This second condition, which is highly associated with overweight, should be prevented from childhood onwards. Personalized nutrition could bypass the problem, reaching a scientific conclusion on the individual subject. This article focuses on childhood and adolescent diabetes, aims to provide a narrative summary of nutrition over the past century, and promotes the concept of personalized nutrition to pediatricians and pediatric diabetologists as a possible tool for the treatment of type 1 diabetes and the prevention of type 2 diabetes.After the discovery of insulin, nutrition has become central in the management of diabetes in order to limit glycemic rise after meals, optimize metabolic control, and prevent complications. Over the past one hundred years, international scientific societies have consecutively refined nutritional needs and optimized food intake for the treatment of diabetes. In particular, over the past century, nutrition applied with pumps for the administration of insulin and continuous glucose monitoring have allowed substantial advancement in the treatment of type 1 diabetes mellitus. The role of some substances, such as vitamin D and  Type 1 diabetes mellitus (T1DM) is the most common endocrine disease in childhood. Its incidence is increasing in all Western countries and is showing a younger age of onset . T2DM inT1DM has a genetic basis related to autoimmunity, which accounts for 50–60% in the determinism, and T2DM has a polygenetic basis, which approximately contributes 50% to the disease. Because genes are expected to be stable over time, it is presumed that environmental determinants and gene–environment interactions are responsible for the increase of both diseases. Nutritional factors favoring or protecting from diabetes, overweight, obesity, and lifestyle, might act as environmental determinants. It is essential to detect each one as a protective, trigger, or accelerator factor of diabetes, because those determinants can be modifiable.In this article, the evolution of nutrition in diabetes in the past century is summarized and light is shed on new perspectives to pediatricians and youth diabetologists regarding type 1 and 2 diabetes mellitus .International Scientific Societies have provided updated guidelines for T1DM and T2DM based on evidence, which represent the state of shared knowledge on diabetes care, including lifestyle and nutrition ,8. RecenThe nutritional intake of carbohydrates, fats, and proteins, which are macronutrients, are defined in n-6 PUFA) and fish, olive oil, and vegetables (n-3 PUFA). Oily marine fish, rich in n-3 PUFA, is recommended once or twice weekly in amounts of 80 to 120 g , and in 2014, born in Italy, 5.1 years [IQR 2.2–7.7] vs. born in their original countries, 7.8 years ) ) .n-3 PUFAs since the clinical onset of T1DM [n-3 PUFA  and DHA (Docosahexaenoic Acid) ratio 2:1). After one year of follow-up, this child showed a partial remission with persistent fasting of C-peptide (marker of endogen secretion of insulin), and a low daily insulin need (<0.05 IU/Kg/day) with optimal metabolic control [n-6 PUFAs. After one year of supplementation of n-3 PUFAs , a decreased insulin demand (p < 0.01) was found, particularly as pre-meal boluses (p < 0.01), and a decreased insulin demand adjusted for HbA1c . . .n-3 PUFAs) supplementation during the first year of life was associated with a lower risk of childhood-onset T1DM . Norris et al., 2007, in Colorado (US) [n-3 PUFAs was associated with a reduced risk of islet autoimmunity , and more recently in 2021, Löfvenborg et al., in a large European study (EPIC-InterAct Study) [n-3 PUFAs plasma levels. In this study, individuals with high levels of GAD65 (≥ 167 IU/mL) and low n-3 PUFAs plasma levels had a higher risk of incident diabetes . This suggests that the intake of dietary fish or high n-3 PUFAs levels may prevent or delay diabetes onset in GAD65 antibody-positive individuals.Finally, Stene et al., 2003, in a large population-based, case-control study in Norway  showed tado (US)  in DAISYt Study) , found aIn conclusion, pending further knowledge, we speculate that some nutritional interventions could assign advantages over T1DM.Specifically, vitamin D supplementation (i) in immigrant children from Africa, possibly since infancy and already during pregnancy in their mothers, (ii) in the siblings of those with T1DM if vitamin D deficiency is found, (iii) at onset of T1DM according to the above-mentioned study of Treiber et al. While a level of vitamin D of 25(OH)D, 30 ng/mL is generally considered adequate for the prevention of skeletal diseases and CVD, the threshold for other diseases, including T1DM, should be higher, in the range of 40 or 60 ng/mL . Those ln-3 PUFAs as supplementation, or fish intake, (i) in individuals with GAD65 autoantibodies, (ii) at the onset of T1DM, when there is a proved deficiency, in co-supplementation with vitamin D, for favoring a partial remission, probably counteracting the process underlying the selective autoimmune destruction of β-cells. The n-3 PUFAs supplementation should be coupled with diet, increasing n-3 and lowering n-6 PUFAs intakes. In planning a clinical intervention on the shortage of n-3 PUFAs, the n-6/n-3 ratio is crucial. Given that n-3 PUFAs, n-6 PUFAs, and saturated fats compete for the common metabolic pathways regulated by the same enzymes for the synthesis of eicosanoids with opposite functions, the ratio n-6 PUFAs: n-3 PUFAs is the best reference, and it should be optimal, ≤4:1. [n-3 PUFAs in plasma or in red blood cells, or simpler AA: EPA (Arachidonic Acid n-6: Eicosapentaenoic Acid n-3 PUFAs) ratio, are quite different and non-interchangeable parameters for investigations. The evaluation of PUFAs levels in red blood cell membrane phospholipids possibly represents the most precise approach [Assuming l, ≤4:1. . Note thapproach .One point that remains open is whether these nutritional interventions represent a delaying onset or mitigation of the underlying autoimmune process of T1DM, a question that should be assessed through specific RCTs.Recent research points to glucose continuous monitoring (CGM) for categorizing glycemic response to diet, which should be introduced to personalized nutrition non-diabetic people , and pron-3 PUFAs alone or in combination with vitamin D, are not accurately assessed, at least in child and adolescent T1DM, individual glucose variability should be tested. AGP and TTP after consuming meals should be utilized as adequate tools, both under controlled and free-living conditions, as was carried out for macronutrients [Counteracting the post-prandial glycemic instability could be the research goal that personalized nutrition should aim for. Because the intake of micronutrients, specifically utrients ,64.n-3 PUFA supplementation . The comparationrison of GCMs during follow-up, 18 months before and 18 months after n-3 PUFA supplementation, saw a significant reduction of mean glucose and GV, and a significant increase in TIR (70–180 mg/dL), without significant hypoglycemics events [One more clinical case in our experience gave further confirmation. It involved a girl with T1DM with severe diabetic ketoacidosis at onset without any remission, and C-peptide levels below the limit of the test. Four years beyond the clinical onset of the disease, this patient showed a metabolic benefit during s events   and the Italian Diabetes Society (SID) have recently released guidelines for adults with T2DM . Both SEIn overt T2DM, nutritional therapy in adolescents overlaps that of adults, such as in macronutrient intake. According to AMD/SID guidelines, the Mediterranean diet has greater benefits in terms of HbA1c and weight reduction, rather than a low CHO consumption diet . Of notehttp://www.genome.gov/gwastudies, accessed on 28 September 2022), of which only one group has been linked with the known phenotypes of β-cells dysfunctions. Furthermore, one point of misunderstanding of a specific determinant is the interaction between various environmental factors, mostly the physical activity that limits or abolishes the biological effect of a nutrient on the genetic predisposition [The understanding of specific gene–nutrient interactions is theoretically investigable with the contribution of T2DM-associated SNPs loci in GWAS. This procedure represents a first step in identifying measurable phenotypes as markers of this complex disease . Quantitative trait locus analysis (QTL), identifying the gene-traits recurrently associated with a specific phenotype, allows one to define which genetic traits are most frequently linked to the disease. Through this method, more than 500 gene variants (SNPs) were found to be associated with T2DM , and T2DM could be identified previously in youth. Possible factors in this role are dyslipidemia, hepatic steatosis, and imbalance of PNPLA3 rs738409 genotype at risk of obesity, Met-S, and T2DM, with a dietetic reduction of the n-6/n-3 PUFAs ratio [n-6/n-3 PUFA ratio in the diet ameliorates the non-alcoholic fatty liver disease (NAFLD) of obese adolescents, and a reduction in high n-6/n-3 PUFA level ratio to normal 4:1 should be pursued to counteract NAFLD in obese youth.As an example, a decrease in hepatic steatosis was documented in cases of obese teenagers carrying the As ratio . In thisn-6 and low n-3 PUFAs intake contributes to obesity, but there are conflicting results of trials on the use of n-3 PUFA to reduce weight [n-3 PUFA supplementation plays a role in this specific pathway. It is certain that n-3 PUFAs decrease hypertriglyceridemia and limit the synthesis of LDL-cholesterol, but in the complex dyslipidemia underlying obesity [The recent literature suggests that the Western diet’s imbalance between high e weight . Given te weight ,85, the  obesity  and T2DM obesity , it is nn-3 PUFA supplementation should be entered as personalized nutrition in a patient, coupled with the reduction of n-6 PUFA in the diet, if a high n-6/n-3 PUFA ratio was found, referring to 4:1 as the target [n-6/n-3 PUFA levels, it is important to counteract inflammation of the obese state [Furthermore, e target . Finallyse state .n-3 and n-6, their level ratios must be of reference for clinicians. The n-6 and n-3 PUFAs determinations in red blood cell membrane phospholipids represents the most precise way for investigations [n-6/n-3 PUFA is not the only expression of nutritional contributions, because genomic investigations found different haplotypes of fatty acid desaturases, which are key enzymes in the synthesis of long chain PUFAs [Practically, given the opposite effects of igations ,83. FinaGiven the wide span of different clinical conditions underlying or coexisting with overweight, it is probable that better results are gained with an articulated path of interventions. Therefore, in the assessment of personalized nutrition, it is important to consider in each patient the degree of obesity and the concomitance of associated problems  that might refer to specific guidelines , and a pn-6/n-3 PUFA balanced