ecotype individuals from LE and ME groups on the first principal component is evident . This suggests that the inspected SNPs are not in a strong linkage with genes involved in local adaptation. Given that the Norway spruce genome size is 19.6 GbGST, Jost's D, and Hendrick's GST) displayed significant differences for the LE–HE and ME–HE ecotypic groups . Romšáková et al.36 detected considerable differentiation among two altitudinally contrasting populations (700 versus 1450 m a.s.l). Subsequently, Hrivnák et al.89 replicated Romšáková's methodology for an altitudinal transect of several populations, reported genetic distinction only for individuals originating from the highest elevation stand (1500 m a.s.l).Several studies investigated Norway spruce geographical variation along altitudinal gradient based on single nucleotide polymorphisms. Scalfi et al.90. In our study, we did not detect individual SNPs contributing significantly to genetic grouping, hence we demonstrated the cumulative SNPs effect for ecotypic clustering by multivariate analysis methods.It is essential to point out that several studies indicate that most variation in adaptive traits is complex and controlled by many loci of minor effects91 are also attributable to genetic variation, but this effect remained hidden in our study as the GBS approach could not detect them. Although epigenetic modifications are frequently overlooked50, it should be more reflected in genetic studies as not all of the variance in the phenotype can be accounted for those coded in DNA sequence92. In practice, that can be overcome only by using epigenetics mechanism-sensitive genotyping platforms.Additionally, the effects of epigenetic mechanisms3. Medium-to-long distance pollen transport is commonly reported93, with the potential of pollen distribution hundreds of kilometers3. This fact reflects the shallow population differentiation revealed by genetic markers16. Conversely, the reproductive success of adjacent potential pollen donors is often limited94, which has negative consequences on seed orchard production96. According to Bucci and Vendramin97, the increase of the genetic divergence attributable to geographically formed variation is estimated in hundreds of kilometers (approximately 1800 km in Norway spruce). The distance between the sampled populations in our study is limited to circa one hundred kilometers without any natural geographical barriers  in between. Therefore, we do not assume the effect of distance and geographical isolation to be factored but most likely selection mechanisms occurring in the HE group. Undoubtedly, also seed dispersal affects population structure, but its effect is more local98.Particularly in coniferous species, pollen gene flow is one of the most critical factors influencing genetic structure43 characterized Norway spruce as a highly translocated species for at least three centuries throughout Europe, including the Czech Republic. Sowing of Norway spruce seeds in the Jizera mountains (area of ME ecotype) has been recorded around 1820, but with local provenances. Although since the 1860s, the use of allochthonous forest reproductive material increased, cultivations were performed up to 1000 m a.s.l.99. In this regard, the HE ecotype might be considered more likely locally autochthonous. To minimize targeting non-natural regeneration in all three ecotypic groups, we intentionally placed sampling transects on less accessible slopes and rocky outcrops.Although we have made every effort to target autochthonous Norway spruce populations, we could not entirely omit the historical effect of the human-mediated stand composition. Jansen et al.100, in Eucalyptus flowering asynchrony build a natural reproductive barrier101. In spruce, local synchrony of pollen production and receptivity might promote short-distance pollination and preserve local-adapted genotypes102. Skrøppa and Steffenrem103 found clinal variation related to mean annual temperature. The intrapopulation variation was considerably larger in low altitude as compared to those from high-altitude populations. Thus, although the mean annual temperature difference of 2.3 °C between LE and ME ecotype is higher than the difference between ME and HE ecotype (1 °C), the narrower biological window of HE pollen receptivity might act as a barrier preventing non-local pollination.An additional aspect involved in the genetic differentiation of a given ecotypic group might be connected to reproductive phenology. In oak species, for example, phenological asynchrony driven by microclimatic variability determines the size of acorn crop104. Unfortunately, the information about the survival rate of lowland provenances planted in higher altitudes is scarce. Jurásek and Martincová105 observed a strong selection effect on Norway spruce seedlings in higher elevations with high mortality levels of lowland-originated seedlings compared to those from higher elevation (1000 m.a.s.l and above). Similar findings were reported by Hrdlička106 on a high-elevation testing plot (1070 m.a.s.l.) planted with two seedling sources. After a decade, seedlings originated from lower altitudes (560 m.a.s.l.) exhibit 95% mortality, whereas those from the higher altitude (970 m.a.s.l.) survived seamlessly.During the twentieth century, numerous international Norway spruce provenance experiments were established across Europe, known as the Internation Union of Forest Research Organization (IUFRO) provenance trials107, higher nitrogenous content in needles108, and significantly different patterns in dehydrin gene expression109.Also in our study, we assume intensive natural selection among high-elevation individuals during the early growing stages as an intrinsic selection mechanism that eliminated unfavorable genotypes for adverse climatic conditions. As we focused on mature trees that persist on the stand despite the ontogenetic selection, we probably largely genotyped individuals that bear distinctive genetic pattern favorable to higher altitudes. Additionally, several ecophysiological studies indicated the resilient specific status of high-elevation ecotype such as enhanced drought toleranceThe current study aimed to dissect the genetic architecture of three Norway spruce ecotypic forms using SNP markers. We focused on presumably autochthonous stands composed from trees of morphologically predominant morphotypes, a.k.a ecotypes in a limited area of the Czech Republic. Due to such stringent selection criteria, we identified only one experimental area for LE, one area for ME, and two stands for high-elevation ecotypic form.The data analysis revealed a conspicuous partition into two groups—low- and medium-elevation ecotypes being the one, and the high-elevation forming the second group. Being aware of the experimental limitations that do not allow to distinguish between local adaptation and IBD, we may only hypothesize reasons for such separation.Although we did not detect any apparent biological  or geographical  barriers, we assume the observed distinctive genetic structure of the high-elevation ecotype is a consequence of a natural selection. Target trees (> 100 years old) have already undergone not only phylogenetic but also ontogenetic selection pressure. Consistently to the long-term observations of forestry practitioners, we assume the intensive ontogenetic selection in a high-elevation ecotypic group promoting local paternal contributors mirrored in the distinctive overall genetic makeup of the extant population. Although our genome coverage was sparse to detect significant genotype-environment associations, we believe we obtained robust outputs in connection to ecotypic distinction. Our findings bring a vital message to forestry management practices. Under the current situation with bark beetle outbreaks across Europe causing increased demand on forest reproductive material for reforestation, some forestry practitioners appeal to relax forest reproductive material transfer rules. That should be considered with caution, especially in locations with extreme climates, as indigenous populations (seed donors) might have previously undergone an intensive selection and adaptation processes and be better adapted to these unfavorable conditions.Supplementary Information.


