Since its clinical implementation, microvascular surgery has depended on the continuous improvement of magnification tools. One of the more recent developments is a high-definition three-dimensional (3D) digital system (exoscope), which provides an alternative to the state-of-the-art operating microscopes. This study aimed to evaluate the advantages and disadvantages of this technology and compare it with its predecessor. The study included 14 surgeons with varying levels of experience, none of which had used a 3D optical system previously. Six of these surgeons performed five arterial and five venous anastomoses in the chicken thigh model with both the VITOM 3D exoscope-guided system and the Pentero operating microscope. These anastomoses were then evaluated for their quality and anastomosis time. The participants and the other eight surgeons, who had used the digital 3D camera system for microsurgical training exercises and vascular sutures, answered a questionnaire. The anastomosis time and number of complications were lower with the conventional microscope. Participants rated the image quality with the conventional microscope as higher, whereas the field of view and ergonomics were favorable in the digital 3D camera system. Exoscopes are optics suitable for performing simple microvascular procedures and are superior to classical microscopes ergonomically. Thus far, they are inferior to classical microscopes in terms of image quality and 3D imaging. Leading characters in the development of a modern microscope were Zeiss, Abbe, and Leitz. The close affiliation between technical and physical sciences, together with manufacturing skills, led to the design and development of modern microscopes in the late nineteenth century, being manufactured in the so-called Carl Zeiss Werke in Oberkochen, Germany9 The first recorded application of a monocular microscope for operative procedures in humans was done by the ear, nose, and throat specialist Nylén in 1921, which was shortly after modified by his head of department Holmgren by attaching a binocular Zeiss microscope11 Other disciplines, such as ophthalmology and plastic and reconstructive surgery, followed and adopted the operating microscope. Since then, further adjustments and modifications were made, improving the magnification and facilitating the utilization of these microscopes. This desire for even more refinements continues until the present day. Specifically, developments in digital image processing allow unimagined application possibilities in the field of microsurgery.Microsurgery, especially microvascular surgery, has been implemented in a broad spectrum of surgical disciplines. The precondition for its application is always the improvement and diversification of the required materials and instruments. Specifically, the development of suitable magnification tools is necessary to operate on small structures precisely. The first records of the discovery and understanding of magnification date back to centuries before Christ and are linked to names such as Archimedes, Ptolemaeus, or Seneca, describing the phenomena of the burning glass, magnifying, or refraction features of waterA more recent development is a three-dimensional (3D) camera system that has been adopted in multiple aspects of daily and professional lives. This high-definition visually guided 3D system has cameras outside the body at a distance of 25–75 cm from the surgical site. Compared with a modern surgical microscope, this digital 3D camera system is characterized by a smaller and more convenient device and allows ergonomically working with the help of 3D glasses.13 Modern robotic systems such as the “Symani surgical system” do not have optics but are compatible with both classic microscopes and exoscopes. The concept of robot-assisted surgery in the field of (super-) microsurgery depends on excellent magnification and image quality, which should ideally be based on digital data that can be transferred to remote screens/goggles to take full advantage of the remote robotic setup. Especially, for interventions in body cavities that are difficult or impossible to access by conventional means , exoscope-based optics would be the ideal partner for robots. However, reservations remain regarding the exoscope’s image quality and 3D perception compared with the conventional binocular microscope.In addition to magnification, another big issue in (super-) microsurgery has now been solved, i.e., physiological tremors. In vascular and lymphogen anastomoses of the lumina with a diameter of < 1.0 mm, eliminating physiological tremors facilitates the performance of this demanding procedure. Most recently, robot-assisted microsurgery has evolved as the final missing puzzle piece in this highly specialized fieldThis study aimed to evaluate the possible advantages and disadvantages of an exoscope-guided 3D system in comparison with a state-of-the-art microscope in an in vitro setting and consider the results in the context of the emerging robotic era in microsurgery see Fig. .Figure 1This study included 14 surgeons with varying levels of experience, and none of them had any previous experience with exoscope-guided microsurgery. Six of them (two advanced and four highly experienced microsurgeons), who had previously used a standard operating microscope in both research-related and clinical practice, followed a standardized surgical procedure, performing a total of 20 end-to-end anastomoses each, which were then objectively evaluated. The other eight surgeons were considered as microsurgery trainees. They were participants of a 3-day microsurgery training course or were members of the department of Oral and Maxillofacial Surgery of the Technical University of Munich, Klinikum rechts der Isar, with basic microsurgical experience levels. They were given the opportunity to perform microsurgical training exercises and vascular sutures and answered the same questionnaire given to the former six surgeons.Each participant received instructions regarding the general properties and various components of the digital 3D camera system, ensuring that they knew how to operate it.14 All participants used the same microsurgical instruments and equipment required for anastomosis . Refrigerated chicken thighs (EDEKA Bio WWF Chicken Thigh 1 kg) were obtained in a nearby supermarket and disposed in an appropriate container after successfully completing the surgical procedures and evaluations.Surgical procedures were performed and evaluated according to a standardized protocol see Fig.  and accoThe time in minutes per arterial and venous anastomoses and the number of sutures performed were registered, and the time per suture in minutes was calculated. Each of the anastomoses was then randomized and evaluated independently by a blinded microsurgeon based on its patency and whether the back wall had been sewn on and the adventitia had been sewn in see Fig. . The patEach of the participants was asked to describe their experience with an operating microscope, and their opinions on the two systems were evaluated with a questionnaire consisting of 10-level Likert scale items  regarding handling, vision, ergonomics, magnification, and practicability . Figures were generated with Excel® . The Wilcoxon test was used to test the hypothesis between anastomoses performed with the operating microscope type OPMI® Pentero® and VITOM® 3D exoscope and participants’ answers regarding those questions that stood in a direct comparison of the two systems. All statistical hypothesis testing was performed on exploratory two-sided 5% significance levels. To identify any tendencies among participants’ answers, a qualitative evaluation was performed on the rest of the questionnaire.All methods were carried out in accordance with relevant guidelines and regulations.The median duration by each of the six participants for anastomosis and per suture along with their corresponding range and the incidence of back wall catch or intraluminal adventitia with each modality are displayed in Table med = 10.4 min [8.9–29.4]) than with the operating microscope (med = 9.2 min [6.9–28.5]). According to the Wilcoxon test, a significant difference with a strong effect was found . Similarly, participants required more time on average for the venous anastomoses with the digital camera 3D system (med = 13.2 min [9.8–34.5]) than with the operating microscope (med = 12.0 min [7.1–34.9]). Unlike the arteries, no significant difference was found   than with the digital camera 3D system (med = 1.3 min [1.2–3.2]), and a significant difference with a strong effect was observed . Regarding venous anastomoses, participants were also faster per suture when using the operating microscope (med = 1.3 min [0.9–3.1]) than when using the digital camera 3D system (med = 1.5 min [1.2–2.9]), but the difference was not significant .Accordingly, participants required less time per knot when performing arterial anastomoses with the operating microscope (med = 0.0 [0.0–1.0]), only one vessel had the posterior wall sewn on. With the operating microscope (med = 0.0 [0.0–0.0]), this was never the case. A significant difference was not found . For venous anastomoses, participants suture the posterior wall more often when using the digital camera 3D system (med = 1.5 [0.0–3.0]) than when using the operating microscope (med = 0.0 [0.0–2.0]). Again, the difference was not significant .Of all arterial anastomoses on the digital camera 3D system (med = 1.0 [0.0–3.0]) than when the operating microscope was used (med = 0.5 [0.0–1.0]), and no significant difference was found . In venous anastomoses, no clear tendency regarding the superiority of one of the systems could be determined: operating microscope (med = 1.5 [0.0–3.0]) and digital camera 3D system (med = 1.0 [0.0–4.0]). No significant difference was found in this aspect .The adventitia of the arteries was sewn on more often when the digital camera 3D system was used (med = 7.0 [2.0–9.0]) than with the operating microscope (med = 6.5 [1.0–9.0]), but no significant difference was found . The field of view of the digital camera 3D system (med = 8.0 [7.0–10.0]) was also rated better than that of the operating microscope (med = 8.0 [4.0–10.0]), and a significant difference was found .All 14 participants answered the 23-item questionnaire. The participants found the general handling more pleasant with the digital camera 3D system (med = 9.0 [7.0–10.0]) than with the digital camera 3D system (med = 7.0 [3.0–8.0]). The difference proved to be significant, and the effect was strong .All participants rated the image quality higher with the operating microscope (med = 9.0 [4.0–10.0]) than with the operating microscope (med = 6.5 [3.0–8.0]). This also showed a significant difference with a strong effect .Ergonomics were better with the digital camera 3D system (med = 8.5 [7.0–10.0]) than on the digital camera 3D system (med = 7.5 [6.0–9.0]). This difference was also significant, and the effect was strong .However, the participants found it easier to recognize different tissue layers on the operating microscope (med = 3.0 [1.0–8.0]) and the digital camera 3D system (med = 3.0 [1.0–10.0]). Accordingly, no significant difference was found   was higher when using the digital 3D camera system, although not statistically significant. These results may indicate that the digital 3D camera system is inferior to the operating microscope in terms of valuable operating time and avoidable complications. However, the implementation of a new operating system may prolong the operating time and may aggravate the conduction of known procedures in first-time applications with consequent complications. This consideration is corroborated by the results of Sassu et al., who performed several anastomoses in a rat’s femoral artery and encountered a reduction in operating time of > 30% compared with the first-time applicationThe questionnaire survey of the study participants revealed different results  endoscope. Their combination also resembled another step in the history of robotic surgery generally and robotic telesurgery specifically12 Although the field of its application is still limited, separating the body of expertise, i.e., the surgeon, from the area of operation entirely is made possible by replacing a standard microscope with a digital 3D camera system.Other studies have reported the same impression25 Specifically, we examined whether the back wall of a vessel was sewed on and if the adventitia, normally surrounding the vessel circumferentially, was sewed inside the vessel lumen by mistake. The finding that the back wall was sewed on 13 times and the adventitia was sewn 27 times inside the vessel lumen (Table 26.Besides our statistical results, this study furthermore emphasizes the need for intense microsurgical training as a foundation, and with the help of which, such a high level of microsurgical proficiency can be achieved. For an objective assessment of the anastomoses, we evaluated their patency in a standardized manner, which is frequently considered an objective qualitative parameter used as a surrogate to describe anastomosis success28 Further research is necessary to evaluate the comparability between 3D systems and conventional microscopes in their clinical application, ideally involving a greater number of surgeons who have had experience with both systems leading up to the study.Nevertheless, this study has some limitations. The limited number of surgeons and their subjectively perceived impressions and opinions may not be permitted to produce definite conclusions to issue a clear recommendation for either of the two optical systems, although the surgical data suggest a certain advantage of the conventional optical microscope. Regardless, no inconsiderate transmission to an application for a microsurgical operation on a patient may be made because we investigated the use of a digital 3D camera system in an experimental setting outside the operating room and its special conditions, even though multiple studies have unveiled promising reports and results hereofThe tools used for microsurgery have undergone continuous development, and the digital 3D camera system will not be the last. However, technical improvements and prolonged implementation in practice appear to be necessary before it can take its place as a state-of-the-art piece of equipment in the operating room. With these adjustments, its ergonomic advantages, especially when paired with other technologies such as operating robots or implemented endoscopes, could justify an equally coexisting or at least partially a replacement of the standard surgical microscope.