diet would have the most success in conditions of “metabolically healthy obesity”, as shown by the research of Rondanelli et al. [n-6 and n-3 PUFAs and other metabolic indices, including insulin resistance, were found. Instead, in obese youths carrying the PNPLA3 rs738409 genotype with NAFLD, the intention of the precise therapy is to increase the intake of n-3 PUFA and to decrease n-6 PUFA, glucose, sucrose, and fructose derivates from soft drinks, with physical activation and, according to N. Santoro, with “the dear and old diet, always a fundamental and irreplaceable NAFLD therapy” [For example, we expect that an i et al. . In thostherapy” .Vitamin D is another micronutrient of interest in T2DM, Met-S, and prediabetes because it can interfere both in β-cell insulin synthesis and in its release through a rise in intracellular calcium concentration . Serum 2In the complex interplay between nutrients and genes, the intestinal microbiome probably plays major roles in mediating the biological effects of nutrients. Given that micro-organisms present in the intestinal tract promote the breakdown and digestion of food-derived fragments by fermentation, the composition of the gut microbiota is expected to determine the intestinal derived products. Significant amounts of short-chain fatty acids (SCFAs) are generated from the breakdown and fermentation of complex carbohydrates, such as acetate, propionate, and butyrate, which contribute to glucose and lipid metabolism. Butyrate and acetate are lipogenic, becoming substrates for cholesterol and PUFA synthesis, and propionate prompts gluconeogenesis . SCFAs, Firmicutes-to-Bacteriodetes ratios [Firmicutes-to-Bacteriodetes ratio, suggesting that the metformin effect is mediated by gut microbiota [What is significant is that both the obese state and T2DM are characterized by lowering s ratios ,106. Whacrobiota .n-3 PUFAs supplementation to repair the microbiota, would be suggestive. Moreover, among infants genetically susceptible to T1DM, it was found that a reduced diversity of microbiota and an expanded Bacteroidetes representation characterized those who developed the disease compared to those who did not [Firmicutes levels , whereas having a greater amount of Bifidobacterium in the gut  was a protective factor for T1D [n-3 PUFAs produced protection from endotoxemia caused by chemotherapy [A possible approach to a cure for obesity and diabetes through microbiota manipulation is still in its infancy. The possibility of diversifying the intestinal bacterial flora with pro- and pre-biotic nutrients, and  did not . Consist for T1D . Moreove for T1D . In animotherapy , antibiootherapy , and necotherapy , probablotherapy . Therefon-3 PUFA must be coupled with a reduction of saturated fat and n-6 PUFA in the diet. The correction of vitamin D deficiency must be supported, at least in overweight children and adolescents, with supplementations to achieve an optimal 25(OH)D level in the range of 40–60 ng/mL, which is safe and inexpensive. The n-6/n-3 PUFA ratio of 4:1, must be pursued primarily with diet, limiting both saturated fats and food rich in n-6 PUFA, and if necessary, with supplementation.In conclusion, in T2DM therapy and in its prevention, particular attention should be paid to total caloric intake and to the distribution of macronutrients and micronutrients in accordance with the Mediterranean diet model. The intake of fiber, vitamin D, and Precision medicine is defined as “an emerging approach for disease treatment and prevention that takes into account individual variability in genes, environment, and lifestyle for each person” (Medline Plus. What is precision medicine?). Personalized nutrition aims to transpose the same objective in relation to nutrients, not necessarily linked to gene–nutrient relationships, which instead, according to the recent literature, is better identified by the adjective “precise” . PersonaConsequently, in the topic of this lecture, several nutrients could be entered as personalized nutrition of type 1 and type 2 diabetes, and their prevention.A personalization process according more to pathways than to genes might (i) include the most patients , (ii) find markers that can allow monitoring of the course, and (iii) achieve measurable endpoints . The roa


SCN7A, SCN9A, MYADM-like, CCDC67, ITGA9, MGAT5, SLC19A1, AMPH, NTRK2, MSRA, SLC35F3, SIRT6, CREB3L3, and NAV3 genes as well as three undefined regions  were suggested as candidates. Most of the identified genes were involved in basic biological processes that are essential to immune system function and cellular growth; specific functions include cellular transport, histone deacetylation, cell differentiation, erythropoiesis, and endocytosis. The top significant SNP for HCT, MCH, and MCHC was found within a genomic region mainly populated by the MYADM-like gene family. This region was previously suggested to be under historical selection pressure in many sheep breeds from various parts of the world. These results have implications on animal breeding program studies due to the effect that the genetic background has on blood parameters, which underlying many productive and wellness related traits.The aim of this study was to estimate genomic heritability and the impact that genetic backgrounds have on blood parameters in Akkaraman sheep by conducting genome-wide association studies and regional heritability mapping analysis. Genomic heritability estimates for blood parameters ranged from 0.00 to 0.55, indicating that measured phenotypes have a low to moderate heritability. A total of 7 genome- and 13 chromosome-wide significant SNPs were associated with phenotypic changes in 15 blood parameters tested. Accordingly,  Ovis aries) were one of the earliest species to be domesticated by humans1. This domestication can be seen spread across various geographical settings and expanded over many continents. Native sheep breeds are important genetic resources due to their adaptive capacities built up for centuries and today they have excellent gene-environment interaction in terms of survivability2. Among the domestic sheep breeds in Türkiye, the Akkaraman sheep is one of the most widespread fat-tailed breeds, owing to its features that make it well-suited for arid environments and utilization of low-quality pastures like various other fat-tailed sheep8.Sheep , leukocytes  and platelets 12.Complete Blood Counts (CBCs) allow for the examination of blood parameters and are widely used to obtain information on the general health status of mammals13. In addition to these, genetics also play an important role in the blood parameters of sheep14. Genomic regions contributing to the determination of quantitative phenotypic characters, such as blood parameters, are termed as quantitative trait loci (QTL)16. Although, studies on the genetic mechanisms responsible for alterations of blood parameters in sheep are quite scarce, the release of a sheep reference genome (Oar_v4.0) enabled researchers to identify genetic loci underlying changes in CBC values at the whole-genome level19. One such study completed a genome-wide association study for red blood cell phenotypes in three domestic sheep breeds, namely Columbia, Polypay, and Rambouillet, where a significant single nucleotide polymorphism (SNP) was found to be associated with mean corpuscular hemoglobin concentration. Further analysis determined that variants within the MYADM-like gene family were likely responsible for the red blood cell (RBC) abnormalities seen in sheep enrolled in the study15. A separate genome-wide association study (GWAS) assessed erythrocyte-related traits in Alpine Merino sheep, where two particular genes  were found to be associated with hematopoiesis, or the production of cells contained within blood14. The aim of this study was to investigate the genetic basis of 18 phenotypes associated with erythrocytes, leukocytes, and platelets in Akkaraman sheep. For this purpose, genetic parameter estimates, a genome-wide association analysis and regional heritability mapping analysis were performed using phenotypes obtained from CBCs and genotypes obtained from a 50 K ovine SNP panel.Environmental factors such as regional pathogens, nutrition, and altitude are known to have a significant impact on blood parameters in sheepOutliers in the phenotypic observations for each trait were removed from the data. Table 9/l). Lastly, heritability estimates for platelet related traits ranged between 0.00 and 0.13, where the former was found for PCT (ng/ml) and the later for PLT (109/l). The variance components and genomic heritability estimates with their standard errors are presented in Table Heritability estimates for blood parameters ranged from 0.00 to 0.55, indicating a low to moderate heritability for measured phenotypes in Akkaraman sheep. Erythrocyte traits had heritability estimates ranging from 0.07 to 0.23, while leukocyte related traits tended to have a broader range with 0.02 for EOS (%) and 0.55 for WBC  were observed to have significant effects on WBC and RDW_CV traits. Concurrently, the live weight of animals was found to be statistically significant for NEU, LYM, EOS, and MCH. Finally, the interactions between herd and feeding type as well as, sex and feeding type, were found to be important for WBC and LYM respectively. Therefore, these were included in the GWAS models.The quantile–quantile (Q-Q) plots of the observed test statistics for each SNP after and before genomic control were compared with the expected test statistics under the null hypothesis of no association . A Q-Q plot and the estimated inflation factor lambda (λ) were obtained for each phenotype which was set to 1 with genomic control applied. The corrected p-values for each trait were obtained as a result of the GWAS and were presented on –log10 scale in the Manhattan plots given in Figs. RHM analysis results using 5 Mb windows are presented in detail in Supplementary Table 1. A total of 30 regions, 22 of which were genome-wide significant, were found to be associated with blood parameters. The genome-wide significant regions on OAR1  and OAR18  were found to be common for MCH and MCHC traits. In addition, the region between positions 50,907,002 and 53,876,295 on OAR6 was found to be significant for NEU, LYM and NEU/LYM traits. Regional heritabilites for windows were ranged from 0.05 to 0.26. While LRT values for the traits were varied between 13.99 and 49.15, p-values of the significant windows at -log10 scale were ranged from and from 4.03 to 11.92, respectively.rs428421565 and rs424356478) were found on Oar_v4.0 via NCBI genome data viewer. As a result, 11 genome-wide and chromosome-wide significant SNPs were observed to be within specific genes, namely Sodium Voltage-Gated Channel Alpha Subunit 7 (SCN7A), Sodium Voltage-Gated Channel Alpha Subunit 9 (SCN9A), Myeloid-associated Differentiation Marker-like (MYADM-like), Coiled-Coil Domain-Containing Protein 67 (CCDC67), Integrin Subunit Alpha 9 (ITGA9), Alpha-1,6-Mannosylglycoprotein 