Species domestication is generally characterized by the exploitation of high-impact mutations through processes that involve complex shifting demographics of domesticated species. These include not only inbreeding and artificial selection that may lead to the emergence of evolutionary bottlenecks, but also post-divergence gene flow and introgression. Although domestication potentially affects the occurrence of both desired and undesired mutations, the way wild relatives of domesticated species evolve and how expensive the genetic cost underlying domestication is remain poorly understood. Here, we investigated the demographic history and genetic load of chicken domestication.We analyzed a dataset comprising over 800 whole genomes from both indigenous chickens and wild jungle fowls. We show that despite having a higher genetic diversity than their wild counterparts , the red jungle fowls, the present-day domestic chickens experienced a dramatic population size decline during their early domestication. Our analyses suggest that the concomitant bottleneck induced 2.95% more deleterious mutations across chicken genomes compared with red jungle fowls, supporting the “cost of domestication” hypothesis. Particularly, we find that 62.4% of deleterious SNPs in domestic chickens are maintained in heterozygous states and masked as recessive alleles, challenging the power of modern breeding programs to effectively eliminate these genetic loads. Finally, we suggest that positive selection decreases the incidence but increases the frequency of deleterious SNPs in domestic chicken genomes.This study reveals a new landscape of demographic history and genomic changes associated with chicken domestication and provides insight into the evolutionary genomic profiles of domesticated animals managed under modern human selection.The online version contains supplementary material available at 10.1186/s12915-021-01052-x. All organisms carry a certain level of deleterious mutations in their genomes, which can potentially affect their fitness , 2. The Chicken is believed to have been domesticated from the red jungle fowl (RJF). Early genomic studies have identified a number of variants that differentiate chicken from RJF, facilitating our understanding of the genetic changes underlying chicken domestication , 23. It G. g. spadiceus, G. g. murghi, G. g. jabouillei, G. g. gallus, and G. g. bankiva) ranging across South and Southeast Asia where they may have been hybridizing with village hens [Measuring the magnitude of domestication bottleneck by direct comparison of genetic diversity in current domesticates with their true wild progenitors is often impossible  or complicated by several factors , 25, 26.age hens . The thiage hens , 37.https://bigd.big.ac.cn/chickensd/), through sequencing and analyzing 863 genomes from both jungle fowls and indigenous chickens sampled across South, Southeast, and East Asia as well as Europe , we demonstrated that all domestic chickens were monophyletic, derived from an RJF lineage of G. g. spadiceus (GGS) whose present-day range is predominantly in northern Thailand, southwestern China, and Myanmar, and then regained genetic diversity via introgression from additional RJF subspecies and jungle fowl species during their dispersals out of the domestication center [In the first phase of our 1K Chicken Genomes Project  for each population. The average π for all domestic chickens was 3.26e−3, slightly higher than that for GGS   approachHowever, PSMC has limited power to reveal the recent Ne within 10 kya. We then used MSMC , a methodadi [dadi analyses suggested that domestic chickens experienced a continuous Ne decline since their splitting off from GGS, indicating a bottleneck in chicken domestication. We noted that admixture with local jungle fowl or other domestic chicken populations is pervasive [Because PSMC, MSMC, and SMC++ do not take into account the possible effects of admixture , 47, thedadi  to fit tervasive , which lervasive , so the To measure whether the domestication-associated bottleneck could have induced the rise of high-impact SNPs (hSNPs) in chicken genomes, we analyzed the variants called in domestic chicken and GGS genomes among our 1K CGP. We found that protein-coding regions accounted for ~4.2% of the chicken genome, and 1.6% of the genomic variants  were present in these exonic regions . A total of 346 protein-coding genes were retrieved, and they are involved in multiple functional GO and HPO categories, including abnormalities of nervous system physiology, growth, bone and muscle development and morphology, cardiovascular and respiratory system, metabolism/homeostasis, vision, and immunity  showed the strongest signal of selection, with one missense mutation  being nearly fixed in domestic chickens compared to RJFs . A recent paleogenetic study, however, showed the timing of selection on this gene in ancient and modern European chickens and concluded that the dramatic rise in frequency to modern ubiquity only began 1.1 kya [TSHR-Gly558Arg has a PROVEAN score at −6.981, to be indicative of potentially high effect (< −2.5). Therefore, it is interesting to assess the potential biological function of this mutation as a potential proof of concept to test all our predictions.In an early investigation , thyroid 1.1 kya . Our rec 1.1 kya . Should TSHR is associated with development and metabolism in mice [TSHR-558Arg knock-in mice (matched with mice TSHR-559Arg) to test whether this mutation has any biological effects  bodies in both sexes than the wild-type mice  between the TSHR-558Arg homozygous and wild-type mice , calorie consumptions, and carbon dioxide production (VCO2) than the wild-type mice . This result suggests that TSHR-Gly558Arg is biologically functional in chickens. However, such validation for a single mutation cannot stand the effect of all mutations; further experimental validation for additional and specific variants is warranted.Because of the challenge in editing an avian genome efficiently and precisely, several studies have employed transgenic mouse or human cell lines  or zebra in mice  and glyc in mice , 59, we r P < 0.0 bodies iice Fig. c. We alsr P < 0.0 bodies iP = 0.01865, Wilcoxon signed-rank test; Fig. P = 1.83e−6, Wilcoxon signed-rank test; Fig. P < 2.2e−16, Wilcoxon signed-rank test; Fig. To compare the levels of genetic loads between domestic chickens and GGS, we evaluated the numbers and frequencies of non-synonymous and synonymous SNPs as well as the hSNPs among