Pulmonary arterial hypertension (PAH) is a progressive and devastating disease characterized by pulmonary vascular remodeling which is associated with the malignant phenotypes of pulmonary vascular cells. Recently, the effects of heat shock protein 110 (Hsp110) in human arterial smooth muscle cells were reported. However, the underlying roles and mechanisms of Hsp110 in human pulmonary arterial endothelial cells (HPAECs) that was disordered firstly at the early stage of PAH remain unknown.6 on HPAECs abnormal phenotypes in vitro and pulmonary vascular remodeling of hypoxia-indued PAH rats in vivo by interrupting Hsp110-STAT3 interaction were evaluated.In this research, the expression of Hsp110 in PAH human patients and rat models was investigated, and the Hsp110 localization was determined both in vivo and in vitro. The roles and mechanism of elevated Hsp110 in excessive cell proliferation and migration of HPAECs were assessed respectively exposed to hypoxia. Small molecule inhibitors targeting Hsp110-STAT3 interaction were screened via fluorescence polarization, anti-aggregation and western blot assays. Moreover, the effects of compound 6 as potent Hsp110-STAT3 interaction inhibitor, which effectively inhibited HPAECs proliferation and migration, and significantly ameliorated right heart hypertrophy and vascular remodeling of rats with PAH.Our studies demonstrated that Hsp110 expression was increased in the serum of patients with PAH, as well as in the lungs and pulmonary arteries of PAH rats, when compared to their respective healthy subjects. Moreover, Hsp110 levels were significantly elevated in HPAECs under hypoxia and mediated its aberrant phenotypes. Furthermore, boosted Hsp110-STAT3 interaction resulted in abnormal proliferation and migration via elevating p-STAT3 and c-Myc in HPAECs. Notably, we successfully identified compound 6 could serve as a promising lead compound for developing first-in-class drugs against PAH.Our studies suggest that elevated Hsp110 plays a vital role in HPAECs and inhibition of the Hsp110-STAT3 interaction is a novel strategy for improving vascular remodeling. In addition, compound The online version contains supplementary material available at 10.1186/s12931-023-02600-5. Pulmonary arterial hypertension (PAH) is a progressive and devastating disease, characterized by increased pulmonary arterial pressure (PAP) and pulmonary vascular resistance (PVR) . HemodynHeat shock protein 110  is a stress-inducible molecular chaperone that helps cells survive by preventing the aggregation of misfolded/unfolded proteins , 17. HspAt present, the principles of research targeting heat shock proteins (HSPs) is decreasing protein levels with small-molecule inhibitors or knocking down HSPs with small interfering RNAs . However6 was successfully identified as lead compound against the Hsp110-STAT3 PPI. The in vitro and in vivo results further indicated that compound 6 displayed remarkable inhibitory activities against HPAECs proliferation and migration, and significantly decreased mean pulmonary artery pressure (mPAP) and vascular remodeling as well. Taken together, our work provides new insights into the role of Hsp110 overexpression in early-stage vascular remodeling, with potential to inspire targeting Hsp110-STAT3 PPI for the treatment of PAH.In this work, we first demonstrated the significant upregulation of Hsp110 in the serum of PAH patient as compared with healthy individuals. It’s also observed that Hsp110 expression is markedly increased in pulmonary arteries and lung tissue of PAH rats, which was induced by hypoxia or monocrotaline (MCT). Moreover, it’s shown that human Hsp110 is mainly localized in HPAECs and is abnormally upregulated during the early phase of vascular remodeling. Our studies highlighted that activation of the Hsp110-STAT3 interaction would contribute to the development of pulmonary vascular remodeling, and compound 1–20 were synthesized before  pyridine derivatives that possess anti-vascular remodeling activities [6. Through many in vitro assays such as SPR, DARTS and Co-IP, we have fully demonstrated that compound 6 could manage the abnormal phenotypes of HPAECs by interfering with the Hsp110-STAT3 PPI and then reducing the protein levels of p-STAT3 and c-Myc. Moreover, pharmacodynamic studies in vivo showed that compound 6 could significantly inhibit pulmonary vascular remodeling, improve RVH, and reduce the mPAP in hypoxia-induced PAH rats via disrupting the Hsp110-STAT3 interaction. Taken together, our results in this work preliminarily proved that compound 6 could be a new lead compound for drug discovery against PAH by targeting Hsp110-STAT3 interaction.Based on the existing research results, we screened the library of pyrazolo[3,4-tivities . We comp6 as a promising lead compound that disrupts the Hsp110-STAT3 interaction, resulting in the reduction of p-STAT3 and c-Myc levels. Moreover, compound 6 improved endothelial dysfunction and blocked vascular remodeling in our in vitro and in vivo PAH models. Our results suggest that disruption of the Hsp110-STAT3 association exhibits therapeutic effects against PAH progression, and provide a promising lead compound to develop the first-in-class agent targeting Hsp110-STAT3 PPI for PAH treatment.Our study first validated that Hsp110 was up-regulated in human serum and rat’s tissues with PAH, and overexpression of Hsp110 promoted HPAECs proliferation and migration. Furthermore, knockdown of Hsp110 significantly inhibited the abnormal phenotypes of HPAECs through suppressing the downstream STAT3 signaling pathway. Notably, we successfully identified compound Below is the link to the electronic supplementary material.Supplementary Material 1


The Quadricorna breed exhibit genetic diversity indices comparable with those of most of the other analysed breeds, however, the two populations showed opposing patterns of genetic diversity suggesting different levels of genomic inbreeding and drift (FIS and FROH). In general, all the performed genome-wide analyses returned complementary results, indicating a westward longitudinal cline compatible with human migrations from the Middle-East and several additional genetic footprints which might mirror more recent historical events. Interestingly, among the Italian breeds, the original Quadricorna (QUAD_SA) first separated showing its own ancestral component. In addition, the admixture analysis does not suggest any signal of recent gene exchange with other Italian local breeds, highlighting a rather ancestral purity of this population. On the other hand, both the neighbor-joining tree and the treemix analysis seem to suggest a proximity of the Quadricorna populations to breeds of South-Eastern Mediterranean origin. Although our results do not support a robust link between the genetics of the first wave and the presence of primitive traits, the observed genetic uniqueness together with the inferred phylogeograpic reconstruction would suggest an ancient presence of the Quadricorna breed in the Italian Peninsula. Because of this singularity, urgent conservation actions are needed in order to keep the breed and all related cultural products alive.Livestock European diffusion followed different human migration waves from the Fertile Crescent. In sheep, at least two diffusion waves have shaped the current breeds’ biodiversity generating a complex genetic pattern composed by either primitive or fine-wool selected breeds. Nowadays most of the sheep European breeds derive from the second wave which is supposed to have largely replaced oldest genetic signatures, with the exception of several primitive breeds confined on the very edge of Northern Europe. Despite this, some populations also in the Mediterranean region are characterised by the presence of phenotypic traits considered ancestral such as the policeraty, large horns in the ram, short tail, and a moulting fleece. Italy is home of a large number of local breeds, albeit some are already extinct, others are listed as critically endangered, and among these there is the Quadricorna breed which is a four-horned sheep characterised by several traits considered as ancestral. In this context we genotyped 47 individuals belonging to the Quadricorna sheep breed, a relict and endangered breed, from Central and Southern Italy. In doing so we used the Illumina OvineSNP50K array in order to explore its genetic diversity and to compare it with other 41 breeds from the Mediterranean region and Middle-East, with the specific aim to reconstruct its origin. After retaining 32,862 SNPs following data filtering, the overall genomic architecture has been explored by using genetic diversity indices, Principal Component Analysis (PCA) and  In livestock, SNP genotyping arrays are among the most widely used genetic tools for a wide range of studies including understanding species’ evolutionary mechanisms and domestication processes, discovering signature of selection, disentangling the nature of complex traits through genome-wide association studies as well as improving selection methods for animal production in the so-called genomic selection , 2. SuchSince Neolithic revolution, domestic animals followed human migration routes and spread throughout the world. For many livestock species, this processes have been shown to have occurred in distinct historical periods by following different colonisation routes leading to a vast array of breed diversity well adapted to specific local environments –7, that Ovis aries) have been shown to have retained much higher genetic diversity conforming with a wider heterogeneous pre-domestication populations, combined with less severe post-domestication bottlenecks [Compared to other domestic species such as cattle, goat and dogs, sheep , has been later replaced, generating a well-defined geographic cline from Middle-East to Balkan and continental Europe . HoweverConcerning the Italian peninsula, a high level of genetic diversity with a strong phylogeographic structuring has been observed . All genOvis longipes. On the other hand, other authors, primarily Sanson [Ovis aries asiatica), as described by Lemoigne [Among the most critically endangered Italian local breed is the Quadricorna sheep which is constituted by a very residual population reared in Central-Southern Italy, counting in 2022 only 2 farms, one located in Colliano  and the other in Monte San Giovanni Campano  with 33  and 23  heads respectively. The sheep population called “Pecora Quadricorna” has been registered since 2006 in the Regional Voluntary Register of Lazio established and from 2018 to the National Registry, but currently, the Herd Book is still lacking. The most characteristic trait of the breed is the polyceraty, hence the name Quadricorna which means four-horned. Worldwide, there are numerous sheep populations showing polycerism  being pry Sanson , argued To disentangle such a complexity and to shed light on the origin of the critically endangered Quadricorna sheep, we genotyped the two remaining populations from Central and Southern Italy using the Illumina OvineSNP50K BeadChip. Therefore, the main aims of this study are (i) to explore the genomic architecture of the Quadricorna population, (ii) to compare it with other Mediterranean breeds in order to gather insights into a more complete evolutionary picture and to (iii) assess whether the presence of considered primitive related