6-Beta-N-Acetylglucosaminyltransferase (MGAT5), Solute Carrier Family 19 Member 1 (SLC19A1), Amphiphysin (AMPH), Neurotrophic Receptor Tyrosine Kinase 2 (NTRK2), Methionine Sulfoxide Reductase A (MSRA) and Solute Carrier Family 35 Member F3 (SLC35F3) on OAR chromosomes 1, 2, 4, 18, 19, 21 and 25. In contrast, 3 of the significant SNPs were found in close proximity to Sirtuin 6 (SIRT6), CAMP Responsive Element Binding Protein 3 Like 3 (CREB3L3) and Neuron Navigator 3 (NAV3) on OAR chromosome 3 and 5. Also 3 of the significant SNPs were within unidentified region namely LOC101117887, LOC106991526 and LOC105608461) on OAR chromosome 2, 13 and 20.Regarding the significant SNPs, the nearest genes and/or the genes flanked by two consecutive significant SNPs  and WBC (9.68); however, for PLT (455.0) and HCT (23.25), the values were found to be higher. Finally, the mean value for RBC (5.43) was found to be lower. In another study conducted on Alpine Merino sheep, the means for MCH, MCHC, and RWD_CV were found to be 13.24, 37.74, and 0.39 which are all substantially lower than the means found in the present study . The possible differences for the results can be attributed to different environmental , physiological, age, and health-related conditions as well as different genetic backgrounds of the breeds.There are highly variable results from the few studies conducted on blood parameters in sheep28. Results in the current study were similar for RBC (0.20), PLT (0.13), HCT (0.21), and HGB (0.12), but higher for WBC (0.55). Again, for Santa Inês sheep, the heritability estimation for HCT (0.18) was found to be compatible with our current study29. Additive genetic variance of the traits and, accordingly, heritability is affected by many natural factors such as natural selection, mutation, genetic drift, and genetic migration as well as artificial selection30. Populations exposed to these natural and artificial factors for different periods of time and in various manners can be expected to have differing heritability for the same trait. However, the heritability estimates of many traits in our study were quite similar with the results of previous studies, albeit these studies contained different breeds. On the other hand, comparatively small sample size of our study led to the relatively high standard errors for the heritability estimations which might rather hamper finding differences between estimations from different studies. However, in any case, the concordant moderate heritability estimates of many blood parameters in different sheep breeds might lead one to speculate that there has not been any systematic selection applied to these parameters which might alter the share of genetic variance in the phenotypic characteristics of these animals. Thus, the genetic improvement of these parameters by selection strategies is quite possible.In the present study, low to moderate (from 0.00 to 0.55) heritability estimates were found for blood parameters Table . Studies31. The main reasons for this are suggested to be the relatively short linkage disequilibrium in the sheep genome and the highly polygenic structure of measured traits33.As a result of the GWAS conducted in this study, a total of 20 SNPs were found to be significant for 15 different blood parameters, with 7 being genome-wide and 13 being chromosome-wide. No significant SNPs were found for HGB, RDW_SD, MON and PDW traits. The current study agrees with those prior, which show that the number of SNPs in peaks obtained for complex traits in sheep is low during GWAS analyses34. The present GWAS determined that 6 erythrocyte-related traits were significantly associated with 8 SNPs, which were made up of 2 genome-wide and 6 chromosome-wide SNPs on OAR chromosome 1, 2, 5, 18 and 21  for RBC, HCT, MCH, MCHC, and RDW_CW traits. The top associated SNP regarding all measured CBC traits was observed for MCHC (p = 4.83e-11), which was rs424122039 on OAR chromosome 18  were found to be adjacent to that defined for MCHC. Importantly, these SNPs were within an approximately ~ 52 Kb distance and discovered in the MYADM-like gene family on OAR chromosome 18. Interestingly, the association between the MYADM-like gene family and variable RBC parameters in sheep has been previously reported by Gonzalez et al.15. In the multi-breed GWAS, the discovered region and rs424122039 SNP were specifically found to be associated with MCHC of three sheep breeds namely Columbia, Polypay, and Rambouillet. The region carrying the MYADM-like gene family has been reported to be conserved throughout many mammalian species 36. Protein products synthesized by the MYADM gene family have been reported to be involved in membrane formation and regulatory processes in myeloid cell lines38. In addition to these functions, the increase of MYADM gene expression levels in pluripotent cells is involved in the completion of erythropoiesis38. The genomic region containing the significant SNPs encompasses a nearly 800 Kb window and was found to be a gene-rich region mainly populated by Myeloid-associated Differentiation Marker-like (MYADM-like) protein coding genes, pseudogenes, and other genes with a high number of SNP variants . This same region was also found to be significant in selection signature analyses during the ovine HapMap project, which was carried out with 74 sheep breeds from many parts of the world32 and by Abied et. al. with thin- and fat-tailed sheep breeds from Sudan and China39. The extreme historical selection pressure of the genomic region emphasizes that this region provides superiority in the adaptive and survival capacity of sheep. Although, the top SNP and the region of interest has been previously associated with the MCHC parameter15, this is the first study discovering the association between rs424122039 and other red blood cell traits  in sheep.Erythrocytes are an important cell type as they directly affect performance and growth as well as the health status of animalsSCN7A) and Sodium Voltage-Gated Channel Alpha Subunit 9 (SCN9A), which were found to have pleiotropic effects associated with RBC and RDW_CV traits. This SCN gene family encodes for voltage-gated sodium channel proteins which are mainly involved in ion transport , especially sodium ion transport , in mammalian cells.Other SNPs associated with the erythrocyte-related traits were within genes Sodium Voltage-Gated Channel Alpha Subunit 7  gene on OAR chromosome 19. This gene encodes an alpha integrin protein which mediates cell–cell and cell–matrix adhesion. Involved biological processes include cell adhesion , integrin-mediated signaling pathway , extracellular matrix organization , and neutrophil chemotaxis . In a study of breast cancer in humans, the expression of ITGA9 was downregulated and lost in the majority of breast cancer cell line40. On the other hand, SNP rs419009933 within LOC101117887 on chromosome 20 showed pleotropic effect on NEU and LYM traits. Interestingly, this SNP had a negative effect on NEU with 4.35% of additive genetic variance, whereas it had positive effect on LYM accounting for3.07% of additive genetic variance (Table MGAT5) gene was found to be related to the neutrophil to lymphocyte ratio (NEU/LYM). MGAT5 encodes a protein belonging to the glycosyltransferase family and acts in biological processes such as the positive regulation of cell migration  and the positive regulation of the STAT signaling pathway . Previously, Bahaie et al. determined that deficiency of MGAT5 cause increased neutrophilic inflammation in allergen challenged mice41. This gene also was found to be associated with T-cell glycosylation and plasma composition of the IgG glycome in ulcerative colitis in humans42.Three genome-wide and four chromosome-wide significant SNPs were associated with leukocyte related traits on OAR chromosome 1, 2, 3, 4, 13, 19 and 20 . This SNP was located within the Solute Carrier Family 19 Member 1 (SLC19A1) gene. This gene is involved in many biological processes such as folic acid transport  and vitamin metabolic processes . It is well known that folic acid and other vitamins have a critical role in the production of white blood cells, particularly granulocytes43. Finally, SNP rs417370910, present within the Amphiphysin (AMPH) gene on OAR chromosome 4, was associated with BAS . Amphiphysin is one of the adhesion proteins, and used in many cells that perform endocytosis, which is a process regarding immune system cells  that is important in destroying the antigens 44.SNP rs419857573 was within undefined region LOC105608461 and had pleotropic effects on PLT and PCT, explaining 2.73% and 7.58% of the additive genetic variance, respectively (Table rs424459233 within the Methionine Sulfoxide Reductase A (MSRA) gene on OAR chromosome 2 was associated with MPV with 5.24% of additive genetic variance.Within the platelet traits studied, there were two genome-wide and three chromosome-wide significant SNPs detected for PLT, MPV and PCT on chromosome 2 and 25 Fig. . SNP rs447. In our study, as expected, RHM results validated the result of single step GWAS by sharing significant SNP located in the significant regions. However, some of the significant regions were not include significant SNP detected in GWAS . The regions on OAR16 and 23 were found to be highly significant for MCV and RDW_CV traits, respectively . Interestingly, no SNP or QTL were found around these two regions for these two corresponding traits. Particularly, the region  on OAR16 was associated with the MCV trait with having 45.36 LRT value and 11.08 -log10 scale p-value. Therefore, RDW_CV with the region on OAR23  was found to be significant with the highest LRT and -log10 scale p-value as 49,15 and 11.92, respectively. Unlike single SNP GWAS analyzes, the region  on OAR6 was associated with NEU, LYM and NEU/LYM traits of white blood cells. It was demonstrated that when related SNPs do not have significant effects at the genome-wide level, RHM outperformed a conventional GWA study. In the RHM approach of the estimating heritability for a specific region, the estimate of additive variance is contributed by rare variants in addition to common variants48. Since RHM uses many sources of variance in regional heritability estimation, it allows the detection of loci that could not be found in single step GWAS analyses. In this context, the regions found in OAR16 and 23, which are important for MCV, RDW_CV, traits, have a very high potential to contain the important rare variants.Regional heritability mapping approach was used in addition to the single step GWAS method to determine the existence of the association between specific genomic regions obtained from the genome and blood parameters. The RHM approach is widely used method to detect potentially important regions in the genome. It has been reported that it is a more effective method than the SNP-based GWAS method in detecting genomic regions containing multiple QTLs and rare allele with relatively moderate and small effects or epistatic effects in the