their genomes Fig. . Our resP = 0.00584, Wilcoxon signed-rank test; Fig. P = 0.00596, Wilcoxon signed-rank test) but less homozygous hSNPs . The total number of homozygous synonymous alleles was higher in GGS than in domestic chickens  or LSBL or π-ratio analysis possessed lower numbers of hSNPs  and π-ratio from our previous study  and compChickens are among the few domestic species with their progenitors extant in the wild today, providing an excellent system to address questions about evolutionary changes under domestication. In this work, we conducted systematic genomic studies of the domestication history and landscape of hSNPs using the largest genomic dataset from a worldwide sampling of indigenous chickens and their wild counterparts. Our analyses suggest that domestic chickens share a nearly identical demographic history with their direct wild ancestor, the GGS, before the Holocene. Around 12 kya, domestic chickens and GGS have diverged from each other, which is generally consistent with that assessed previously using MSMC based on 50% relative cross-coalescence rate (~9500 ± 3300 years ago) . SubsequGallus gallus beginning ~80 kya may have induced the accumulation of hSNPs across all lineages at a comparable magnitude, but the domestication bottleneck and/or recent selection for genetic improvement dramatically increased the genetic load of domestic chickens. A similar pattern has already been observed in horses [Our work reveals that domestic chickens carried 2.95% more hSNPs than GGS, a value comparable to those identified in dogs (2.6%)  and ricen horses  and dogsn horses . Our resn horses , 25, 68;We show that heterozygous hSNPs are accumulated more frequently in the genomes of domestic chickens than GGS, while homozygous ones display a contrary pattern. This is not unexpected, because most harmful mutations are at least partially recessive and therefore could only expose their damaging effects in homozygous states . EspeciaTSHR-559Arg homozygous mice displayed a significant difference in metabolism and development compared with the wild-type mice, suggesting that this mutation is biologically functional. This supports the early study that investigated the function of this mutation based on birds intercrossed between the ancestral RJF (wild type) and White Leghorn [TSHR function; however, whether it follows the same biological process in both chickens and mice remains unjustified. Third, despite the pattern of hSNPs in domestic chickens is generally consistent with the observations in other species, our genome coverages are relatively low for both domestic chickens and RJFs, and the levels of heterozygotes and het-hSNPs are likely underestimated. Further validation using higher coverage genomes is warranted. Lastly, our analysis focused on variants in the coding regions; however, non-coding regions are increasingly known to play important regulatory roles, and some variants within these regions likely have significant biological functions [There are several potential caveats in this study. First, our 1K CGP initially aimed to infer the domestication history and evolution of chicken; there is a sampling bias in our study. Our sampling efforts initially focused on diverse and village chickens (which likely present as more “ancient” populations) from Asia (where RJF inhabited) and adjacent regions, while lacking samples from Africa, Oceania, and South America. Even though domestication bottleneck and increased hSNPs are observed in several chicken populations compared with their wild relatives, our samples cannot present the whole genetic diversity of all chickens across the world, and issues on the strength of bottleneck and the number and frequency of these hSNPs accumulated during the early domestication or recent genetic improvement of specific breeds could not be resolved based on our data. Also, there is pervasive gene flow between chickens and other jungle fowls, which also likely result in bias in our estimation of genetic load. Therefore, the magnitudes of bottleneck and genetic load underlying chicken domestication remain open; our analysis provides a result for further testing. Future work by exploring genomes from more heritage chicken lineages and breeds across the world and ancient samples spanning a wide range of periods are necessary to address these questions , 69–71.  Leghorn . Becauseunctions ; future In conclusion, we systematically characterize the existence of a pre-domestication loss of genetic diversity followed by a domestication bottleneck in chickens, leading to the prominence of high-impact alleles across domestic chicken genomes. Through functional trait analyses, we suggest that these high-impact alleles affect behavior, development, and morphology, and our findings indicate that these alleles are partially under artificial selection pressure while the frequencies of detrimental variants are increased due to drift. This study presents a new page in chicken genomics, calling for a sharpened focus on the comparative genomic diversity of specific breeds and wild lineages, and for intensive functional analyses of high-impact alleles, to understand which contribute to domestication and genetic improvement of particular traits and which are maladaptive. This would enable the development of reliable markers for monitoring the concrete impact of genetic improvement and the purging of deleterious mutations on chicken genome evolution. In addition, our dating of the bottleneck and recovery processes in one of the most heavily relied upon domesticated species in the world has broad implications for understanding the biocultural interactions, translocation, and domestication practices affecting suites of species in Eurasia that were exploited in the past. Our study provides a possibility for further investigation using breeding experiments and a larger scale of genomes covering a wider sampling of global chickens and fossils.n = 620) and all five red jungle fowl subspecies , as well as green jungle fowls , Ceylon jungle fowls , and gray jungle fowls . Specifically, G. g. spadiceus was sampled from Thailand and Yunnan province of China, and domestic chickens were sampled from villages in Indonesia, Thailand, Vietnam, China, India, Sri Lanka, Bangladesh, Pakistan, Iran, Afghanistan, and Europe. By combining an additional 76 published genomes [n = 696) and G. g. spadiceus (n = 45). Genotypes for 9 G. g. spadiceus samples (ypt2887–ypt2895) from Thailand and 1 G. g. spadiceus sample from Daweishan (ypt570) were admixed with chicken [G. g. spadiceus samples) was used to perform the genetic diversity and genetic load analyses. The dataset is available at ChickenSD .In our 1K Chicken Genome Project (CGP), we leveraged the Illumina sequencing platform and generated 787 genomes from indigenous chickens and jungle fowls . These s genomes , 74–77 a genomes , 33.4 M https://www.ncbi.nlm.nih.gov/assembly/?term=GCA_000002315.5) using the standard BWA-GATK pipeline [Pair-wise sequential Markovian coalescent (PSMC)  and multpipeline , 79. Seqpipeline .http://samtools.sourceforge.net/). The loci with less than 1/3 or more than 2 times average read depths were deleted, and sites with consensus qualities below 20 were also removed. PSMC was running with parameters set as “psmc -N25 -t15 -r5 -p 4+25*2+4+6”. Input data for MSMC was prepared using the tool generate_multihetsep.py suggested by the author from https://github.com/stschiff/msmc-tools. The genotypes for all samples were phased jointly using Beagle V4.1 [PSMC , MSMC 44, and SMgle V4.1  with defhttp://lh3lh3.users.sourceforge.net/snpable.shtml) with settings “- k=35 and r=0.9”. To maximize more populations to be analyzed, five genomes for each population with coverage over 15-folds were used. Input file for SMC++ was generated using the pipeline as the author suggested (https://github.com/popgenmethods/smcpp). Except that Laos chicken, Emei chicken, and Muchuan chicken have less than five genomes with sequencing depth over 15-folds, we analyzed all populations that were analyzed by MSMC and PSMC above. We further included Jingyang chicken and Pengxia chicken for the SMC++ analysis. SMC++ was ran using default parameters.For running SMC++ (v1.15.2), we sequenced genomes for each of the three GGS samples  reported  previousdadi) [https://www.ncbi.nlm.nih.gov/) and low-complexity regions identified using mdust [Finally, we investigated the population histories by analyzing the joint allele frequency spectra using diffusion approximation for demographic inference (dadi) . Becauseng mdust . Finallyng mdust , as descng mdust . We examdadi were scaled using a generation time (g) of 1 year and a mutation rate (μ) of 1.91 × 10−9 substitutions per site per year [Estimations from PSMC, MSMC, SMC++, and per year . We usedper year  to averaper year  in domeshttp://dec2015.archive.ensembl.org/index.html). The ancestral state of a variant was inferred from the green jungle fowl. To predict the effect of a missense mutation on a protein, we applied the PROVEAN software [https://www.ncbi.nlm.nih.gov/). The prediction was based on evolutionary conservation by comparing the query and target sequences. Similar to the early study [To examine the evolution of hSNPs in chickens before and after their domestication, we followed a similar pipeline as described previously , 61 to asoftware  in searcly study , a mutatTo obtain a global perspective on the functions of genes carrying such hSNPs, we used g:Profiler  to retriG. g. spadiceus, G. g. jabouillei) and LSBL2 , as well as π-ratio (πG.g.spadiceus/πchicken) [Furthermore, we retrieved genomic regions of putatively selective sweeps that were measured by locus-specific branch length (LSBL) statistics  in the cchicken)  from ourchicken) . For sweTSHR-Gly558  in domestic chickens is highly conserved across vertebrates and corresponds to the mice-TSHR-Gly559 (c.1675G) in the 10th exon of transcript Tshr-202 . A C57BL/6 mouse model with a mutation at the mouse TSHR locus  was constructed by CRISPR/Cas-mediated genome engineering . Briefly, Cas9 mRNA, gRNA, and donor DNA were micro-injected into the fertilized eggs of C57BL/6J mice to obtain F0 generation mice with the mutation of the target site  and placed in CLAMS (PRO-MRR-8) to measure the metabolism of homozygous (HO) and wild-type mice at 30 °C, 18 °C, and 5 °C for 72 h. The levels of oxygen consumption (VO2), carbon dioxide exhalation (VCO2), calorie consumption, and water and food intake were recorded every 5 min for each mice (including HO and wild type). All mice had free access to water and food and were subjected to the same day-night cycle during the examination. Statistical significance was measured by Student’s test (two-tailed), and P < 0.05 was accepted to be significant.A Comprehensive Laboratory Animal Monitoring System (CLAMS) was used to monitor and analyze the indexes of metabolism and feed intake of the transgenic mice. Eight-week-old male mice were weighed . Figure S2. Demographic histories for G. g. spadiceus and diverse chicken groups by PSMC. A total of 18 chicken populations were included in this analysis. Figure S3. Four tested demographic models for dadi analysis. Nanc, ancestral population size before the split; T, timepoints; m, migrations; Napop, ancestral population size after split. Ncpop, current population size. Arrows depict migration directions. Figure S4. Comparing observed data and model allele frequency spectrum for the best model (Model 3). Figure S5. Provean-scores for nonsynonymous mutations  in microchromosomes, macrochromosomes, and intermediate chromosomes. Figure S6. Pipeline for constructing the mouse model with a mutation at the mouse TSHR locus . Figure S7. Photograph showing TSHR-559Arg knock-in homozygous (HO) and wild-type mice at 10 months old. Figure S8. Gly558Arg knock-in mice consumed less food than wild-type. *, P < 0.05. Statistical significance was measured by the Student’s t test. N = 8 for both HO and wild-type male mice were used in each test. Figure S9. Number and ratio of high-impact mutations among chicken populations. GGS, G. g. spadiceus; DC, all domestic chickens; WL, White Leghorn; TC, Tibetan chicken, XJ, Xinjiang local chicken; You; Beijing You chicken. Figure S10. Number and frequency of deleterious mutations in the genomic regions of putatively selective sweeps. Table S1. Information for high-coverage genomes used for PSMC, MSMC, and SMC++ analyses. Table S2. Estimations of likelihoods and AIC scores from four demographic models. Table S3. Summary of population histories calculated from 2D–SFS. Confidence intervals (95%) were obtained by bootstrapping all sites and performing parameter inference on each bootstrap dataset with 100 runs. Table S4. Distribution of variants identified in dog, sheep, goat, cattle, pig, and horse. This data is from our previous publication. Table S5. GO enrichment for genes carrying nonsynonymous mutation with provean-score of <–10. Table S6. Guide RNA sequences for the exon 10 of mouse-TSHR.