traits is also accompanied by ancestral genetic diversity.Blood samples were collected from sheep during normal health prophylaxis procedures by the Istituto Zooprofilattico Sperimentale del Lazio e della Toscana, in agreement with the recommendations of European Union Directive 2010/63/EU, to ensure appropriate animal care.A total of 47 blood samples belonging to the Quadricorna sheep breed  were stored and selected for the genotyping. These samples included 22 individuals belonging to Colliano (QUAD_SA) and 25 to Monte San Giovanni Campano (QUAD_FR). The two examined farms pursue similar breeding conditions in a semi-wild state with small additions of flaked feed when fresh forage is scarce.The Illumina OvineSNP50K BeadChip was used to genotype the Quadricorna sheep populations while the raw genotyped data for 41 breeds  were retrieved from previous works , 34–40 .plink v1.9 [Using the software ink v1.9 , all gene) and observed (Ho) heterozygosity and the inbreeding coefficient (FIS) was calculated using the R package dartR [ROH) was calculated for each breed using the following parameters: the minimum length of the ROH was set to 4000 (—homozyg-kb), the SNPs density was set to 100 (—homozyg-density) with a maximum gap between consecutive homozygous SNPs of 1,000 (—homozyg-gap), one missing SNP (—homozyg-window-missing), up to one possible heterozygous genotype was allowed in the ROH (—homozyg-window-het) and the minimum number of SNPs within a ROH was set to 50 (—homozyg-window-snp). Thus, the final inbreeding coefficients (FROH), have been calculated using the autosomal length as implemented in the genome_wide function of the R package detectRUNS [For each breed expected (Hge dartR . In additectRUNS .Ne) for each breed was calculated using the software GONe [Finally, the contemporary effective population size  was performed using plink. The PCA was performed through a supervised approach using the “—pca-cluster” flag in  ggplot2 .admixture , was used to estimate the genetic structure, testing K values ranging from 1 to 50. For each K, we run three independent iterations, setting the cross-validation error procedure to 10-fold in order to choose the most informative number of K repartitions. The number of K before the separation of the two Quadricorna populations, was also used to plot the mean of the ancestral components in a pie fashion into a geographic map.The maximum clustering likelihood approach as implemented in the software The phylogenetic relationships among breeds were inferred using the “dist.genpop” function implemented in the R package adegenet . Startintreemix [To assess the genetic drift and migration events among breeds, the maximum likelihood dendrogram was inferred using the software treemix . The anatreemix  was usede and Ho was observed in the Soay breed, while the highest values were observed in Rasa Aragonesa. Concerning the FIS, the highest value was observed in the Leccese breed while the most negative value was for Sakiz. In general, we observed a higher amount of negative FIS values in ether breeds considered primitive  or with known recent history of admixture . Interestingly both Quadricorna populations showed negative Fis values (ROH) showed the highest values in Jacob and Soay, while the minimum value was observed in the Rasa Aragonesa (RASA) breed. The two Quadricorna populations showed different values of inbreeding, lower in the QUAD_FR than in the QUAD_SA  is reported in The contemporary effective population size , first separated the Soay from all the other breeds, while at K = 3, the Jacob breed is splitted in a different cluster. The two Quadricorna populations separated with all the other breeds at K = 9, however, at this K value, they display a different genetic admixture pattern . Indeed, the QUAD_FR is closely clustered with the Massese breed while the QUAD_SA is placed in a basal position of a group which includes Greek and Middle-East breeds  and from Middle-East  to ROMA. A weaker migration edge is also assigned from SOAY to Northern Italian breeds .Finally, the ic level . In our In a previous work we recently published in this journal, proudly and even a little surprised, we described the alleged ancestry of an endangered native breed (Quadricorna). Multiple convergent historical and morphological evidence moved towards an obvious and charming story to tell. However, only a few days after publication, we recognized a strand flipping error that strongly compromised the results by returning a substantial overestimation of the overall differentiation. Yet, despite the major oversight we also realised how we stumbled over the so-called confirmation bias. Behind this, we have re-analysed the whole dataset and although the divergences have largely diminished, we still found interesting patterns of ancestry which would seem to indicate a particular uniqueness of the Quadricorna breed with genomic relation with South East Mediterranean breeds.o and He), effective population size (Ne) and lower inbreeding coefficients (FIS and FROH). Different patterns of genetic diversity indices between populations of the same breed are not surprising and have been commonly reported in many livestock species including local [The genetic indices for the two Quadricorna populations in terms of expected and observed heterozygosity resulted in line with values of other local breeds in the Italian Peninsula . Also, tng local  and cosmng local , 52. EspOn the whole, the genomic architecture of the analysed breeds resolutely reflects a genetic cline from East to West  compatibOvis aries asiatica). It is also interesting to note that in the admixture analysis, the QUAD_SA population is the first among the Italian breeds in showing its own ancestral component , documenting the presence in the Italian Peninsula of livestock varieties from the Middle-East [The observed genetic uniqueness together with the inferred phylogeograpic reconstruction pointed to a proximity of the Quadricorna populations to breeds of South-Eastern Mediterranean origin. To address such outcomes, a possible hypothesis can be related to a diffusion from the Middle-East probably as a consequence of the increased marine trade from the Hellenistic to the Roman periods. For example, written sources about livestock trade are reported in the dle-East , 56.From a morphological point of view, the Quadricorna shares several primitive traits (policeraty and hair-type) with those breeds that can be traced back to the first wave. Indeed, four-horned sheep were historically widely distributed in Europe, due to advantages in the size and arrangement of the horns in anti-predatory strategies . SubsequS1 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


The use of molecular markers allows for precise estimates of genetic diversity, which is an important parameter that enables breeders to select parental lines and designing breeding systems. We assessed the level of genetic diversity and population structure in a panel of 151 tropical maize inbred lines using 10,940 SNP (single nucleotide polymorphism) markers generated through the DArTseq genotyping platform. The average gene diversity was 0.39 with expected heterozygosity ranging from 0.00 to 0.84, and a mean of 0.02. Analysis of molecular variance showed that 97% of allelic diversity was attributed to individual inbred lines within the populations while only 3% was distributed among the populations. Both neighbor-joining clustering and STRUCTURE analysis classified the inbred lines into four major groups. The crosses that involve inbred lines from most divergent subgroups are expected to generate maximum heterosis and produce wide variation. The results will be beneficial for breeders to better understand and exploit the genetic diversity available in the set of maize inbred lines we studied.The online version contains supplementary material available at 10.1007/s11105-022-01358-2. Zea mays L.) is a widely produced and consumed cereal crop, with worldwide production increasing from 313 million metric tons in 1971 to 1162 million metric tons in 2020 (Statista Maize (Statista . Maize wStatista . It has Statista .In sub-Saharan Africa (SSA), maize is the most important cereal crop as food, feed, and industrial crop, grown on over 40 million hectares of land  was used in the study, which included inbred lines developed from the National Agricultural Research Organization (NARO) in Uganda, and CIMMYT maize breeding programs.http://www.lgcgroup.com/our-science/genomics-solutions/#.WXpE7ITyu70) using the plant sample collection kit from LGC Genomics , gene diversity, and allele frequency were calculated using PowerMarker version 3.25 ) and ΔK, the output from population structure analysis of the 151 inbred lines suggested the presence of either 2 or 4 clusters Fig.  revealedST values for subpopulations 1, 2, 3, and 4 were 0.65, 0.50, 0.63, and 0.24, respectively. The allele frequency divergence values were 0.27 between subpopulations 3 and 1, followed by 0.23 recorded between subpopulation 3 and 2, then 0.17 observed between subpopulations 4 and 1, and the least allele frequency divergence of 0.14 recorded was between subpopulations 2 and 4, and 3 and 4.The expected heterozygosity among inbred lines within the four subpopulations ranged between 0.16 for cluster 1 and 0.30 for cluster 4 with an average of 0.22. The FThe goal of AMOVA was to use SNP markers to assess population differentiation in the maize inbred line populations. The results of AMOVA revealed that differences between and within populations accounted for 3% and 97% of the variation, respectively Table .Table 2APrincipal component analysis results based on the 10,940 SNPs were in agreement with the population structure and NJ clustering, grouping the inbred lines into four subgroups Fig. . The firGenetic diversity is imperative to provide a robust food security system capable of adapting to recurrent biotic and abiotic stresses. This is through identifying alleles that could be used as the source of novel genotypes which are high yielding and resilient for biotic and/or abiotic stresses as well as meeting the end-user demands in plant breeding. In the determination of the genetic diversity of a population, molecular markers are preferred since they are not influenced by environmental factors. In this study, 10,940 SNP markers were used to determine the genetic diversity and population structure of 151 maize inbred lines from different sources. In the present study, the PIC average was 0.33, a value found higher than that reported by Wu et al. , SserumaThe two complementary methods , used to determine the number of groups in this study, both clustered the inbred lines into four separate groups, thus implying there is a high degree of genetic diversity in the inbred lines evaluated in this study. Most of the inbred lines were clustered depending on their ancestry, selection history, and endosperm color Figs.  and 4. HST values observed in the present study ranged from 0.24 to 0.65 and were high; these values were similar to those reported in previous studies on other tropical inbred lines by Boakyewaa Adu et al.  also support the population differentiation. AMOVA revealed major molecular variance within populations rather than among populations implying a high genetic diversity among accessions Below is the link to the electronic supplementary material.