genomeSCN7A, SCN9A, MYADM-like, ITGA9, MGAT5, SLC19A1, SLC35F3 and AMPH) which are important to basic biological functions such as cellular transport, histone deacetylation, cell differentiation, erythropoiesis, and endocytosis44. Some of the identified SNPs  were responsible for pleotropic effects on WBC, RDW_CV, HCT, MCH, MCHC, NEU, LYM, PLT, and PCT measured traits. Furthermore, SNP rs424122039 was found to be associated with MCH, MCHC, and HCT, supporting prior work which linked MCHC measurements to the same gene-rich MYADM-like region15. Previous studies have also shown that this region is under historical selection pressure39. In this context, the MYADM-like gene was suggested by Gonzalez et. al., (2013) as a potential candidate gene associated with erythrocyte related traits ; moreover, the results of our current study confirmed this hypothesis. Our current study is one of the limited number of studies aimed at understanding the genetic components underlying blood parameters in sheep. The current study and future studies of its kind will shed light on the genetic region that effect measured blood traits and how to use these associations to further selection programs.The results of the present GWAS revealed many genes . The results of this study can be utilized as a selection criterion in sheep breeding approaches. Considering that erythrocytes and leukocytes are directly related to adaptation and survivability of sheep, the resultant breeding approaches may produce more resistant and sustainable animals. Finally, in order to gain more sophisticated knowledge on the genetic mechanisms of blood parameters and transfer this knowledge to applications, it is strongly recommended that this and similar studies be conducted on larger numbers and in different populations.As a conclusion, in our study, it has been illustrated that 7 genome-wide and 13 chromosome-wide significance SNPs in GWAS and 30 significant regions in RHM are associated with blood parameters in Akkaraman lambs, which is an important breed in Türkiye. These SNPs were associated with 14 genes namely The study was carried out in the outskirts of Ankara province, Türkiye . The region is known to have poor pasture with a continental climate that has cold, snowy, winters and hot, dry, summers. Annual rainfall, average temperature, and average altitude are 389 mm, 11.7 °C, and 938 m respectively. In the study, 480 Akkaraman lambs , were randomly selected from 3 different farms that are registered to the National Community-based Small Ruminant Breeding Program. Lambs were born between January and February of 2021 and weaned at an average age of 3 months old. After weaning, 372 lambs were grazed on the pasture during the summer period without supplemental feeding, while 114 lambs were subjected to a 90 days of finishing period with 750–1000 g concentrate feed/day. Selection decisions of the studied populations were given around the mating period  based on the phenotypically observed growth rates without any pedigree based selection.V. jugularis in an EDTA-coated vacutainer and transferred to the laboratory in cooling box. Samples were analysed within 6 h of collection to avoid hemolysis of blood. The 18 blood parameters were measured by ZETA Laboratories . Eight of these phenotypes were erythrocyte phenotypes including total red blood cell count , hemoglobin (g/dl), hematocrit (%), mean corpuscular volume (fl), mean corpuscular hemoglobin (pg), mean corpuscular hemoglobin concentration (g/dl), coefficient of variation of RBC volume distribution width, and standard deviation of RBC volume distribution width. Leukocyte phenotypes were total white blood cell count (109/dl), neutrophils (%), lymphocytes (%), monocytes (%), eosinophils (%), and basophils (%). Lastly, platelet (PLT) phenotypes were total platelet count (109/dl), mean platelet volume (fl), procalcitonin (ng/ml) and platelet distribution width (fl). Preceding further analyses, outliers were identified and observations exceeding three standard deviations ± mean for each trait were excluded from the data. Descriptive statistics of these parameters were presented in Table Lambs were monitored from birth until the sample collection, which was around 6 months of age. Additional records including environmental factors  were collected. Animals with a clear sign of an active disease were excluded from the study. 6 ml of blood were taken from the After the analyses for blood parameters, the remaining samples were transferred to the Genetics Laboratory of the International Center for Livestock Research and Training (ICLRT) for extraction of DNA. To minimize contamination, DNA extraction was performed with the Qiacube HT automated device using a commercial kit . Quality control of the extracted DNA was performed before genotyping. Samples that passed the quality criteria  were stored for genotyping at -20 °C. Genotyping was performed using the Axiom 50 K Ovine Genotyping Array with the GeneTitan MultiChannel microarray instrument  at the Genetics Laboratory of the ICLRT. The genotyping procedure was implemented according to the manufacturer’s protocol.51. Accordingly, SNPs with minor allele frequency (MAF) lower than 0.05, a call rate below 95%, and SNPs that are mapped on sex chromosomes were excluded from the data. Additionally, samples with call rate lower than 90% and samples with an Identity By State (IBS) value of more than 95% were removed from the data. Finally, SNPs deviating from Hardy–Weinberg Equilibrium (HWE)  were excluded from the data. The threshold value for the deviation from HWE was determined with the Bonferroni correction. Raw genotype data contained 49,931 SNPs and after performing QC analyses with the described criteria, 40,463 SNPs and 479 animals remained for further analyses.After the genotyping process, filtering was performed with certain QC criteria to decrease Type-I and Type-II error rates. QC criteria adopted by McCarthy et al., (2008), The Wellcome Trust Case Control Consortium, (2007), and Weale, (2010) were considered52. In addition, the genomic relationship matrix (G), described by Astle and Balding (2009) and equivalent of model 2 provided by VanRaden (2008), was calculated to be used in the genetic parameter estimations and GWAS54.Since GWAS does not accommodate missing genotypes, an expected genotype score estimated from the population was used to impute missing genotypes, considering Chen and Abecasis (2007)2) for the traits were implemented using the general generalized linear model below and with restricted maximum likelihood (REML) approach using ‘sommer’ package of R55.Estimation of the variance components and narrow sense heritability  for WBC and RDW_CV traits; the live weight of animals for NEU, LYM, EOS, and MCH; the interactions between herd and feeding type and the interaction between sex and feeding type for WBC and LYM), u is the polygenic background effect obtained from MVN , and e is the vector of random residual errors obtained from MVN . X and Z are the design matrices mapping fixed effects and polygenic background effects to each observation respectively.GWAS were performed with Mixed Linear Models to detect significant SNPs associated with phenotypes. In the model, the additive genetic effect of SNPs was estimated by including a genomic relation matrix in the model to also account for covariance between related individuals and population stratification. The following linear model (1) was used in the univariate analysis implemented with the R ‘GenABEL’ package57. Manhattan plots were used to visualize the –log10  of all SNPs respective to their position at the associated chromosome. Genome-wide and chromosome-wide significance thresholds defined by using Bonferroni correction were applied to avoid increasing Type 1 Error rate due to multiple testing SNPs. Accordingly, the genome-wide significance threshold was set to 1.23e-06 (0.05/40.463) and the chromosome-wide significance threshold was set to 3.21e-05 ((0.05/40.463) *26). Genetic variances regarding the significant SNPs were estimated with the formula below:p’ is the major allele frequency, ‘q’ is the minor allele frequency and ‘The quantile–quantile plots were used to compare the observed test statistics for each SNP to those expected under the null hypothesis of “no association” to detect any inflation in the test statistics due to systematic biases. To avoid higher inflation in the test statistics, genomic control was applied to the p-values as described by Devlin and Roeder48. For this purpose, each chromosome for RHM was divided into 5 Mb windows with 2.5 Mb overlap. After sliding windows, 1038 windows were obtained with average 40 SNPs. The whole genome relationship matrices and regional genomic relationship matrix were constructed with the SNP information by using Genome-wide Complex Trait Analysis (GCTA) software (version 1.94.1)58. Model 2 was used to detect the contribution of each window effect as follow up below:y, µ, β and u were the same as Model 1. r is the background effect of each window obtained from MVN , and Q are the design matrices mapping fixed effects and polygenic background effect of the windows to each observation respectively. A likelihood ratio test (LRT) was used to compare full (2) and reduced (1) model to detect the impact of each window in GCTA software. Bonferroni correction test was applied to set genome-wide (p ≤ 0.05) and suggestive (p ≤ 0.10) significant levels. Regional heritability (h2reg) was calculated as In addition to SNP based GWAS analyzes, regional heritability mapping (RHM) analyzes of traits were performed to evaluate the effect of each predefined region59. Genes that are containing the SNPs were suggested as candidates. If the significant SNP was not within a gene, the nearest genes and the genes flanked by two consecutive significant SNPs were suggested as candidates. Biological information on the identified candidate genes were obtained by using DAVID Bioinformatics Resources 202161. Additionally, previously identified QTL related to sheep blood parameters were checked through Animal QTL Database to see if there is any overlap with the SNPs identified in this study62. Where there is not enough annotation in the sheep genome reference, orthology between species was utilized and annotation of the specific genes from cattle, goats, mice, and humans were used. Finally, the biological processes involved by the genes of significant SNPs were represented with their Gene Ontology (GO) terms, which can be further detailed with QuickGo website63.Positional information of significant SNPs and the nearby genes were obtained from NCBI Genome Data Viewer by using the Oar_v4.0 genome assemblyAll experimental protocols in the study was approved and conducted under the supervision of Animal Experiments Local Ethics Committee of the International Centre for Livestock Research and Training , Ankara, Türkiye, and the methods were also carried out in accordance with the ARRIVE guidelines.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.