Lupinus albus L.), is characterized by exceptional protein content of high nutritional value, competitive to that of soybean (Glycine max) and is well adapted to rainfed agriculture. However, its high seed-quinolizidine alkaloid (QA) content impedes its direct integration to human diet and animal feed. Additionally, its cultivation is not yet intensive, remains confined to local communities and marginal lands in Mediterranean agriculture, while adaptation to local microclimates restrains its cultivation from expanding globally. Hence, modern white lupin breeding aims to exploit genetic resources for the development of “sweet” elite cultivars, resilient to biotic adversities and well adapted for cultivation on a global level. Towards this aim, we evaluated white lupin local landrace germplasm from Greece, since the country is considered a center of white lupin diversity, along with cultivars and breeding lines for comparison. Seed morphological diversity and molecular genetic relationships were investigated. Most of the landraces were distinct from cultivars, indicating the uniqueness of their genetic make-up. The presence of pauper “sweet” marker allele linked to low seed QA content in some varieties was detected in one landrace, two breeding lines, and the cultivars. However, QA content in the examined genotypes did not relate with the marker profile, indicating that the marker’s predictive power is limited in this material. Marker alleles for vernalization unresponsiveness were detected in eight landraces and alleles for anthracnose resistance were found in two landraces, pointing to the presence of promising germplasm for utilization in white lupin breeding. The rich lupin local germplasm genetic diversity and the distinct genotypic composition compared to elite cultivars, highlights its potential use as a source of important agronomic traits to support current breeding efforts and assist its integration to modern sustainable agriculture.Legumes crops are important for sustainable agriculture and global food security. Among them white lupin ( Lupinus albus L.) is considered one of the most important domesticated lupin species, when regarding the nutritional value of seeds [White lupin , France (3600 ha), and Spain (3045 ha) are the main lupin-producing countries [White lupin originates in the southern Balkans, and “graecus-type” natural populations disperse throughout the Eastern Mediterranean basin, where landraces are used for human consumption and fodder, dating back to 2000BC . Howeverountries .pauper gene contain 0.02–0.05% alkaloids of the seed dry weight [pauper locus has a key role in the species domestication and breeding [Modern breeding efforts to improve white lupin agronomic characteristics are very recent . Thus, ty weight . The recbreeding .Other important agronomic characters of white lupin, which attracted the breeders’ attention, are vernalization insensitivity , anthracWhite lupin’s global commercial potential has incited the breeding interest to focus not only in yield boost, but also in expanding its cultivation to agroclimatic regions, other than the Mediterranean basin, as extreme climate-change-related phenomena push the cultivation of some crops northwards . On top Colletotrichum lupini, is mostly identified as the responsible pathogen for lupin anthracnose. Nonetheless, it has been reported in several other crops such as olive [Anthracnose is a global fungal disease, responsible for devastating epidemics, characterized by significant yield losses ,33,34. Tas olive . The patas olive ,39,40,41as olive ,42.Successful breeding significantly depends on the extent of the available genetic resourcesPhenotypic and molecular markers have already been used in few studies, to estimate the genetic diversity between wild and breeding white lupin germplasm, and to enable incorporation of potentially valuable alleles from distantly related wild accessions to the genetic pool of elite cultivars ,44. AddiAlthough such progress will inevitably lead to development of more sophisticated tools to explore genetic variation in white lupin genetic resources, up to now SSR markers have been proved integral tools to investigate species diversity. The Balkan Peninsula represents a yet untapped germplasm diversity center for white lupin, concealing potentially valuable loci in landraces and natural populations that could promote adaptability to climate-change-relevant extreme conditions ,47. In tp = 0.003). A Pearson’s correlation matrix revealed strong correlation between seed area with TSW, perimeter and width (above 0.662) and between mean Gray Value with maximum and median Gray Value (above 0.709), suggesting, that a single character measurement is sufficient to represent highly correlated characters. Circularity showed only small to medium significant correlation with seed width, minimum, and mean and median Gray Value (above 0.323). Integrated density had a negative correlation with the rest of the variables, statistically significant only to height and width.All seed morphological characters’ measurements were statistically analyzed and a summary of the statistics is shown in Partitioning of the variance and grouping of the accessions, regarding seed morphology, were estimated by hierarchical clustering under the Unweighted Pair Group Method with Arithmetic mean (UPGMA) and Principal Component Analyses (PCA). The UPGMA analysis gave formation to a three cladded dendrogram , with moThe molecular marker HRM analysis elucidated 53 distinct allelic patterns, which were assumed as different genotypes. The marker PIC values (Polymorphic Information Content) ranged from 0.1 for GI-F1 to 0.8 for LSSR06a and LSSR41, with an average of 0.5 online . In ordeBased upon this matrix, we calculated the Nei’s genetic distance among all samples and formed a circular dendrogram, according to UPGMA clustering . As presp = 0.068). Possible associations of each morphological trait with the molecular markers were tested by linear regression analysis, but the results did not reveal any significant correlation.Patterns of