ST, 162 molecular markers  were detected among cultivars. Identifying those loci using NGS techniques can reveal the genetic bases and molecular mechanisms involved in the domestication and selection indicators among banana cultivars.Banana plantation has been introduced recently to a temperate zone in the southeastern parts of Saudi Arabia . The introduced banana cultivars were of a clear origin without a recorded genetic background. In the current study, the genetic variability and structure of five common banana cultivars  were analyzed using the fluorescently labeled AFLP technique. Nine different primer pairs combinations yielded 1468 loci with 88.96% polymorphism. Among all locations, high expected heterozygosity under the Hardy–Weinberg assumption was found (0.249 ± 0.003), where Dhamadh was the highest, followed by Fifa and Beesh, respectively. Based on the PCoA and Structure analysis, the samples were not clustered by location but in pairs in accordance with the cultivar’s names. However, the Red banana cultivar was found to be a hybrid between the American and Indian cultivars. Based on Φ Musa, banana and plantain (Musa sp.) belong to the Eumusa section of the genus Musa, family Musaceae and order Zingiberales [Among the edible, vegetatively propagated, monocotyledonous, and herbaceous species of iberales . Bananasiberales . One huniberales . Numerouiberales ,3. Thereiberales ,5. Unlikiberales .Musa to address contemporary food security needs. Clone identification and taxonomic studies have relied heavily on morphological and agronomic characteristics [Unlike their wild relatives, cultivated bananas grow without pollination. Fantastic collections of parthenocarpic mutants have primarily been made by farmers and multiplied and distributed by vegetative propagation of spontaneously occurring mutants . During eristics ,10.Musa acuminata (AA) and Musa balbisiana (BB). They are classified into other genomic groups, including AA, AB, and BBs classified as diploids, AAA, AAB, ABB, and BBBs classified as tetraploids, resulting from interspecific hybridization between M. acuminata and M. balbisiana [Musa species. Hybrid cultivars and wild types exhibit complex genome structures and phylogenetic relationships that require further investigation. Banana cultivation is susceptible to pests and diseases because of its narrow genetic base [Two wild species in the section Eumusa produce different genotypes: lbisiana . Severaltic base . Furthertic base . In ordetic base .Musa species’ genetic diversity. For example, the application of random amplified polymorphic DNA (RAPD) techniques, which provide helpful information and new insights into the taxonomy [It is common practice in plants to use molecular markers to identify genetic differences in germplasm, identify duplicate accessions, and test for genetic fidelity . The avataxonomy , restrictaxonomy , sequenctaxonomy , and mictaxonomy . The AFLtaxonomy ,20. Furttaxonomy . This stThe study was performed in three districts of one department of the southwestern region of Jazan province in Saudi Arabia . Banana cultivars were collected from farms in the main banana-growing agroecological zones of the country. The agroecological zone of the southwestern regions of Saudi Arabia is characterized by three agroclimatic zones and ten subzones defined by geographic location and topography that differ in rainfall and air temperature . High alMusa species and subspecies were used in this study. Three samples of fresh banana leaves of each cultivar were collected from the field, packed in plastic bags, labeled with a site code, and kept in iceboxes until examination. To avoid sampling duplication from the same individual, we did not sample plants located directly next to each other  with a final elution volume of 50 mL. To check the DNA quality, we visually tested 5 uL of each sample by 1% gel electrophoresis. DNA appears as sharp bands when visualized under UV light using the Ingenius3 Gel documentation system . Extracted DNA was stored at −20 °C until required for PCR.AFLP analysis was carried out following the method of Vos et al. , with onTM  and Raw Geno V2  were used to automate the AFLP scoring. The band-binary criterion was applied to the analysis of the AFLP data as the detected bands were codified as 1 when present and 0 when absent. As the total number of samples equals 8, thus a single sample frequency = 12.5%. Bands with a frequency of >87% or <13% are often uninformative or misleading when included in the analyses [Peak Scanneranalyses ,26 and wanalyses .ST was tested with 10,000 permutations for the detected AFLP loci.The Bayesian clustering method was applied by using Structure V2.2  to invesp < 0.00). Band scoring yielded a total number of 1468 bands with 162 monomorphic ones (88.96% polymorphism) for all primer pairs applied to the eight samples  for all bulked samples combined was 1.46 ± 0.006. The expected heterozygosity under Hardy–Weinberg assumption (He) for all bulked samples combined was 0.249 ± 0.003. Samples from Dhamadh scored the highest ne (1.525 ± 0.01) and the highest He (0.292 ± 0.006) when FIS = 1. Samples from Beesh yielded the lowest ne (1.39 ± 0.013) and the lowest He (0.195 ± 0.006), while samples from Fifa scored 1.47 ± 0.010 for ne and 0.261 ± 0.006 for He . HoweveM. balbisiana population, where a similar He of 0.241 was estimated, even though wild specimens usually record much higher diversity than the cultivated ones [A high number of variable markers is possible with the AFLP technique, allowing genome-wide analysis of genetic variability. In our study, based on nine AFLP primer pairs combinations, 1468 loci were detected, compared to Opara et al. , who yieted ones .Molecular data consisting of unlinked markers are used by Structure software to infer population structure using model-based clustering. In Jazan locations, a genetic structure was detected, even though it was proven to be influenced by the genetic background of the cultivars rather than the sampling locations. Patterns of phylogeography have been tested for banana plants in China by Ge et al. , and allThere is increasing interest in identifying genes or outlier loci that underlie adaptations to different factors in several species or in finding signatures of selection and domestication ,40,41. O


Colletotrichum gloeosporioides is a species complex of agricultural importance as it causes anthracnose disease on many crop species worldwide, and strong impact regionally on Water Yam  in the Caribbean. In this study, we conducted a genetic analysis of the fungi complex in three islands of the Lesser Antilles—Guadeloupe , Martinique and Barbados. We specifically sampled yam fields and assessed the genetic diversity of strains with four microsatellite markers. We found a very high genetic diversity of all strains on each island, and intermediate to strong levels of genetic structure between islands. Migration rates were quite diverse either within  or between islands , suggesting important roles of vegetation and climate as local barriers, and winds as an important factor in long-distance migration. Three distinct genetic clusters highlighted different species entities, though there was also evidence of frequent intermediates between two clusters, suggesting recurrent recombination between putative species. Together, these results demonstrated asymmetries in gene flow both between islands and clusters, and suggested the need for new approaches to anthracnose disease risk control at a regional level. Colletotrichum is a widespread pathogen of cultivated plants /2, adapted from Wright [C. gloeosporioides species being haploid, we divided by 2 where 4 is used in diploid populations, see [The sample size was 560 sample strains in total from 58 yam fields, from 16 fields in Guadeloupe , 18 fields in Martinique  and 24 fields in Barbados . Since there was a dramatic variation in strains sampled by fields, the field was not further analyzed as a structuring level for genetic diversity. Genetic analysis was run with hierfstat  in R 4141. We rem Wright  by correons, see ). Lastlyons, see , to explThe extant of genetic diversity was very high for all populations, with the four loci demonstrating high allelic richness , a high share of these alleles among islands see . Most alColletotrichum samples, up to 61 strains in total. Most identical multilocus genotypes occurred in pairs or triplets . Clonality was thus representing about 10.89% of total samples, spread similarly among islands. Interestingly, few clones were actually sampled within fields , while many clones were distributed in different fields within populations . In a few cases, clones we sampled between different populations: three times between fields in Basse Terre and Grande Terre, and once between Grande Terre and Barbados. The latter situations probably represented recent migration events.As a consequence of this dramatic diversity level, most strains were characterized by a unique genotype, and we counted only 20 multilocus genotypes shared among s was lower than Ht or Ht′, and both Dst and Dst′ show an important share variation between populations for all loci, st, Fst′ and Jost Dest estimates give evidence of a geographical effect of Archipelago condition, with signs of moderate to strong genetic structuring of C. gloeosporioides , and situations where local dispersal was greater than long-distance migration  . We can Colletotrichum species from C. gloeosporioides complex in yam fields in the Caribbean  (work in progress). All islands presented their share from two clusters (origins are interspersed in both), in approximately similar proportions save for Martinique which demonstrated no samples from the leftward cluster . Allelic diversity was rich enough to demonstrate both diagnostic alleles, sometimes to field level, and importantly shared genetic components between islands. Clonality was nevertheless relatively low, suggesting asexual multiplication is not contributing strongly to the local structure at the field level, but that contamination occurs via many sources, most probably from local vegetation. Genetic structure was strong, indicating that study populations indeed function as distinct entities at least partially, yet also highlighted the importance of long-distance migration , often with rates greater than local dispersal . Lastly, PCA highlighted three distinct genetic clusters, indicative of the sampling of three putative species within the complex, with one cluster fully differentiated while two clusters exhibited numerous intermediate genotypes thus hinting to casual recombination between strains. Clusters were sampled in all the study islands. We will discuss these results in the light of anthracnose disease management on yams.Our results showed astonishingly high levels of genetic diversity of Colletotrichum) [Colletotrichum as prevalence in natural flora was shown to be particularly high [Genetic diversity levels were high, as expected given the propensity of microsatellite markers to mutate. Furthermore, at field and population levels, allelic diversity was more important than clonality, and most sampled strains had distinct genotypes. This study is confirming earlier results based on RAPD markers in the same patho-system (Yam/trichum)  or in ottrichum) . This sttrichum) ,45,46). rly high , thus reColletotrichum species may be seen as “storm riders” and following dominant winds (northwards) as migration roads. This reinforces the importance of