Waxy (Wx) and ALKALI DEGENERATION (ALK), which are able to enhance the eating and cooking qualities. Moreover, only about one fifth of all varieties harbors favorable the allele of fragrance gene Betainealdehyde dehydrogenase (BADH2). Together, these results give an overall view of the genetic constitution of the target traits, which provide useful information for future genetic improvement in breeding practices.Traditional breeding strategies mainly focus on the evaluation of trait performance, but pay less attention to the changing genetic background. A comprehensive understanding of the genetic diversity of germplasms is crucial for the deliberate improvement of specific traits. A collection of 154 highland rice varieties were collected as the initial genetic resource in our breeding program to improve the pathogen resistance and eating and cooking qualities. These varieties were analyzed using a whole-genome SNP array and were clustered into three groups. Further analysis revealed that the favorable alleles of pathogen resistance genes are mostly absent in our collected varieties. However, it showed that most varieties possess favorable alleles of  Oryza sativa L.) is one of the most important staple crops that feeds approximately half of the population in the world [Rice  markers have gradually become the ideal markers used for genetic discovery research and molecular breeding. The investigation of the rice genome has revealed millions of SNPs ,8,9,10. Waxy (Wx) and ALKALI DEGENERATION (ALK)/starch synthase IIa (SsIIa) [Wbx in Nipponbare is a weak allele conferring a moderate amount of amylose accumulation and better quality [ALK in Minghui 63 is an unfavorable allele that increases the gelatinization temperature and results in a lower eating quality [Betainealdehyde dehydrogenase (BADH2), badh2.1 is associated with the rice fragrance [badh2.1 was used as functional nucleotide polymorphism for both breeding and further research.In recent years, consumer preferences have shifted towards rice with better eating and cooking qualities. Although each country or region have a particular preference of rice with a specific combination of quality traits, a common trait across most countries in Asia is the soft texture of freshly cooked rice, and its maintenance during storage . This tr (SsIIa) ,22,23. P quality , and ALK quality . Furtherragrance . SNP mutIn this research, a total number of 154 cultivated rice varieties were genotyped with 44,263 high-quality SNP markers throughout all 12 chromosomes. The genetic structure of these varieties was analyzed. By using the SNP markers located in the cloned and characterized genes [Oryza rufipogon, Oryza meyeriana and Oryza officinalis) and Nipponbare as the control variety were analyzed in this study. All the materials were planted in the experimental plot located in the Songming county, Kunming city, Yunnan province, China. The longitude and latitude were 102.41 E and 25.05 N, respectively. The sowing date was March 17th 2019, and the transplanting date was between the 14th and 16th of May.A total number of 154 local rice germplasms from the Yunnan province, China, together with 3 wild rice accessions (Flag leaves of each variety were collected for DNA isolation. DNA was extracted from the frozen leaf tissues of each variety through the CTAB (Cetyltrimethylammonium bromide) method .A total of 44,263 high-quality SNP markers  > 0.05) were obtained after genotyping the germplasm collection of 4726 cultivated rice. The distances between any two lines were measured by the ratios of polymorphic SNPs. The phylogenetic tree was constructed based on an improved version of the neighbor-joining algorithm method  using thThe genotypes of rice varieties were visualized using a program written in R scripts. The program translated the SNP data information into color bars according to the length of chromosomes. Chromosome fragments with homozygous SNP genotypes are shown as empty bars with a green border, whereas those with heterozygous SNP genotypes are shown as solid blue lines or bars.The SNP array platform was designed to cover the causal SNP(s) for most cloned rice genes according to the information in public publications . To deteA SNP array platform was designed with a total number of 44,263 high-quality SNP markers to cover the entire rice genome. These SNPs are almost evenly distributed on the 12 chromosomes with an average of 11 SNP markers per 100 kb A. The gaOryza rufipogon, Oryza officinalis and Oryza meyeriana) and the control variety Nipponbare and was subjected to whole-genome analysis with an SNP array. Based on the genotypes of the SNP markers, these varieties were clustered into three groups, named as groups I, II and III  showed heterozygous genotypes, although to a different extent  shared the functional allele of  variety . For genectively . The pro variety .To further understand the genetic background with regard to the bio-stress resistance in the collection of varieties, the number of favorable alleles possessed by different varieties was counted. About half of the varieties contain no favorable alleles of genes conferring rice blast resistance A, and moWx and ALK regulating amylose content as well as BADH2, which controls rice fragrance. Interestingly, 94.2% and 68.8% of our varieties possess the favorable alleles of Wx and ALK, respectively ). This result suggests that the other four varieties, sharing a more common background with Yunhui 290 in group II, may represent indica varieties. Likewise, the varieties of group III grouped together with Nipponbare may represent japonica varieties. Introduced varieties, such as Dozikara-1, Yuanbai and Linyou 22, are clustered in group III, but into different subgroups. This result implies that these introduced varieties have been well utilized and introgressed into local varieties, therefore creating several new varieties. However, the varieties Lvmi 3, Lvmi1, SMGR 4, Lvmi 2, Nanjing 5055 and Xuebai were clustered together, which suggests that these introduced varieties share a highly common background and have not been well utilized by local breeders.The collection of varieties was clustered into three groups . Group IOryza rufipogon, Oryza officinalis and Oryza meyeriana contain different levels of heterozygous fragments in their genome  genes for rice bacterial leaf blight resistance [Bph genes are almost absent in all the tested varieties, except one variety that contains the favorable allele of Bph18 (Xa21, and no varieties harbor the favorable allele of xa13, Xa23, xa5 and Xa7. These results reveal the fact that rice breeding during the last few decades pursued a high grain yield and paid less attention to bio-stress resistance. Nowadays, beyond obtaining a high grain yield, new varieties should be adapted to the growing conditions with less pesticides to meet the goals of sustainable agriculture. Thus, our results could provide essential information for both candidate parent and favorable allele selection for target genes in further breeding progress.Thanks to the advances in functional genomics in rice, a series of genes regulating bio-stress resistance were cloned, such as sistance , Pi genesistance  and Xa (sistance . Interesof Bph18 . For genWx and ALK genes, which confer better eating and cooking qualities to rice. These results provide useful information for the future genetic improvement of rice in breeding practices.In order to obtain a better understanding of the genetic background of the collection of varieties cultivated in the Yunnan province, China, a collection of 154 varieties cultivated in that region were subjected to a genetic background analysis with high-density SNP array, which revealed the complicated genetic diversities represented by the distinguishing background and high percentage of homozygous genomic fragments. Further analysis showed that the favorable alleles of pathogen resistance genes displayed a relatively low frequency of existence in this collection of varieties. However, most varieties possessed the favorable alleles of


This platform was used in diversity analysis of 50 rice varieties. Of the 2565 SNP markers, 2341 (91.3%) produced useful polymorphic genotype data, enabling the production of a phylogenetic tree of the 50 varieties. The mean number of markers polymorphic between any two varieties was 854. The platform was used for QTL mapping of preharvest sprouting (PHS) resistance in an F8 recombinant inbred line population derived from the cross Odae × Joun. A genetic map comprising 475 markers was constructed, and two QTLs for PHS resistance were identified on chromosomes 4 and 11. This system is a powerful tool for rice genetics and breeding and will facilitate QTL studies and gene mapping, germplasm diversity analysis, and marker-assisted selection.The development of efficient, robust, and high-throughput SNP genotyping platforms is pivotal for crop genetics and breeding. Recently, SNP genotyping platforms based on target capture sequencing, which is very flexible in terms of the number of SNP markers, have been developed for maize, cassava, and fava bean. We aimed to develop a target capture sequencing SNP genotyping platform for rice. A target capture sequencing panel containing 2565 SNPs, including 1225 SNPs informative for  Oryza sativa) is an essential component of the diet of over 3.5 billion people [http://www.fao.org/faostat, accessed 18 January 2022). The predicted increase in the global population to ~10 billion by 2050 demands urgent improvements in rice productivity. At the same time, rice breeding programs must develop varieties that require less fertilizer and are resistant to the intensifying biotic and abiotic stresses resulting from global climate change [Rice (n people . WorldwiSingle-nucleotide polymorphism (SNP) markers are now the most commonly used DNA marker type for assisting crop genetics and breeding. SNPs possess several characteristics useful in DNA markers, being abundant, codominant, and evenly distributed across plant genomes. Mapping of genes or quantitative trait loci (QTLs), genome-wide association studies (GWAS), germplasm diversity analysis, marker-assisted selection, and genomic selection studies are nowadays most frequently performed using SNP genotyping. Therefore, the development of efficient, robust, and high-throughput SNP genotyping platforms is pivotal for crop genetics and breeding.japonica rice varieties consisting of 768 SNPs [Arrays and next-generation sequencing (NGS) are two major high-throughput SNP genotyping platforms . Various768 SNPs ; two Ill768 SNPs ; two 50 768 SNPs  and OsSN768 SNPs ; and the768 SNPs . These aindica rice breeding pools [Zea mays) [Vicia faba) [Manihot esculenta Crantz) [Genotyping by sequencing (GBS) is an NGS-based SNP genotyping platform that has been widely used for genetic studies, including GWAS, gene and QTL mapping, and diversity analysis, because it is highly efficient and cost-effective. GBS-based approaches require considerable bioinformatics support, however . Anotherng pools . Other Sea mays) ,15. In aia faba) , and a 2 Crantz) . Moreove Crantz) . japonica rice varieties [japonica rice varieties. These markers, however, were not useful for analyzing variation in indica rice due to their low polymorphism in indica varieties. This motivated us to develop an SNP genotyping platform suited to both japonica and indica varieties.We previously developed 1225 KASP markers based on SNPs detected by resequencing 13 Korean temperate arieties ,20,21. Tarieties , seed siarieties , and spiarieties ,27,28 injaponica and indica, based on target capture sequencing technology. We developed a target capture sequencing panel including 2565 SNPs: the SNPs used to develop the 1225 KASP markers for temperate