genetic relationships among the studied germplasm are visualized by PCoA analysis, with the first two axes explaining the 55.49% of the observed variance . Four diThe 45 accessions under examination were further characterized for presence of marker alleles identified in other studies to be linked to agronomic traits important for white lupin breeding. For resistance to anthracnose, the accessions were screened with TP222136 and TP338761 molecular markers . Most ofSEPALLATA 3 locus, all cultivars apart from Sulimo, breeding line GR29, as well as landraces GR3, GR7, GR12, GR16, GR19, GR21, and GR26, bring upon the early flowering allele  in order to detect the restriction site of AciI, which was present in landraces GR5, GR10, and cultivar Frieda . Overall, the approach used allowed to tentatively annotate sixteen alkaloids, including angustifoline, lupinine, epilupinine, sparteine, etc. online  that aren online . Relativpauper allele.Interestingly the partitioning of the 16 alkaloid compounds assessed in each sample varied between accessions, indicating that each alkaloid biosynthesis product may accumulate in different amounts in different genotypes online . FurtherGenetic diversity of white lupin genetic resources in Greece was assessed using morphological and molecular markers. This is the first report for molecular characterization of local white lupin germplasm in a country that is considered as a center of genetic variation for this crop.Seed morphological characteristics were selected because heritability of seed morphological traits and ease of access to seeds may provide a reasonable alternative to estimate the distribution of genetic variation . Considegraecus form of lupin, while all the others were grouped together regardless of geographic origin. Interestingly, all the commercial cultivars were grouped in one cluster.It is conceivable that as valuable as they may be, agronomic traits and morphological characteristics as genetic markers are of limited resolution power for genetic diversity assessment, compared with molecular markers. In this study, molecular polymorphism was considerable. Relatively moderate to high PIC values, indicating a high level of genetic diversity in the germplasm assessed. The markers were suitable for discrimination of most landraces that clustered apart from the commercial cultivars indicating a unique diversity. Specifically, landraces were grouped into two clusters with those originated from Andros island forming a distinct group representing the wild Molecular markers have been proved valuable tools for germplasm diversity studies in many plant species. Yet, limited number of studies on white lupin molecular diversity are cited in the literature. To the best of our knowledge, genetic diversity using molecular markers has been estimated for local germplasm from Ethiopia ,52 and EL. albus diversity. Such isolated regions, justify divergence of local landraces, explaining the finding that landraces from Andros and Lemnos islands and from Mani peninsula are clustered separately from the cultivars examined. Additionally, landraces from Andros island are distinctly diversified from the rest accessions, implying that Andros constitutes a genetic pool of unique genes, potentially useful for white lupin breeding.The Greek landscape is characterized by a mountainous terrain, with numerous peninsulas and islands, and is considered a hotspot of Eventual exchange of landraces between Greek farmers and dispersal of their cultivation farther than their local point of origin may explain landrace sub-clusters on the dendrogram . It is apauper being thoroughly investigated thus far, on account of its contribution to QA synthesis regulation [pauper marker was detected in all cultivars, in breeding lines GRKML and GRKAL, and in GR26 landrace. Absence of the “sweet” pauper marker from the vast majority of the landraces is in accordance with their high QA content. However, presence of the marker in high QA genotypes, like the cultivar “Multitalia”, makes this marker a weak predictor of low QA content with broad applicability.Quinolizidine alkaloids are predominantly found in high levels in natural populations and landraces, making lupin a repulsive choice for lambs and goats, that feed on pasture . That jugulation ,48,57. RSEPALLATA 3 and FLOWERING LOCUS T early flowering alleles, implying a vernalization independence-promoting regional microclimate at the collection sites [SEPALLATA 3 was detected in all commercial varieties, except for Ulysse. The inability of FTa1 primer set to hybridize in three landraces from Andros island and in cultivars Frieda and Celina, suggest contingent mutations located at the primer binding site, in the genomes of those accessions  was calculated, based on the weight of 100 seeds in three independent replicates. Seed morphological diversity was estimated, by subjecting 10 seeds of each sample to examination according to standard criteria (IBPGR 1981) using image analysis as previously described . Calcula® 96 , using KAPA HRM FAST PCR Kit , in a total reaction volume of 12 μL, containing 5 ng genomic DNA template, 1X KAPA HRM FAST Master Mix, 0.2 μM of each primer, 2.5 mM MgCl2, and PCR-grade H2O. The PCR amplification program constitutes an initial denaturation step at 94 °C/4 min, followed by 35 cycles of denaturation at 94 °C/30 s, primer annealing at 58 °C, and extension at 72 °C/40 s. The HRM step includes denaturation at 95 °C/60 s, annealing at 40 °C/60 s, gradual denaturation from 65 °C to 97 °C by 0.05 °C/s, and fluorescence detection 20 times per °C.For every accession, the molecular genetic diversity was assessed on bulked samples of 5 individuals. Genomic DNA was extracted from seeds, applying the QIAGEN DNeasy Plant Pro Kit . The yield of the extracted DNA was estimated with Qbit 4 Fluorometer  and normalized to 5 ng/μL for downstream applications. Genotypic analysis was performed using 6 polymorphic microsatellite markers online , based oThe samples were assigned to groups, according to the amplicons melting temperature (Tm), the shape of the