monitoring source populations for disease risk estimation. A further hypothesis regarding wind-based long-distance dispersal, not accounted for in the case of anthracnose to the best of our knowledge, is that the Caribbean region is also casually and seasonally subjected to sand mists originating from Sahelian West Africa  [Colletotrichum species in the Caribbean, and this phenomenon should be the focus of further research, especially focusing on Ivory Coast where D. alata is also the dominant yam cultivated as in the Caribbean islands [Migration rates were reasonably high, yet varied considerably between constitutive populations, segregating situations where the intra-deme dispersal was lower than long-distance migration, and conversely, situations where local dispersal was greater than migration. Overall, these results suggest strong metapopulation dynamics , with soor Lent) . Since sor Lent) , West Af islands . In summ islands , and canColletotrichum spp. are known to casually recombine [C. gloeosporioides and C. acutatum) [Colletotrichum species are indeed notoriously hard to define, and while the approach of morpho-species developed by von Arx [Colletotrichum species . Our team usually favours a morpho-species approach to understand the ecology of C. gloeosporioides species complex , and, andCollC. gloeosporioides complex sampled in Water Yam fields in the Lesser Antilles were genetically highly diverse and demonstrated a dominance of sexual reproduction over clonality and asexual multiplication. Lesser Antilles populations are structured, with important long-distance migration, viscosity in local dispersal probably due to vegetation acting as natural barriers. Some populations (Barbados) are propagule sources at a regional scale. Three species coexist on Yams, but there is strong evidence of recombination between some of them, furthering the importance of sex events in the dynamics of recombination in the Genus and increasing diversity in rich reservoir pools, thus raising anthracnose disease risk. Potential metapopulation functioning in the Caribbean suggests that anthracnose control will be difficult to sustain only by increasing genetic resistance in varieties, though potential solutions exist to manage risk include: i/careful monitoring of strain skill in inoculating yams aggressively, especially in source populations; ii/increasing viscosity of dispersal in the landscape by increasing vegetation/tree cover; and iii/a regional varietal scheme allowing rotation of cultivars with different resistance levels to avoid local matching of Colletotrichum strains and yams.Strains from the


Vulpes lagopus), and to a lesser extent in red fox populations (Vulpes vulpes). Red foxes are thought to be responsible for sporadic southward movement waves of the ARVV outside the enzootic area of northern Canada. In this study, we wanted to investigate whether red foxes displayed notable levels of genetic structure across the Quebec-Labrador Peninsula, which includes portions of the provinces of Quebec and Newfoundland-Labrador in Canada, and is a region with a history of southward ARVV movement waves. We combined two datasets that were collected and genotyped using different protocols, totalling 675 red fox individuals across the whole region and genotyped across 13 microsatellite markers. We found two genetic clusters across the region, reflecting a latitudinal gradient, and characterized by low genetic differentiation. We also observed weak but significant isolation by distance, which seems to be marginally more important for females than for males. These findings suggest a general lack of resistance to movement in red fox populations across the Quebec-Labrador Peninsula, regardless of sex. Implications of these findings include additional support for the hypothesis of long-distance southward ARVV propagation through its red fox reservoir host.Rabies is a lethal zoonosis present in most parts of the world which can be transmitted to humans through the bite from an infected mammalian reservoir host. The Arctic rabies virus variant (ARVV) persists mainly in populations of Arctic foxes ( Rabies is a lethal zoonosis in mammals, including humans, present in most parts of the world , 2. The Vulpes lagopus) to dogs and people [Vulpes vulpes) is thought to be a competent reservoir species for the ARVV, although the virus strain does not seem to persist to the same extent in red fox as it does in Arctic fox populations [Rabies has been documented in the Canadian Arctic, Greenland and Svalbard for a long time, known to be transmitted by Arctic foxes  and the striped skunk (Mephitis mephitis) [Understanding how pathogens spread over the environment is of crucial importance for implementing disease management efforts during outbreaks of zoonoses , 21. We ephitis) –24. TherInvestigations of the population genetics of Arctic and red foxes suggest a low degree of genetic differentiation in both species, and almost no effect of geographic distance on gene flow –27. SeveFor this study, we used two sets of red fox samples collected as part of two different projects, totalling 675 individuals. The first dataset includes 365 individuals collected from two sources, from 2012 to 2017 across the Quebec-Labrador Peninsula and southern regions of Quebec . A portiFor both datasets, DNA was extracted from tissue samples using the DNeasy Blood & Tissue Kit , according to the manufacturer’s instructions. Across the two datasets, we used four thermocyclers: Mastercycler Pro S, Mastercycler X50a , 2720 Thermal Cycler and SimplyAmp  to execute the Polymerase Chain Reaction (PCR) amplification of 15 microsatellite markers developed for a range of canid species, including domestic dogs, Arctic fox and red fox. Specific PCR program and recipe used to genotype both datasets are detailed by Alanazi, 2020 . For theWe selected four locations  > 1, we assigned the most likely cluster to each individual, and we calculated Hedrick’s G’K), using 100,000 iterations, and a burn-in period of 10,000 iterations. We executed 10 runs for each K value between 1 and 10. We used the admixture model, individual alpha for each population, allele frequencies correlated among populations and inferred a separate lambda for each population. We kept all other parameters at default value. We used the CLUMPAK software [K. We executed the analysis a second time, using the most probable K determined in the prior analysis, to calculate membership probability of each individual to each identified genetic cluster.First, we used the Bayesian clustering algorithm implemented in the Structure 2.3.4 software . We did software , in whicsoftware  method, Secondly, we ran an analysis using the ‘adegenet’ package in R 4.2.1 , which iWe also looked for evidence of isolation by distance across the study area, using a matrix-based linear multiple regression approach. We measured the effect of geographic distance on genetic distance. We calculated Smouse and Peakall’s  individuUsing data from Sites 1–4 n = 252; , we founUsing data from 13 markers, we found no significant deviation of Hardy-Weinberg equilibrium in Sites 1–4, at a threshold α = 0.05 after Bonferroni correction . We obseAcross the combined dataset, average number of alleles was 8, observed heterozygosity was 0.658, and expected heterozygosity was 0.700. All these values are similar to those calculated for Sites 1–4 .Using the combined dataset of 675 red fox individuals, our genetic clustering analysis using both approaches  showed highest probability of a total of two clusters, when analyzing all individuals, females only and males only . There wST value between the two genetic clusters was 0.225, 0.248 and 0.272, for males only, all individuals, and females only, respectively. Analysis of molecular variance showed 3.1%, 3.9% and 5.5% of genetic variation was among genetic clusters, for males only, all individuals, and females only, respectively. These results suggest low but notable levels genetic differentiation between the identified two genetic clusters for all three analyses.Hedrick’s G’Our multiple regression on distance matrix analyses revealed a significant effect of geographic distance on genetic distance, for all individuals, for females only and for males only . The sloOur study investigated the genetic structure of red foxes across the Quebec-Labrador Peninsula and southern regions of Quebec. Based on previous reports, we expected weak genetic differentiation and weak spatial genetic structure across the study area, despite its large extent , 42–44. Our findings of a marginally lower slope estimate of isolation by distance for males, and slightly lower genetic differentiation among genetic clusters for males, suggest the presence of slightly male-biased dispersal. Altogether, the huge geographic distances surveyed in our study did not seem to affect either sex to a large extent. In most mammal species, we observe female philopatry and male-biased dispersal , althougOur results also highlight the larger long-distance movement potential in red foxes compared to two other mesocarnivorous rabies reservoir species such as striped skunk and raccoon, which showed a larger slope estimate of isolation by distance across a much smaller but partially spatially overlapping study region , 23, 51.One major limitation of our study is low sampling in the interior of the study region. Reasons for this sampling bias include lack of human settlements away from the coast in northern Canada, and lack of transportation options to reach the interior areas. However, the typical long-distance dispersal behavior seen in red foxes in general and our findings of a single genetic cluster across most of the northern portion of the Quebec-Labrador Peninsula give us confidence that we were able to capture most of the genetic variation present in this area. Another limitation is our use of two datasets collected for different studies and genotyped using different methods. However, our site-specific analysis was unable to parse the two datasets into separate populations, across four sites. Altogether, we are confident that using two datasets for our study did not reduce the representativity of our results. Finally, almost all red fox samples used in this study either tested negative for rabies, or were not tested, and rabid animals may display a hyperexcitable or a paralytic behavior, and may therefore hypothetically disperse over larger or smaller distances than non-rabid individuals, respectively, during these disease stages. However, rabies symptoms only appear in the late infection stages several days before death occurs. Therefore, infection status is unlikely to have a significant effect on genetic structure, as hypothesized by Talbot et al.’s  study onAs noted by Nadin-Davis et al.’s  study, rS1 TableGenotypes at 15 microsatellite markers, geographic coordinates rounded to the second decimal, year of collection, species and sex for all specimens used in this study. Missing data are labelled “NA”.(XLSX)Click here for additional data file.S1 AppendixGraphs showing likelihood and Delta K values for each value of number of genetic clusters (K) for red fox samples from Sites 1–4, for all red fox samples, for females red fox samples and for male red fox samples (Outputs 1–10), and graphs showing various results from the spatial principal components analyses for all red fox samples, for females red fox samples and for male red fox samples (Outputs 11–16).(DOCX)Click here for additional data file.