japonica varieties [indica varieties; and an SNP located in the semi dwarf 1 (sd1) gene [japonica and indica varieties. It provides an important contribution to rice genetics and breeding that will facilitate QTL and gene mapping, analyses of germplasm diversity, and marker-assisted selection. In this study, we aimed to develop a novel SNP genotyping platform for rice that is applicable to both arieties ,20,21; tarieties  and the arieties , which ad1) gene . This sySeeds of the 50 rice varieties listed in japonica varieties [indica varieties were also present, the 895 SNPs included in the 1k-RiCA [indica SNPs were included in the panel. Finally, an SNP from sd1 [In total, 2565 SNPs were included in the target capture panel design. These included the SNPs used to develop 1225 KASP markers for temperate arieties ,20,21. T 1k-RiCA  and the  1k-RiCA  were incfrom sd1  was inclGenomic DNA was cut into approximately 250 bp fragments using a Bioruptor Pico Sonication System  and processed for Illumina sequencing by the following steps: end-repair, dA-tailing, adapter ligation, and pre-PCR for indexed NGS library. The prepared gDNA library and capture probes were hybridized to capture target regions using the Celemics target enrichment kit . Target-captured libraries were amplified by post-PCR to enrich the amount of sample and sequenced on an Illumina NextSeq550 instrument  using the read layout 2 × 150 bp. The raw sequencing data were analyzed using the CLC Genomics Workbench program version 21 .PIC) value of markers was calculated using the following equation, which is applicable to inbred varieties [The polymorphism information content (arieties :PIC=1−∑iThe population structure of the 50 varieties was determined using the STRUCTURE (version 2.3.4) ,32 progrFor a QTL analysis of PHS resistance, 162 RILs derived from an Odae × Joun cross were grown in a greenhouse with maximum/minimum temperatures of 32 °C/22 °C and light/dark periods of 14 h/10 h. Seeds of both parental varieties and 162 RILs were sown in 50-hole seedling raising trays. Seedlings were transferred to pots  approximately 4 weeks after sowing; a total of six seedlings per line were transferred to two pots (three plants per pot). During plant growth, the main culm was retained on each plant, but all tillers were removed. This ensured PHS was measured in panicles from main culms of plants, thus eliminating variation in PHS caused by different tiller positions. PHS rates were evaluated according to the method of Cheon et al. . The heaOur target capture panel contained, in total, 2565 SNP markers: 1225 SNPs used to develop KASP markers for temperate japonica rice varieties; 1339 SNPs from the 1k-RiCA , which cWe analyzed 50 rice varieties  scores of these QTLs were 4.06 and 14.74, respectively . The LODjaponica and 21 indica), providing a mean of 854 for every combination of two varieties tested. Most previous QTL or gene mapping studies in rice have used traditional markers such as SSR and RFLP and involved 100–300 polymorphic markers. Our novel platform thus contained a sufficient number of polymorphic markers for QTL and gene mapping studies, MABB, and germplasm diversity analysis. In addition, the mean PIC value of markers in our platform was fairly high (0.356), while all PIC values were in the range 0–0.5  in the rice platform described here. Most of the indica varieties used in this study were Korean Tongil-type varieties, which have low genetic diversity as there is far less breeding of Tongil-type rice than of japonica rice in Korea. The requirement to develop new lines of Tongil-type rice is increasing in Korea, however, as these varieties are suited to diverse uses, including livestock feed and raw material for processed foods such as rice noodles and cookies. It is therefore important to ensure a sufficient number of suitable markers is available to enable breeding of such indica rice varieties. This will involve the development of markers capable of detecting polymorphisms within Tongil-type varieties by using genome sequence analysis to identify highly polymorphic SNPs within representative Korean Tongil-type varieties.A target capture sequencing SNP genotyping platform is highly flexible because it is very easy to add liquid probes to an existing panel ,15,16. T8 RIL population derived from a cross between two Korean temperate japonica rice varieties, Odae and Joun. Joun is resistant to PHS, whereas Odae is moderately resistant to PHS. We constructed a genetic map comprising 475 SNP markers  and high PVE (28.66%), it may be productive to identify the gene(s) responsible for its effect using map-based cloning approaches. However, this QTL mapping was performed with PHS rate data measured in only 1 year. Therefore, PHS rates must be measured in another year or environment for complete QTL mapping.PHS occurs when seeds break dormancy under warm and humid circumstances prior to harvest. It is observed in many cereal crops, including wheat, barley, and rice . PHS is  markers  and foun4 region . No QTL 8 RIL population derived from a cross between two Korean temperate japonica rice varieties, Odae and Joun.We developed a target capture sequencing SNP genotyping platform containing 2341 SNP markers that were polymorphic across 50 rice varieties. The mean number of polymorphic markers for any combination of two rice varieties was high (854), which indicates that this platform has the potential to become a powerful tool for diverse types of genetic studies, including QTL and gene mapping analyses and the development of novel breeding programs. This platform was used to identify two QTLs for PHS resistance with an F


The Principal Components, Bayesian clustering and Treemix analyses split the Portuguese breeds in two main groups which are consistent with historical records: one comprising Campaniça and Serra da Estrela together with other European and transboundary dairy breeds; and another of the well-differentiated multi-purpose Merino and Merino-related breeds. Runs of homozygosity analyses yielded 1,690 ROH segments covering an average of 2.27 Gb across the genome in all individuals. The overall genome covered by ROH segments varied from 27,75 Mb in Serra da Estrela to 61,29 Mb in Campaniça. The phylogenetic analysis of sheep mitogenomes grouped the Portuguese native breeds within sub-haplogroup B1a along with two animals of the Akkaraman breed from Turkey. This result provides additional support to a direct influence of Southwest Asian sheep in local breeds from the Iberian Peninsula. Our study is a first step pertaining to the genomic characterization of Portuguese sheep breeds and the results emphasize the potential of genomic data as a valid tool to guide conservation efforts in locally adapted sheep breeds. In addition, the genomic data we generated can be used to identify markers for breed assignment and traceability of certified breed-products.As the effects of global warming become increasingly complex and difficult to manage, the conservation and sustainable use of locally adapted sheep breeds are gaining ground. Portuguese native sheep breeds are important reservoirs of genetic diversity, highly adapted to harsh environments and reared in low input production systems. Genomic data that would describe the breeds in detail and accelerate the selection of more resilient animals to be able to cope with climatic challenges are still lacking. Here, we sequenced the genomes of 37 animals from four Portuguese native sheep breeds  and 19 crossbred sheep to make inferences on their genomic diversity and population structure. Mean genomic diversities were very similar across these breeds (.30 ≤ H Ovis aries) quickly became a valuable resource for the production of meat, milk, wool and leather products   . These bovinos/) . Among tovinos/)  (Supplemovinos/) . SE is tovinos/) . Howeverovinos/) . The MB,ovinos/) . The popovinos/) , due to In the last decade, the enormous progress in high-throughput sequencing (HTS) technologies  providesGenetic diversity is a key factor underlying the adaptive capacity and resilience of livestock populations under changing conditions . The purAnimal handling and blood collection were performed during routine veterinary check-ups, following the official animal healthcare program guidelines and under the consent of breeders.n = 6), Serra da Estrela (n = 11), Merino Branco (n = 10) and Merino Preto (n = 10)] were collected at 15 farms throughout the country. Crossbred sheep (CB) sampled at other 10 farms distributed across the Alentejo region were also included in the analysis for comparison purposes [Crossbreds (n = 19)]. The animals were randomly selected from each herd. Ten milliliters of blood were collected from the jugular vein by vacuum puncture and stored at −20°C in collection tubes containing EDTA. Genomic DNA was extracted using the DNeasy Blood & Tissue Kit  according to the manufacturer’s instructions. DNA concentration and quality were assessed with a Nanodrop spectrophotometer  and 500 ng of each sample were used for preparation of genomic libraries for resequencing  which was used as an outgroup. Whole-genome HTS data were retrieved from the NCBI database . Details on breeds, locations and accession numbers are shown in We used two complementary strategies to investigate breed relationships and infer the population structure of Portuguese sheep breeds by integrating our HTS data with: 1) whole-genome data publicly available for other European, Asian, African, Australian and transboundary commercial breeds; and 2) Illumina Ovine 50 K SNP genotype data obtained for other Iberian breeds within the Sheep HapMap Consortium . The population structure analysis of worldwide sheep included a total of 48 animals representative of 18 breeds and the Asiatic mouflon (Bioproject ID: PRJNA414087) was performed using BWA MEM v.0.7.15-r1140 (R2 = .966) were filtered based on quality (minQ > 30), SNP coverage per genotype (minDP ≥ 7) and genotype quality (minGQ > 20) using VCFtools v.0.1.17 ; genomic inbreeding coefficient (FIS) and fixation index (FST). First, autosomal SNPs were filtered based on MAF (--maf .05), calling rate (--max-missing .1) and Hardy-Weinberg equilibrium (--hwe .001). SNPs that did not pass these quality criteria were excluded from the analysis. The nucleotide diversity was estimated as the average number of nucleotide differences per site within 10 Kb windows  across the genome. The expected and observed heterozygosities, as well as the inbreeding coefficients, were estimated for each population using the functions (--hardy) and (--het), respectively. For pairwise breed comparisons, FST values were calculated following Weir and Cockerham’s  was calculated either for each chromosome or genome-wide in each population as the ratio of the total length of ROH for each individual and the total length of the autosomal chromosomes. The R v.4.0.5 (https://CRAN.R-project.org/package=detectRUNS) was used to obtain summary statistics and visualize the results.A genome-wide detection of runs of homozygosity (ROH) was carried out in Portuguese native sheep in a sliding-window approach using PLINK software v.1.90b5.2 . Briefly v.4.0.5  package Ovis orientalis)  and integrate the autosomal HTS data obtained for the Portuguese sheep with: 1) whole-genomes of 45 worldwide sheep and three Asian mouflon  ; and 2) entalis) . To deteentalis)  were estentalis) . Individsoftware  was usedsoftware .http://tree.bio.ed.ac.uk/software/figtree/).The Maximum Likelihood (ML) phylogeny of sheep mitogenomes was inferred under the TN93 + R evolutionary model selected using the Akaike Information Criterion (AIC)  in the Pm = 1–10) to add to the tree were determined using the OptM package  and the crossbred sheep population. On average, the total number of SNPs per individual ranged from 9,348,138 SNPs in Merino Branco to 9,581,638 SNPs in Serra da Estrela. Based on the Kruskal-Wallis statistical inference analysis for the n breeds .