normalized melting curves, and the difference plots, generated by the HRM analysis, with different groups denoting dissimilar genetic profiles, and a binary matrix was formed, for further analyses. Genetic distances between all 45 accessions were calculated based on Nei’s genetic distance , and a d®, Waltham, MA, USA), using KAPA Taq ReadyMix PCR Kit  in a total reaction volume of 15 μL, containing 20 ng genomic DNA, 1X KAPA Taq ReadyMix Mix, 0.2 μM of each primer, 1.5 mM MgCl2, and PCR-grade H2O. PCR amplification was performed under the following conditions: initial denaturation at 94 °C for 4 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing for 30 s, extension at 72 °C for 30 s, and final extension at 72 °C for 4 min. In order to detect the presence of the target allele of each marker, all samples were subjected to post-PCR restriction enzyme digestion, following the appropriate protocol provided by New England BioLabs . The presence or absence of the target alleles was then verified by 3% agarose gel electrophoresis (1× TAE) on 100 volts for 40 min.Germplasm evaluation on agronomically important traits, was addressed by investigating for the presence of alleles, that control low alkaloid content, resistance to anthracnose and vernalization requirement, applying a set of recently developed, specifically designed dCAPS molecular markers  18,19,4,419,48. 2O, 4:1 (v/v) solution (10 mL) containing 0.1% formic acid was vortex mixed (1 min), and then extracted with an ULTRA-TURRAX homogenizer  for 4 min in total  at 4 °C. Consequently, after centrifugation the supernatant was filtered with Nylon filters (0.22 μm), collected, and a 1:100 dilution was applied to furnish the final extract working solution. The latter was injected to the UHPLC-HRMS system (Ultra-High Performance Liquid Chromatography)—coupled to Q-Orbitrap High Resolution Mass Spectrometry.Sample preparation was adopted from a recently published study , incorpov/v) formic acid (A) and 0.1% (v/v) formic acid in acetonitrile (B). The gradient elution was: T = 0 min, 5% B; T = 3 min, 5% B, T = 21 min, 95% B, T = 23 min, 95% B, T = 24 min, 5% B; T = 30 min, 5% B. The flow rate was 0.22 mL/min and the injection volume 3 μL. The ionization was performed using heated electrospray ionization (HESI), in the positive ion mode. The conditions for the HRMS were set as follows: capillary temperature, 350 °C; spray voltage, 2.7 kV; S-lense Rf level, 50 V; sheath gas flow, 40 arb. units; aux gas flow, 5 arb. Units; aux. gas heater Temperature, 50 °C. The analysis was performed in the full scan ion mode, applying a resolution of 70,000, with a mass range of 100–1200 m/z while acquisition of the mass spectra was performed in the centroid mode.Samples were analyzed with a Dionex Ultimate 3000 UHPLC system  linked to Q-Exactive Orbitrap HRMS . A Hypersil Gold UPLC C18  reversed phased column  was used for the separation that was maintained at 40 °C. Gradient elution of analytes was carried out with aqueous 0.1% , methanol (MeOH), and formic acid of LC-MS grade were obtained from Merck . Ultra-pure water was produced from SG Milipore apparatus. Nylon filters (0.22 μm) were obtained from Macherey-Nagel . m/z.Post-acquisition data analyses were performed using Compound Discoverer 2.1 software . The software was used to manipulate data and apply procedures of chromatographic processing region selection, application of baseline correction, peak detection, deconvolution, peak alignment, deisotoping and gap filling. For the putative annotation of the compounds a custom library was prepared based on the expected compounds of the genus of Lupinus using as a source the Dictionary of Natural Products applying a tolerance of 5 SEPALLATA 3 vernalization unresponsiveness-allele, as well as 2 landraces possessing both anthracnose-resistance alleles. These landraces could provide a significant genetic resource, to be harnessed in white lupin breeding programs. Expansion of white lupin cultivation to various hardiness zones, would promote legume reintroduction to the European farming systems, endorsing agriculture and environment sustainability in support of green farming.Legumes are pivotal for the sustainability of farming and food systems, by promoting soil fertility and environmental protection, in addition to food security. White lupin represents an emerging crop, providing both agricultural and nutritional benefits, yet in demand of extensive breeding. This study highlighted the presence of highly variable Mediterranean landraces, concealing potentially valuable genetic loci for adaptation in the local conditions, important for lupin breeding. The landraces evaluated are considered bitter-seeded, as they possess high-alkaloid content and probably the relative allele(s) in their genome. Therefore, they require debittering processing prior to consumption. Further breeding, addressing the reduction of high-alkaloid seed content, could yield lupin seeds ready-to-use in food industry and in forage as well. Moreover, 8 landraces were identified harboring the


In this study, genetic purity, parent-offspring relationship and diversity among the inbred lines were assessed using 92 single-nucleotide polymorphism (SNP) markers. A total of 188 maize genotypes, comprising of 26 inbred lines, four doubled haploid (DH) lines and 158 single-cross maize hybrids were investigated in this study using Kompetitive Allele Specific Polymerase Chain Reaction (KASP) genotyping assays. The bi-allelic data was analyzed for genetic purity and diversity parameters using GenAlex software. The SNP markers were highly polymorphic and 90% had polymorphic information content (PIC) values of > 0.3. Pairwise genetic distances among the lines ranged from 0.05 to 0.56, indicating a high level of dissimilarity among the inbred lines. A maximum genetic distance of (0.56) was observed between inbred lines CKDHL0089 and CML443 while the lowest (0.05) was between I-42 and I-40. The majority (67%) of the inbred lines studied were genetically pure with residual heterozygosity of <5%, while only 33% had heterozygosity