For both evaluation years combined, genetic gain through RCRGS reached 12.3% from cycle C0 to C3 and realized gain was 0.28 ton ha−1 per cycle with a GY from C0 (6.88 ton ha−1) to C3 (7.73 ton ha−1). RCRGS was also associated with some changes in important agronomic traits that were measured  but not selected for. To account for these changes, we recommend implementing GS together with multi-trait prediction models.Genomic selection (GS) in wheat breeding programs is of great interest for predicting the genotypic values of individuals, where both additive and nonadditive effects determine the final breeding value of lines. While several simulation studies have shown the efficiency of rapid-cycling GS strategies for parental selection or population improvement, their practical implementations are still lacking in wheat and other crops. In this study, we demonstrate the potential of rapid-cycle recurrent GS (RCRGS) to increase genetic gain for grain yield (GY) in wheat. Our results showed a consistent realized genetic gain for GY after 3 cycles of recombination (C GS signhttps://www.cimmyt.org) has explored GS as a new applied breeding tool since 2009 , and recycling them based on total genetic values.The International Maize and Wheat Improvement Center  and its incorporation in the statistical analyses of multi-environment trials by modeling G × E interaction with the factor analytic model as shown in The CIMMYT Global Wheat Program started implementing GS as a routine breeding tool in 2013, and, since then, has made important contributions by developing and testing several new genome-enabled prediction models  consisted of 1,609 F4 lines derived from 14 F2 families, which were based on 16 parents from the CIMMYT spring bread wheat breeding program. Eleven F2 families comprised 94 to 95 F4 lines and 3 F2 families included 186 to 190 lines. F2 individuals were genotyped with the Infinium iSelect 90K SNP genotyping array (https://www.illumina.com). The F2 individuals were phenotyped as F4 lines at the Campo Experimental Norman E. Borlaug (CENEB) in Ciudad Obregón, northern Mexico. The F4 trial was sown as an augmented block design with 2 replications, including the line “BORLAUG100 F2014” as a repeated check. The phenotypic data included grain yield , days to heading , and plant height . Agronomic traits (DTH and PH) were only measured in one replication. Best linear unbiased estimators (BLUEs) for GY were assessed for all genotypes. A numerical relationship matrix (A) derived from the pedigree was also available for all individuals in the training set. This relationship matrix was computed by the “coefficient of parentage (COP)” using the BROWSE software (The training population (Csoftware .0), the 10 highest yielding lines of 6 F4 families each were selected as parents to form cycle 1 (C1). The 6 F4 families were selected based on several criteria: their rank in GY (BLUEs) in the training population, GY heritability, and the estimated genomic prediction ability within and between F4 families. The agronomic traits (DTH and PH) were not considered when making selections. The 60 selected F4 lines were planted in the greenhouse on 3 different dates (3 pots with 6–7 plants per F4 line at each date). C1 was formed by intermating the F4s (4 family (36 crosses) and 10 crosses between 11 pairs of F4 families (110 crosses), which were chosen based on the average genomic prediction ability between them. Each F4 family was used in intercrosses at least 3 times. The F1 seed of each cross was harvested and threshed to form C1.In cycle 0 . DNA was extracted from bulked tissue and shipped to TraitGenetics GmbH, Germany, for genotyping with the Illumina 20K microarray (https://www.traitgenetics.com). GEBVs were calculated for all 136 F1s. The top 26 C1F1s were selected and intermated to form the cycle 2 (C2) population. Like C1, crosses were performed within and between families , C2F1 seed of each cross was harvested and threshed. In C2, the recombination protocol was repeated. The top 29 C2F1s were intermated to form cycle 3 (C3). The number of F1s planted per cycle, the number of parents selected for next cycle recombination, and the number of crosses performed are shown in 2 recombination, C3F1 were genotyped and GEBVs calculated, but they were not recombined.In C1s from each cycle (C1 to C3) were selfed to derive F6 lines. Not all F1s within a selection cycle had sufficient seed for selfing. Therefore, a distributed set of F1s was chosen. The GEBVs of the selfed F1s ranked between 1 and 58 in each cycle, and about 50% of F1s were used in recombination, while the residual F1s were not crossed . Lines included 101 F6 lines derived from the recurrent GS cycles and 17 of the 60 C0 parents and the check. Means of GY BLUEs of each recurrent selection cycle were compared, and differences were determined using the least significant difference (LSD at 5% significance). The heritability of the trials was estimated from the variance components using the equation:A total of 118 lines were field tested together with the cv. “BORLAUG100 F2014” as standard check for 2 crop cycles (2020–2021 and 2021–2022) at CENEB. The lines were grown in 2 trials of 60 entries (59 lines + check) in an incomplete block design with 2 replications. The evaluations were conducted under optimal conditions, i.e. 500 mm of irrigation water, mechanized and chemical control of weeds, diseases, and pests. The 240 plots were of dimension 2.8 × 1.6 m and sown at a seed rate of 120 kg har = number of replications, e = number of environments (years), σ2 = error variance, with1 plants with the lower-density Illumina Infinium 20 K wheat SNP array. A total of 7,815 markers overlapped between platforms. We imputed missing marker genotypes at random according to allele frequencies and subsequently removed monomorphic markers and markers with a minor allele frequency smaller than 0.05. After this quality control, 7,139 markers were available for further analysis. The 101 derived F6 lines were also genotyped with the Illumina Infinitum array (TraitGenetics) to compute the genetic diversity maintained in each selection cycle.As described above, the initial training population was genotyped with the Infinium iSelect 90 K SNP genotyping array for wheat and the recurrent selection F1s in each recurrent cycle: (1) the genomic best linear unbiased prediction model (GBLUP), (2) GBLUP including the pedigree information (P + GBLUP), (3) Reproducing Kernel Hilbert Spaces with Kernel Averaging (RKHS-KA) method, and (4) Reproducing Kernel Hilbert Spaces with Kernel Averaging and Pedigree (P + RKHS-KA). While all models were computed, selections during the RCRGS scheme were based only on Model 4.We considered 4 different prediction models to fit the training population for selecting the best parents to be crossed and initiate the RCRGS as well as for selecting the best FFor the prediction (GEBV) and selection of the best parental candidates for the next cycle, we focused on (1) assessing additive effects by including the pedigree (P) information (numerator relationship matrix) and thus emphasizing between family variance, and (2) including genotypic values of individuals where both additive and nonadditive are included on the genomic information (RKHS-KA). Using pedigree and marker information together has been successful in decreasing the interval cycle at the early stages of population improvement .The GBLUP model has become widely used in genomic prediction e.g. . The mody is a vector with the response variable of dimension n × 1 (phenotypes), μ is an intercept, 1 is a vector of ones, K = MM′/p is a genomic relationship matrix derived from markers  and the genomic relationship matrix derived from markers described before  derived from the coefficient of co-ancestry between the wheat lines and wherei, i′ , the bandwidth parameter controls how fast the covariance function drops as a function of the distance. The estimation of the bandwidth parameter is computationally demanding and to overcome this problem, The Gaussian kernel commonly used in genomic prediction is e distributed independently, with 3 different Gaussian kernels computed with bandwidth parameters m that corresponds to the median squared Euclidean distances between lines without including the diagonal entries  were were4) w0 and the phenotypically evaluated F6 lines from the different selection cycles C1, C2, and C3. Principal component analysis (PCA) was performed to assess the genetic relationship between lines. The matrix of genetic distances and PCA were generated with the packages “adegenet” and “ggplot2” using the R Software  to an 2h of 0.73 for family 4 (NELOKI//KACHU/KIRITATI). To select the parents for rapid cycle recombination, we implemented random cross-validation within and between the F4 families. Prediction abilities using the P + RKHS-KA model within the families were significantly higher than between the families but varied widely. The highest prediction ability within families was 0.496 for family 12 (MUTUS*2/JUCHI/6/COPIO) and, between families, it was 0.310 for family 3 (NELOKI//KFA/2*KACHU). We further assessed the COP between families. Mean COPs had an overall lower range compared with prediction abilities, the most distinct family being family 2 (NELOKI/WAXBI) with a value of 0.483  with the overall highest GY heritability and good prediction ability within and between families. The 10 highest-yielding F4 lines of each family were selected as parents in C0  and declined in the subsequent cycles. Correlations also declined in the selected subsets of F1s in comparison to the entire F1 population.In addition to the P + RKHS-KA model, which was the only model applied for the selection of parents in each recombination cycle, GEBVs using the RKHS-KA model without the numerical relationship matrix  setting . The GEB0, C1, C2, and C3 and the repeated check were used for field evaluation at CENEB across 2 crop cycles. The mean GY for each cycle and the average gain per cycle are shown in −1, but not significantly different from Year 2 (2021–2022) with an average of 7.51 ton ha−1  had the lowest GY, with an average of 6.88 ton ha−1 across years. The same 17 parents revealed an average GY of 7.31 ton ha−1 in the original training population, which was the same as the 60 initial parents used in C0 (7.31 ton ha−1) and higher than the average GY (6.71 ton ha−1) across all entries in the training population . In Year 2, C0 entries showed a higher GY average than C1 and C2 entries , while in the subsequent cycles, PH slightly increased, but with no significant change. For all 3 traits, C0 showed nonsignificant values compared to the check.In the training population, genetic correlations of PH, DTH, and DTM with GY were in general low  and high levels of indirect selection were not expected. The effects of GS on the unselected agronomic traits PH, DTH, and DTM are presented in 0 were higher than between the F6 lines in each recombination cycle, but mean distances between the F6 lines did not significantly decline from cycle C1 to C3. The largest genetic distance was observed between the group of C0 parents and the F6 lines in C3. Principal component analysis at a 2-dimensional scale depicted 3 groups for the initial 60 parents (4 family (families 2 and 12). The larger group (group 2) included the parents of the 4 additional F4 families  characterized by their common parent “KACHU.” Lines in each of the selection cycles follow approximately the same, but wider patterns driven by intercrossing.The genetic diversity in each of the selection cycles computed by Nei’s standard genetic distance is displayed in  parents . The 2 s1s that originated from 14 CIMMYT crosses, based on 16 elite breeding lines.Accelerating the genetic progress of major cultivated crops such as wheat, maize, and rice is necessary to increase production in response to the global food crisis , it is necessary to account for the number of cycles per year (2–3 cycles per year in this study) and for the time from the initial cross to the last cycle . Therefore, given that GY from C0 (6.88 ton ha−1) to C3 (7.73 ton ha−1) increased by 12.3%, the average genetic gain of 0.28 t/ha per cycle is equivalent to 0.187 ton ha−1 year−1 [i.e. (3 · 0.28)/5] under our conditions and equivalent to 0.242 ton ha−1 year−1 under optimal theoretical conditions. −1 year−1 in optimally irrigated environments relative to a widely grown cultivar “ATTILA” and 0.044 ton ha−1 year−1 relative to several local checks. −1 year−1 under irrigated and rainfed (limited drip irrigation) conditions, respectively. Therefore, the short-term genetic gain from RCRGS observed in the populations used in this study (0.187 ton ha−1 year−1) is higher (up to six times) than observed in previous CIMMYT studies under phenotypic selection, which, however, were longer-term studies between 8 and 50 years and were achieved in national trials in the case of To further compute the realized genetic gain per year . In this study, we balanced crosses within and between bi-parental F1s for each recombination cycle, generating 3 times more crosses between bi-parental F1s than within F1s, which we propose to be a reason that genetic diversity remained at a similar level throughout recombination cycles.High rates of inbreeding per breeding cycle with GS have been observed in simulations and empirical studies  in our study. Lines in cycle Celection . It willA (P + RKHS-KA) for the selection of new parents in each cycle. We favored this model because it demonstrated a significant positive correlation between observed yields of F2:4 lines and predicted GEBVs of F2 single plants in the study of 2s in We calculated GEBVs based on the nonlinear Gaussian kernel function, including the relationship matrix 1 individuals was limited in several cases. Being potential new parents, F1s were sown at 3 dates for subsequent crossing, and insufficient seed remained for selfing. It could, therefore, be recommended to apply GS at the F2 or F3 breeding generation to bypass the limited amount of seed for selfing (Rapid generation advance or speed breeding can achieve up to 6 generations by year for spring wheat (Different GS strategies are likely to be relevant in individual breeding programs and each program must determine which strategy is the best choice. This will be specific to the biological specificities of a crop, the breeding organization itself, and its economic context. Wheat breeding programs tend to use GS to control for G × E interaction, predicting the total genetic values of individuals, where both additive and nonadditive effects determine the final commercial value of the lines. Practical implementation of rapid-cycling GS strategies in wheat is still lacking and our study shows the potential of RCRGS to increase genetic gains for GY. Further work is needed to evaluate and optimize these GS strategies in wheat and other crop species in order to support the acceleration of current breeding progress.jkad025_Supplementary_DataClick here for additional data file.