–3 (SE) to π = 2.032 × 10–3 (MP). The comparisons between breeds of expected (He) and observed (Ho) heterozygosities were not significantly different . On average, He varied between .3 (MB and MP) and .35 (CAM) with an overall mean of .32, and Ho ranged from .30 (MB and MP) to .34 (CAM and SE) with an overall mean of .31. The genomic inbreeding coefficient estimated from SNP data was relatively low in all breeds , with the highest value observed in CAM (.038). Weir and Cockerham’s mean pairwise FST was used as a measure of breed differentiation across breeds  to .037 (CAM and MB), showing a close genetic relationship between Portuguese native sheep. CAM and the dairy breed SE had the highest mean FST across pairwise comparisons, consistently with their relative geographic isolation, and in the case of the latter also selection for milk production.The distributions of SNPs across MAF categories in each population are summarized in s breeds . The paiR2 = .8239). CAM showed the highest average number of homozygous segments per animal (nROH = 44.5) comprising on average 61.29 Mb, whereas the lowest values were observed in the dairy breed SE (nROH = 20.9) comprising about 27.75 Mb per animal. The Merino breeds showed an intermediate number of ROH segments (MB: 29.5 and MP: 28.5) and mean lengths  per animal  and SE the lowest (.010). FROH estimates across chromosomes varied within and between breeds (The ROH-based inbreeding coefficient (FROH) was estimated for each population . On avern breeds , which sThe PCA based on HTS data was conducted to assess the population structure of Portuguese native and worldwide sheep breeds and infer the proportion of the total genomic variation explained by each PC. The first two PCs, which account for the highest variation of the data set, depicted: an east-to-west cline of sheep breeds ; and the differentiation between the Asiatic Mouflon, domestic sheep and the inbred Suffolk and East-Friesian breeds  . The PorFor a fine resolution, the population structure of Iberian breeds was also assessed in a PCA by merging our HTS data collected for Portuguese sheep with Illumina 50 K SNP genotyping data available for nine Spanish breeds . The firADMIXTURE analysis allowed us to infer ancestry contributions underlying the gene pool of Portuguese native sheep. The results of the model-based clustering approach are shown in Phylogenetic relationships inferred from the sheep mitogenomes are depicted in To evaluate the phylogenetic relationships and historical genetic drift events among Iberian breeds, we built a maximum likelihood (ML) tree based on the population allele frequency covariance matrix and rooted in the Asian Mouflon using TreeMix . When onWe conducted the first whole-genome sequencing analysis of Portuguese native sheep in which four breeds and a population of crossbreds were characterized in the context of other Iberian and worldwide sheep. Improving knowledge on the genomic diversity and population structure of local sheep is especially important to disclose signatures of adaptation and improvement, but also to implement appropriate management and conservation strategies. To date, few genetic studies have been carried out on Portuguese native sheep and were based on either mitochondrial data or a limited number of microsatellite markers . We repoo ≤ .39; .31 ≤ He ≤ .38) (–3 to 3.1 × 10–3)  with slight differences between breeds. However, a significantly high proportion of fixed SNPs (MAF = 0) was observed in CAM which can be due to a recent bottleneck from replacement by high-output transboundary breeds , or to te ≤ .38) . Nucleot × 10–3) .IS) estimated for the Portuguese native breeds were relatively low (ranging from .005 to .038). Even the highest FIS value estimated for CAM is considerably lower than the observed in some European sheep breeds (between .04 and .42) (ST .03).In livestock populations, high inbreeding could result in an overall decrease of their performance, which may impact economically important traits . For insand .42) . As oppoROH) with a large number of SNPs . The FROH values obtained for Portuguese sheep were similar to those reported for the Spanish breeds Castellana, Ojalada, Ripollesa, Segurena and Xisqueta (.008 ≤ FROH ≤ .025)  from recent inbreeding and low genetic diversity  . In our  of SNPs . The FRO ≤ .025) . OverallPopulation structure and breed relationships were investigated considering worldwide and Iberian sheep breeds by integrating the genomes we generated with publicly available whole-genome and SNP array data, respectively. Congruent results were obtained from complementary PCA, Admixture and phylogenetic analyses. In the PCA, Portuguese sheep split in two clusters according to breed histories, i.e., Merino populations were genetically close whereas CAM and SE belong to a distinct group of breeds. The Swiss White Alpine is a Merino-derived European breed and in agreement with previous analysis was also included in this group . When onhttps://www.merina.pt), national authorities promoted the upgrading of local Merino sheep with Merino animals from Spain, as well as Rambouillet and Merino Precóce from France. The Rambouillet is itself derived from Spanish Merino flocks (http://afs.okstate.edu/breeds/sheep/rambouillet), while the latter breed originates from Rambouillet and Île-de-France (https://www.fondazioneslowfood.com/en/ark-of-taste-slow-food/precocious-merinos-goat/). The results of our ADMIXTURE analysis show that MP and MB animals as well as crossbreds, share some ancestry with the Swiss White Alpine, a breed which derives from a cross between the Swiss White Mountain and Île-de-France . Indeed, whole-genome 600 K SNP array data confirm the close relationship of Rambouillet and Merino breeds from Spain and France , as well as the tight clustering of the Portuguese Merino populations. For K greater than six, some sub-structure within CAM, SE, MP, and MB starts to emerge, which could be due to an effect of the herds from where the samples originate. Overall, our results are consistent with low levels of breed differentiation and a complex genetic background observed in sheep from the Iberian Peninsula . The wead France , also ded France . The MP via the Mediterranean dispersion routes (Additionally, the phylogenetic analysis of sheep mitogenomes showed that Portuguese native breeds and two animals of the Akkaraman breed from Turkey are genetically close which provides support to a direct influence of Near-Eastern stock having reached the Iberian Peninsula n routes . Our resn routes . Consistn routes . The clun routes .We examined the patterns of genomic diversity and population structure of four Portuguese native sheep within other Iberian and worldwide breeds. Our results suggest these Portuguese breeds are not genetically compromised, showing moderate diversity and negligible inbreeding. Expanding our study to a larger number of animals and farms should allow for more comprehensive inferences on Iberian sheep biodiversity to define management and conservation plans. The population structure analyses depicted the Iberian Merino sheep as a well differentiated breed group. The Merinos are thought to have been developed in the Iberian Peninsula and their selection for wool, meat and adaptive traits for local conditions appear to have resulted in a distinct genetic make-up. The gene flow between the Portuguese and Spanish Merino breeds depicted in the phylogenetic analysis could be explained by traditional transhumance routes which increase the chance for crossbreeding. Portuguese native breeds formed a tight clade within major haplogroup B in the phylogeny of sheep mitogenomes. This is the first study of Portuguese native sheep using whole genomes and sets the ground for defining ancestry informative SNPs for breed-specific admixture analysis, i.e., a powerful tool for breed assignment and traceability of certified breed-products, but also for genome-wide association studies. In addition, the genomic data we generated will be most valuable for a combined analysis of sheep genomes retrieved from historic and archaeological specimens to investigate the origins, evolution and modes of improvement of native Iberian sheep.


Thuja plicata), an ecologically and economically important conifer species of the Cupressaceae. The genome assembly, derived from a uniquely inbred tree produced through five generations of self-fertilization (selfing), was determined to be 86% complete by BUSCO analysis, one of the most complete genome assemblies for a conifer. Population genomic analysis revealed WRC to be one of the most genetically depauperate wild plant species, with an effective population size of approximately 300 and no significant genetic differentiation across its geographic range. Nucleotide diversity, π, is low for a continuous tree species, with many loci showing zero diversity, and the ratio of π at zero- to fourfold degenerate sites is relatively high (approximately 0.33), suggestive of weak purifying selection. Using an array of genetic lines derived from up to five generations of selfing, we explored the relationship between genetic diversity and mating system. Although overall heterozygosity was found to decline faster than expected during selfing, heterozygosity persisted at many loci, and nearly 100 loci were found to deviate from expectations of genetic drift, suggestive of associative overdominance. Nonreference alleles at such loci often harbor deleterious mutations and are rare in natural populations, implying that balanced polymorphisms are maintained by linkage to dominant beneficial alleles. This may account for how WRC remains responsive to natural and artificial selection, despite low genetic diversity.We assembled the 9.8-Gbp genome of western redcedar (WRC;  Picea glauca), Norway spruce (Picea abies), loblolly pine (Pinus taeda), sugar pine (Pinus lambertiana), and Douglas fir (Pseudotsuga menziesii), have been sequenced  . Coniferequenced . Such efn = 11 in Cupressaceae; n = 12 in Pinaceae) (Sequoiadendron giganteum) and the hexaploid coast redwood (Sequoia sempervirens), have been published  , genome inaceae) , and likublished .Thuja plicata) is an ecologically, economically, and culturally important species in the Cupressaceae. Endemic to the Pacific Northwest of North America and ranging from Northern California to Southern Alaska, WRC is a stress-tolerant, slow-growing tree prized for its durable, lightweight, and rot-resistant wood  and its complement, outcrossing (1 – s), are of interest to evolutionary biologists owing to their implications for genetic diversity and fitness and have been investigated extensively over the past century , with a mean outcrossing rate of ∼70% , 2004, a century . Althoug century , which,  century . Researc century . Charact century . The exc century .Here we introduce the first genome sequence for WRC and present unique features of the genome in the context of genetic diversity and the evolutionary history of WRC. We further explore the effects of extreme selfing on heterozygosity and selective pressures in multiple selfing lines (SLs).Supplemental Table S1) expected to be >98% homozygous. The S5 reference genome assembly was generated from a combination of short-fragment paired-end reads, large-fragment mate-pair reads, and linked-reads from large molecules, using 13 libraries and 28 lanes of Illumina sequencing, with a sequencing read length of 2 × 151 bp . Overall genome depth of coverage was estimated to be 77×.The assembly of conifer genomes remains challenging partly owing to their large size  