levels of >5%. Inbred lines, which were not pure, require purification through further inbreeding. Cluster analysis partitioned the lines into three distinct genetic clusters with the potential to contribute new beneficial alleles to the maize breeding program. Out of the 68 hybrids (43%) that passed the parent-offspring test, seven hybrids namely; SCHP29, SCHP95, SCHP94, SCHP134, SCHP44, SCHP114 and SCHP126, were selected as potential candidates for further evaluation and release due to their outstanding yield performance.Assessment of genetic purity of parental inbred lines and their resultant F Zea mays L.) is the principal crop for food security and nutrition for many people in sub-Saharan Africa (SSA) and Latin America . Gen. Gen3]. th locus , were usGenetic relationships within and among the inbred lines were assayed with a neighbor-joining algorithm, using the unweighted pair group method (UWPGM) in DARwin 6.0 software . PairwisIS values. Nearly 39% of the SNPs had FIS values between 0.10 and 0.40 (o) values ranged from 0.0 to 0.56 with a mean of 0.40 (data now shown). SNP markers PZA00793_2 and PHM2350_17 had Ho value of 0.0 indicating the alleles of these SNPs were 100% fixed among the maize genotypes, however, 97% of the SNPs had Ho values exceeding 15%. SNP markers PHM2350_17, and PZA00793_2 showed inbreeding coefficient value of 1.The distributions of values for polymorphic information content, gene diversity, inbreeding coefficient and minor allele frequencies of the 92 SNPs estimated on the 188 maize genotypes are shown in and 0.40 . The SNPand 0.40 . Approxiand 0.40 . The minand 0.40 . More th0 values ranging from 39% to 49%. The gene diversity values for the inbred lines ranged from 0.42 to 0.49, with a mean gene diversity of 0.45. However, no significant differences were observed in PIC and gene diversity values among the ten chromosomes. The mean inbreeding coefficient (FIS) was significantly higher for inbred lines ranging from 0.75 to 0.85, with a mean value of 0.80. The hybrids on the other hand, revealed very low FIS values ranging from -0.02 to 0.17, with a mean value of -0.05.The genetic diversity parameter estimates of the 92 SNPs used in this study summarized per chromosome are presented in The percentage of missing data per SNP in this study was below 3% and varied from 0 to 2.17%, with the overall mean of 1.16%. Based on the 92 SNPs, genetic purity among the 30 inbred lines varied from 0.0 to 57.6, with an overall mean of 10.43 . All theThe population structure for the parental lines was assessed using distance-based cluster analyses. Cluster analysis based on Jaccard’s genetic distance values classified the 30 parental lines into three distinct clusters . The disParent offspring verification test revealed that out of 158 single-cross hybrids tested,16 hybrids had 0% contamination, 96 hybrids registered contamination level within the range of 0.54% to 4.89% (< 5%) and 46 hybrids had contamination greater than 5% . Further-1 for SCHP115 to 7.33 t ha-1 for SCHP17 and a mean yield of 5.55 t ha-1 was observed. The parent-offspring test was performed based on the criteria of < 5% genetic contamination on at least one of the parents and their hybrids. Based on the above criteria, of the 15 top performing hybrids, seven hybrids  passed the test and represented 47% of the hybrids. Similarly, among the bottom 15 performing hybrids, five hybrids  fulfilled the requirement. Notably, hybrids SCHP29 and SCHP115 among the top 10% and bottom 10% performing hybrids, respectively, exhibited genetic contamination of 0%.The hybrids were evaluated for grain yield performance and the top and bottom 10% performing hybrids are presented in In cross-pollinated crops such as maize, maintaining genetic purity for the inbred lines and the hybrids is vital for successful hybrid breeding and seed production. Significant changes in the genetic constitution of the inbred lines affect both the quality of newly developed hybrids and hybrid seed. The routine quality control genotyping in this study was done to detect any contamination which could have happened during hybrid development and inbred lines maintenance. Inbred lines are regarded pure or fixed if the proportion of heterozygous SNP loci does not go beyond 5% . Inbred The remaining 14 inbred lines  with 100% genetic purity were generated through conventional system. This affirms that the maintenance of these inbred lines was carefully done for several generations of selfing for seed increase and they are suitable for high quality hybrid production. Similar findings were reported by , reporteThe rationale for doing parent-offspring test is to confirm if the particular hybrid is the true resultant F1 hybrid derived from the original inbred lines with no pollen contamination or within acceptable contamination levels . Thus, t-1) in this study. This is because parental lines for the majority of top yielding hybrids passed purity test . This could be attributed partly to the use of genetically impure parental inbred lines or partly due to pollen contamination during hybrid production. The results revealed that the resultant hybrids derived from segregating inbred lines exhibited higher levels of genetic contamination of greater than 5%. Daniel et al.  reportedity test  and the Polymorphic information content (PIC) gives an estimate of the discriminatory ability and effectiveness of molecular markers with respect to the number of alleles that are expressed and their relative frequencies . Lander 0) of 0.09 and mean inbreeding coefficient of 0.80 reported in this study among the inbred lines indicate that the majority of the lines attained an appreciable level of homozygosity and were fixed. The low level of residual heterozygosity and high inbreeding coefficient in the present study further confirmed the existence of high level of inbreeding [IS values ranging from -0.02 to 0.17, with a mean value of -0.05 validating an excess of heterozygotes, which was expected for hybrids.The mean observed heterozygosity Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.