Fagopyrum esculentum and Fagopyrum tataricum  using 37 agro-morphological traits and 24 SSR markers. A wide range of variation was observed in both species for most of the traits analysed. The two species differed significantly in most traits, with traits related to seeds and flowering contributing most to differentiation. The accessions of each species were divided into three major phenoclusters with no clear geographic clustering. At the molecular level, the polymorphic SSR markers were highly informative, with an average polymorphic information content (PIC) of over 0.65 in both species. Genetic diversity, as determined by Nei’s expected heterozygosity (He), was high  and differed significantly between species (p = 0.03) but was homogeneously distributed between regions, confirming the lack of genetic structure as determined by clustering approaches. The weak genetic structure revealed by the phenotypic and SSR data and the low fixation indices in both species suggested frequent seed exchange and extensive cultivation and selection. In addition, 93 and 140 significant (p < 0.05) marker-trait associations (MTAs) were identified in both species using a general linear model and a mixed linear model, most of which explained >20% of the phenotypic variation in associated traits. Core collections of 23 and 13 phenotypically and genetically diverse accessions, respectively, were developed for F. esculentum and F. tataricum. Overall, the data analysed provided deep insights into the agro-morphological and genetic diversity and genetic relationships among F. esculentum and F. tataricum accessions and pointed to future directions for genomics-based breeding programs and germplasm management.Characterisation of genetic diversity is critical to adequately exploit the potential of germplasm collections and identify important traits for breeding programs and sustainable crop improvement. Here, we characterised the phenotypic and genetic diversity of a global collection of the two cultivated buckwheat species  Fagopyrum spp.) is a dicot pseudo-cereal belonging to the Polygonaceae family and native to China, where its domestication dates back ∼4000 years [Fagopyrum includes 20 species, of which Fagopyrum esculentum (common buckwheat) and Fagopyrum tataricum (Tartary buckwheat) are the most important species cultivated worldwide [F. esculentum is outcrossing and has a relatively larger genome (∼450 Mbp) compared to F. tataricum, a predominantly self-fertile species [F. tataricum has been shown to contain duplication events that allow this species to better adapt to harsh environments [F. esculentum being widely cultivated on all continents, while F. tataricum cultivation is limited to China and India in Asia and to several areas in Europe [Buckwheat  and F. tataricum (Tartary buckwheat), with contrasting mating systems using a large set of agro-morphological traits and SSR markers in order explore in details the genetic relationship between the two species and contribute to better understanding of the role of mating systems on the patterns of their genetic diversity and structure. Importantly, this comparative analysis will aid in the development of breeding programs that efficiently utilise the available germplasm of these two main cultivated buckwheat species. Hence, the major aims of this study were (i) to provide an in-depth characterisation of phenotypic and genetic diversity and structure and (ii) to identify marker-trait associations (MTAs) related to important agro-morphological traits. In addition, the data obtained were used to develop phenotypically and molecularly informed core collections for both species to facilitate future genetic studies and breeding programs for buckwheat. However, the importance of our study is also providing trait-focused information about the two most widely cultivated buckwheat species for breeders all over the world, to implement the most interesting genetic material into breeding programmes considering variable breeding objectives and finally to exploit the precious genetic potential of buckwheat germplasm.In-depth characterisation of the agro-morphological and genetic diversity of diverse germplasm is indispensable for identifying genomic regions controlling important agronomic traits and elite accessions holding favourable alleles that can be used for future marker-assisted breeding programs. In this study, we performed a detailed phenotypic and molecular characterisation of a diverse worldwide collection of the two closely related buckwheat species, F. esculentum and F. tataricum, respectively. Similar patterns of variation were observed for most traits in both species. The highest variation was observed in the number of flower clusters per cyme, with a CV of 26.07% and 26.09% for F. esculentum and F. tataricum, respectively. The remaining traits showed moderate (10% ≤ CV ≤ 20%) variation in both species, except for days to flowering (CV = 7.26%) and seed length (CV = 6.63%) in F. esculentum and days to flowering (CV = 6.26%) in F. tataricum (p < 0.0001). However, the ranges of values (minimum and maximum) overlapped for most traits , was observed between accessions in most quantitative traits of the two species studied, with CV averaging 15.17% and 15.58% for ataricum . On the t traits .H′) was similar in both species, with mean values of 0.77 and 0.75 for the 25 observed traits in F. esculentum and F. tataricum, respectively , inflorescence compactness (cyme loose), and seed surface (irregular). Among the polymorphic traits, the highest degree of diversity was observed for leaf blade shape (H′ = 1.61) in F. esculentum and for leaf margin colour (H′ = 1.59) in F. tataricum, while seed surface and time to maturity were the least polymorphic in both species, with H′ values of 0.03 and 0.10, respectively . Although the direction of the relationship (positive or negative) was the same in both species, the strength was generally higher in F. tataricum. In both species, the strongest positive correlations were observed between cotyledon anthocyanin colouration and cotyledon/seedling leaf colour (>0.85) and between pairwise seed-related traits such as 1000-seed weight, seed width, seed length, and seed surface area (0.62–0.82), most likely reflecting the strong developmental relationships between these traits. In contrast, weak to very weak significant negative associations were observed between the pair formation of all the remaining traits in F. esculentum, except for the moderately negative correlation between the number of nodes per stem and plant height (ρ = −0.53). In F. tataricum, the most relevant negative associations were observed between growth and branching form and the number of nodes per stem with flower-related traits.The associations between all pairs of traits of both species  showed tF. esculentum and F. tataricum were clearly distinguished from each other along the first dimension, which explained 9.4% of the total variance, based on the data from the 37 traits analysed  were grouped in a large phenocluster (cluster 3) characterised by a branched inflorescence with semi-compact cyme, a high number of flower clusters, a stem with many nodes, a long leaf blade and cyme, and tall plants  and Asia (13) . The 190 accessions of l plants A. The ficlusters B. The fic region C. Howevesia (13) D.F. esculentum and F. tataricum. Of all the alleles detected, 104 alleles were unique common buckwheat, only 18 alleles were unique Tartary buckwheat, while 100 alleles were shared between the two species.The 24 SSR markers analysed in our study were transferable to common and Tartary buckwheat and were highly polymorphic in both species, except for the marker Fem1303, which was monomorphic in Tartary buckwheat. A total of 205 and 118 alleles were detected in F. esculentum, the maximum number of alleles (Na = 25) and the highest allelic richness (Ar = 15.47) were observed in SXAU048, while the lowest Na and Ar values were observed in TBP_6 with Na and Ar mean values of 13,95 and 8.64, respectively, across all loci. Major allele frequency (MAF) varied from 0.18 in Fem1322 to 0.65 in Fem1303, with a mean of 0.35  and Ft3_572 , suggesting that these two loci were the most polymorphic. In contrast, the SXAU060 572 loci  appeared to be the least polymorphic. At all loci, except the monomorphic locus Fem1303, the average Na, Ar, MAF, PIC, Ho, and He values were 6.38, 6.34, 0.44, 0.61, 0.74, and 0.66, respectively. As with F. esculentum, most of the markers analysed showed a negative fixation index with values ranging from -0.85 at Ft2_1743 to 1 at SXAU060 and an average value of -0.12 across all 23 polymorphic loci.In  of 0.35 . With va of 0.35 . With vaF. esculentum appears to be more genetically diverse than F. tataricum. This was confirmed by analysis of variance, which revealed that the two species differed significantly in allelic richness , observed number of alleles , and expected heterozygosity . Although Africa and Eurasia were not represented in F. tataricum, non-significant differences were found among regions within the species for all indices of genetic diversity, indicating that there was little variation in genetic diversity among regions  and that genetic variation was not significantly affected by region and country of origin of the accessions  was conducted to estimate patterns of genetic variation at the species and accession levels. AMOVA revealed that although most of the genetic variation was explained by differences within accessions (78.09%), significantly high variation occurred between the two species (21.09%), while no variation was detected between accessions within species, indicating substantial genetic differentiation between the two species . Furthercessions .F. esculentum and F. tataricum accessions were clearly divided into two genetically distinct groups across the first axis, while the second axis appeared to reflect genetic differentiation within the species  appeared as an outgroup genetically close to F. tataricum. UPGMA clustering based on Nei’s genetic distance confirmed the genetic relationships among the analysed accessions and separated F. esculentum from F. tataricum with 100% bootstrap support  greater than 0.94 for F. esculentum and 0.99 for F. tataricum , which explained 19.52% and 6.23% of the genetic variation, respectively, showed that the  species A. The F. support B. Howeve support B. The gr support C, indicaataricum D, with FQ = 0.81 D.F. esculentum, PCoA analysis and UPGMA clustering were not geographically clustered  ranged from 0.001 to 0.07, with an average of 0.01, indicating a very low LD. One hundred and eighty loci pairs had r2 > 0.01, while only thirty pairs were in significant linkage disequilibrium at p < 0.001  and SXAU089 and SXAU060   in  < 0.001 A. In F. < 0.001) B.F. esculentum using both approaches at the p < 0.05 probability level (R2) varied from 7.78% to 49.59% in GLM and from 7.49% to 44.60% in MLM, with an average across all markers of 24.99% and 24.67%, respectively, in both models. In GLM, the highest number of MTAs was recorded for seed length (eleven), followed by seed colour and plant growth habit (seven), while seven traits had a single MTA, such as germination duration, days to flowering, plant height, cyme length, and leaf blade shape. In the MLM, the highest number of MTAs (four) was associated with branched inflorescences and seed length, while the lowest number (one) of MTAs was recorded for eight traits such as number of cymes per plant, number of flower clusters per cyme, stem diameter, stem anthocyanin coloration, seed width, and 1000-seed weight. We also found that most SSR markers were simultaneously associated with multiple traits (multi-trait MTAs). The highest number of associations was recorded for Ft4_2725 (ten traits), followed by Ft1_114 and Ft3_572 (eight traits), Ft5_2899 and Ft8_605 (seven traits). The strongest associations were observed for the number of nodes in the stem (SNN) with Ft3_572 , SXAU138  and TBP_6 , and for the branched inflorescence (BINF) with Ft5_2899   and a general linear model (GLM). A total of 93 significant marker-trait associations (MTAs) were detected in ty level . The 73 ty level . The phe 31.10%) .F. tataricum, GLM and MLM identified 119 and 21 MTAs, respectively (p < 0.05), with 22 markers associated with 27 traits and 16 SSRs associated with 21 traits. Among all MTAs detected, there were only five overlapping associations derived from both models (R2) ranged from 6.96% between SXAU048 and seed shape to 73.18% between Ft4_2725 and seed width, with an average of 31.74%. On the other hand, MLM analysis revealed that seed shape was associated with the highest number of loci (four), followed by number of flower clusters per cyme (three), seed colour (two), and number of nodes per stem (two). Among the detected MTAs, the R2 varied from 14.68% for the association between Fem1322 and germination duration to 74.08% for the association between Fem1840 and seed shape  and Ft6_2849 . Seed width showed a very strong relationship with GB_FE_054 , TBP_5 , and Ft4_2725 . A very strong relationship was found between days of flowering and Ft6_2849 . Seed colour was strongly associated with SXAU129  and Ft2_1743  (In h models . In the ed shape . Notably 39.72%) .F. esculentum , comprising 13 accessions from Europe, six from Eurasia, three from Asia, and one with unknown origin. At the molecular level, the retention rates of Ar, Na, Ho, and He between the core collection and the original collection were 63.97%, 66%, 97.70%, and 100%, respectively. At the agro-morphological level, the coincidence rate (CR %) and the variable rate of the coefficient of variation (VR %) for the 12 quantitative traits were 87.51% and 129.90%, respectively, while the class coverage of the 26 qualitative traits was 91.85%. For F. tataricum, the defined core collection consisted of 13 accessions , of which five were from Europe, four from Asia, three from America, and one accession of unknown origin. In this core set, 87.36%, 88.24%, 99.15%, and 100% of Ar, Na, Ho, and He, respectively, remained from the original collection. In addition, CR, VR and CC of the preserved collection are 85.63%, 108.56% and 97.11%, respectively.Based on the analysis of SSR and agro-morphological data with PowerCore, a subset of 23 accessions was selected to form a core collection of Fagopyrum esculentum Moench (common buckwheat) and F. tataricum (L.) Gaertn. (Tartary buckwheat) are cultivated in various countries as dual-use pseudocereals with excellent nutritional and medicinal value [Optimal use and management of plant genetic resources and identification of accessions suitable for breeding programs rely on thorough analysis of genetic variation and structure of germplasm collections stored in genebanks . The twoal value . Currental value . Howeveral value . Indeed,al value , India [al value ,31,32, Nal value , and Koral value ,35. Someal value ,36 or onal value , SSR maral value  and genoal value . To overFagopyrum species could be due to various factors such as massive seed exchange, spontaneous variation, genetic drift, natural selection, and artificial selection through the domestication process.Our results indicate that the buckwheat germplasm analysed in our study exhibits great agro-morphological diversity that can be used to select elite accessions for future breeding programmes. The diversity observed in our study was similar to that observed under field conditions for common and Tartary buckwheat collections from China , Nepal ,40, IndiPrevious studies have shown that genotype–environment interactions significantly affect the agro-morphological variation in buckwheat ,57, indip = 7.03 × 10−6). These results suggest that genetic control of flowering time is very simple in both buckwheat species, making it an important trait for selection and breeding programs. Similarly, we found that plant height was associated with Ft1_114 only in common buckwheat. In Tartary buckwheat, the same marker was strongly associated with plant height and explained > 65% of the variation. This result suggests that this marker may be an optimal locus for marker-assisted breeding in both buckwheat species. In addition, our study identified numerous SSRs with pleiotropic association with seed traits and stem-related traits, providing a genetic explanation for the strong positive correlations between these traits. However, since MTAs are affected by environmental fluctuations [The high genetic diversity and weak population structure observed in the common and Tartary buckwheat collections, as well as the low linkage disequilibrium among the SSR markers analysed, indicate that the germplasm studied and the results obtained meet the requirements for marker-trait association analysis useful for marker-assisted breeding. In common and Tartary buckwheat, few studies focused on identifying molecular markers associated with agronomic traits ,27,60,70tuations , the strtuations , these cF. esculentum and 51 accessions of F. tataricum  per accession were grown to maturity during the 2021 growing season at the Agricultural Institute of Slovenia in Ljubljana, Slovenia . Seeds were sown on May 10 in 7-L plastic pots filled with Potgrond H substrate  in a glasshouse heated to 22 °C. One month later, the pots were moved outdoors. All accessions were phenotyped for 12 quantitative (Fagopyrum spp.) according to the International Plant Genetic Resources Institute (IPGR) [n = 5) were used to calculate the mean values of the accessions for the quantitative traits, and a representative plant was selected to characterise the accession for the qualitative traits.The plant material used for the study consisted of the seeds of 190 accessions of ataricum . The gertitative  and 25 qtitative  agro-more (IPGR)  and the e (IPGR) . For pheFem [SXAU [Ft [GB-FE [TBP [Genomic DNA was extracted from a young, healthy leaf tissue using the DNeasy Plant Pro kit  without the addition of PS buffer at the homogenisation step, as described in Pipan et al. . ExtractFem  . The package “pastecs” [diverse” [stats” package with p ≤ 0.05 as the significance threshold. The association between pairwise analysed traits was estimated via Spearman correlation coefficient (ρ) using the package “CorrPlot” [FactoMiner” and “Factoextra” [fviz_mfa  function, and the contribution of each trait to variation among accessions was graphically presented based on each contribution to the first three MFA dimensions. The same packages were then used to perform further MFA analyses on the data for each species, and hierarchical clustering based on principal components (HCPC) was applied to the scores of the three MFA dimensions to divide the accessions into phenotypic groups (phenoclusters) with similar agro-morphological traits using the function fviz_dend .To evaluate the variability within and between the common and Tartary buckwheat species, the data of agro-morphological traits were analysed by univariate and multivariate approaches using the statistical programming environment R version 3.4.4  using the R package “poppr” [ade4” [poppr” and GenAlex v.6.5.2 [poppr’ package [F. esculentum and F. tataricum were genetically differentiated and to infer the genetic structure pattern at the species level. Each Bayesian clustering analysis was performed with the admixture model and the number of inferred clusters (K) set as 1–10, with the application of a burn-in period of 10,000 steps followed by 100,000 Monte Carlo Markov Chain replicates for each independent run. STRUCTURE outputs were then analysed using Structure Harvester v0.6.94 [SSR data for both species were first analysed using PowerMarker version 3.25  in ordererfstat” . Variati “poppr”  on the S” [ade4” . Further v.6.5.2 , respect package . Additio package  was used v0.6.94  based on v0.6.94  to deterr2) using the program TASSEL version 2.1 [p < 0.05, and the marker R-square (R2) was used to estimate the amount of variation explained by the SSR locus.For each species, the linkage disequilibrium (LD) in its significance between pairwise SSR markers was evaluated through the squared allele frequency correlations  and F. tataricum (L.) Gaertn. (Tartary buckwheat) were given. The obtained results showed that the analysed collections have a great genetic diversity that can be used in future breeding programs. The genetic diversity of common buckwheat was significantly higher than that of Tartary buckwheat. However, for both species, this diversity was not significant between regions, indicating frequent seed exchange and/or recent extensive cultivation and selection. In addition, LD was very low between the SSR markers analysed, which is a desirable feature for using this germplasm panel for association studies and deciphering genetic control of agronomic traits of interest. The variation in agro-morphological traits analysed in our study was controlled by many genomic regions with pleiotropic and multipolygenic effects. To further explore the genetic potential of the buckwheat germplasm studied, core collections were established for both species for further genetic studies and future breeding efforts.This work has provided a deep insight into the agro-morphological and genetic diversity and structure of a worldwide collection of the two cultivated buckwheat species,