and highSupplemental Fig. S1). We calculated approximately one single-nucleotide variant (SNV) every 4.6 kbp, for an estimated genome-wide heterozygosity of 0.000216, an exceptionally low estimate, highlighting the value of SLs for genome sequencing and assembly.The WRC genome was previously estimated to be 12.5 Gbp in size across 11 chromosomes . GenomeSSupplemental Table S3). Benchmarking universal single-copy ortholog (BUSCO) analysis .We assembled 7.95 Gbp of the estimated 9.8-Gbp genome to produce a draft assembly with an N50 of 2.31 Mbp, the largest scaffold being 16.3 Mbp. This assembly comprises 67,895 scaffolds >1 kbp , and an additional 26,150 alternative transcripts. A total of 25,984 gene models had Pfam protein family annotation, 31,537 had transcriptome support over their full length (100%), and 19,506 had peptide homology coverage support ≥90% . Intron length ranged from 20 bp to 148.3 kbp, which is consistent with estimates from other conifers, with maximum lengths ranging from 68 kbp (Norway spruce) .Genome annotation using evidence from Iso-Seq full-length cDNAs resulted in the identification of 39,659 gene models supported by the alignment of unique primary transcripts ( spruce)  up to 57 spruce) . Scott e spruce)  and gian spruce) . Single-Supplemental Table S4B). We further validated the completeness of the genome assembly and annotation using a panel of 59 full-length WRC sequences from GenBank, of which 48 were reliably identified in the genome annotation. We also searched for a set of 33 WRC terpene synthase (TPS) transcripts, of which we reliably (>90% identity) identified 15  in n = 112 unrelated trees from across the geographic range of WRC (range-wide population [RWP]) . Trees were grouped into three subpopulations: Northern-Coastal (n = 77), Central (n = 26), and Southern-Interior (n = 9)  and 2,186,998 invariant sites (see Methods). Total mean SNP depth was 34.3×.We estimated nucleotide diversity, short-range linkage disequilibrium (LD), population structure, genetic differentiation, and effective population size ( (n = 9) A. Using Supplemental Data Set S6; Supplemental Table S8).We annotated 17,728 SNPs across 2886 genomic scaffolds using the Ensembl variant effect predictor (VEP)  threshold of 0.05 to avoid bias owing to rare alleles . The mean of all pairwise r2 estimates was 0.299 with a median of 0.151. The half-decay value  was 0.118 Mbp. LD decayed to an r2 of 0.2 at 0.751 Mbp and an r2 of 0.1 at 2.17 Mbp (r2> 0.8) appears to exist for SNPs millions of base pairs apart . The ΔK method of We analyzed STRUCTURE  results n = 13,427 SNPs from SCO and intergenic regions. Cross-validation for DAPC with a priori cluster definitions optimally retained 22 PCs and two discriminant functions capturing 30.3% of the conserved variance; however, de novo k-means clustering failed to resolve any clusters, identifying an optimal K of one . PCA revealed a latitudinal gradient of differentiation along the first principal component (PC), with some separation of the Southern-Interior subpopulation along the second PC  , and no significant isolation by distance was found by our Mantel test for subpopulations  and individuals  .We applied nonparametric approaches of discriminant analysis of principal components (DAPC) and principal component analysis (PCA) using a set of econd PC C, mostlydXY, between subpopulations using all SCO and intergenic SNPs. Average π (SD) across 10,631 SCOs was 0.00272 (0.0122) ; 1411 genes had a π of zero. Across 10-kb windows, average π was 0.00204 (0.0141), indicating diversity is similar in coding and noncoding regions. Average dXY was not significantly different between any pair of subpopulations nor was it significantly different from π in SCOs  .We estimated nucleotide diversity, π , in the 0/π4, the ratio of π in zerofold to fourfold degenerate sites. We found a π0 of 0.00158 (0.0146) and a π4 of 0.00485 (0.0147), yielding a π0/π4 of 0.325. The site frequency spectrum (SFS) for fourfold SNPs appeared to decay slower than the SFS for zerofold and for all SNPs, supporting evidence of a recent bottleneck and indicating that there may be stronger positive selection at these sites .To assess the efficacy of purifying selection, we estimated πNe can be defined as the idealized population size expected to experience the same rate of loss of genetic diversity as the population under observation .ervation . We estistimator . SNPs weNe from about 500,000 beginning ∼2 MYA, accelerating from ∼40,000 yr ago down to under 300 by present day, consistent with one or more bottleneck events during the recent glacial maximum . Our estimates of Ne are extremely low for a continuous tree population; for example, species in Picea  or S5 (n = 11), retaining n = 151 trees . We used all filtered SNPs for these analyses .To examine the effect of complete selfing on heterozygosity and selection in WRC, we selected 189 trees from the 15 FS families, forming 41 SLs for SNP genotyping. The process of SNP calling is error prone, and despite filtering for multiple quality criteria, errors are likely to remain in any SNP data set. Using SLs, we were able to correct for erroneous genotyping calls and impute missing genotypes for SLs up to S4 , whereas median observed heterozygosity was significantly lower than expected beginning in generation S3  . Mean heterozygosity at FS was 0.296, whereas in the RWP, it was 0.219 . In comparison, Pinus contorta) and white spruce, respectively.Under selfing in diploids, heterozygosity is expected to decline by 50% in each generation purely through genetic drift. Mean heterozygosity declined slower than expected  and is a measure of reduction in heterozygosity owing to inbreeding. We estimated F for each sample in the SLs and the RWP using the approach of FUNI). Mean F increased from 0.00569 at FS to 0.801 at S5, significantly less than expected , indicating that the observed reduction in heterozygosity cannot entirely be attributed to inbreeding . Mean F in the RWP was 0.331, further emphasizing the degree of inbreeding in wild populations .The inbreeding coefficient Supplemental Data Set S9). Of these, 15 fixed for the reference allele and two fixed for the alternate allele more often than would be expected under drift alone; meanwhile, all outlier SNPs had a higher proportion of heterozygous alleles by S4 than expected under drift. Outlier SNPs were present on all putative LGs in the genome, and mean depth was similar to the mean depth of the total SNP set . VEP predicted effects for 67 outlier SNPs, 14 (16.9%) of which were in coding regions . Gene Ontology (GO) annotation was available for 30 genes containing outlier SNPs; 10 GO categories were overrepresented in outliers compared with the entire SNP set  .Following the expectation of a 50% decline in heterozygosity per generation under complete selfing and assuming a model of only genetic drift, we anticipated that 25% of SLs would become fixed for one allele at any given locus and 25% for the other in each generation. Thus, by generation S4, 46.875% of SLs should fix for each allele, and 6.25% should remain heterozygous. We identified 83 SNPs that deviated from expected proportions of fixation at a false-discovery rate threshold of 0.05 (hereafter: outlier SNPs) (−5) to be overrepresented in outlier SNPs, whereas synonymous variants (P = 0.0274) and 3′ untranslated region (UTR) variants (P = 0.00993) were underrepresented . In the RWP, the minor allele for these SNPs was nearly always the alternate allele, namely, the allele inducing the change. This pattern suggests that balanced polymorphisms may be maintained by selection favoring linked dominant alleles, namely, associative overdominance  found that selfing over several generations can reduce genome size by up to 7.9%  long-read sequencing or linked-reads . WRC is  to 7.9% , which mPanicum miliaceum) (Populus species (π = 0.0041–0.011) (Glycine spp.) (Populus qiongdaoensis (π = 0.0014), which is restricted to a single small island and has an estimated Ne of about 500  , weedy bliaceum) , and mos1–0.011) . We founne spp.) , or rarebout 500 . Additio0/π4 ratio (0.33) may suggest weak purifying selection in WRC (0 returns to equilibrium quicker than π4 following a bottleneck event (Ne (about 300) and general lack of population structure, genetic differentiation, or nucleotide divergence between geographic subpopulations despite a relatively wide geographic range and continuous population suggest that much of the variation in WRC was likely eliminated owing to bottlenecks following the last glacial period, a pattern confirmed by our Stairway Plot results and affirming the conclusions of previous studies , and with each successive generation, F increased slower than expected, indicating that IBD does not fully explain the reduction in heterozygosity in WRC   for outlier SNPs and the total SNP set suggest paralog content is minimal. Higher than expected heterozygosity was observed during selfing in eucalypts (grandis)  and maizgrandis) . HoweverWe recognize that use of SLs of single seed descent makes differentiating between patterns of selection and genetic drift difficult, as genetic drift is stronger when there are fewer individuals in a population. The use of multiple cloned seedlings for each SL in future studies could help improve our analysis, with the potential to find more SNPs under selection.Current breeding of WRC focuses on traits such as growth and herbivore and disease resistance; thus, low genetic diversity may have considerable ecological and potential economic consequences. When low genetic diversity is observed in plant or animal populations, conservation strategies may become necessary to maintain existing genetic variation and reduce the risk of extreme inbreeding depression, especially when census population size in the wild is small. Although ours and previous results  indicateWRC's apparent adaptability and potential for range expansion make it an important forest tree in a time of changing climate and environments . Low genWRC is a fascinating example of adaptation in a long-lived conifer, despite very low levels of genetic variation. As our understanding of the genome improves, we will be able to improve prospects for survival and maintenance of this tree as an ecologically and economically significant species and better understand and test how selfing behavior evolves and can be advantageous in wild plant populations.n = 112 individuals originating from across the geographic range growing at the Cowichan Lake Research Station (CLRS) at Mesachie Lake, BC, Canada. Trees were separated into three geographic subpopulations, Northern-Coastal, Central, and Southern-Interior, based on UPGMA clustering of genetic distances  were crossed to create 15 FS families and ensure an initial inbreeding coefficient of F = 0. Each FS line was then selfed for up to five generations (S1–S5) with one generation every 2 yr, facilitated by GA3 hormone treatment. A single S5 seedling of SL 23 (2323-211-S5) was used for genome sequencing. We selected 189 individuals from the 15 FS selfing families for genotyping for the SL analysis .The WRC RWP represented istances . SLs weristances  at CLRS.https://www.biost.com) and sequenced at the Joint Genome Institute (JGI).Foliar tissue was used for DNA extraction for genome sequencing. Purified nuclear genomic DNA was extracted at BioS&T . Detailed DNA extraction, sequencing, and assembly methods can be found in Supplemental Methods.Genome sequencing was executed using three types of libraries: short-fragment paired-end, large-fragment mate-pair, and linked-reads from large molecules using 10x Genomics Chromium. Depth of  k = 128 . We anale v1.0.0 . Paired-We estimated completeness of the WRC genome assembly and other conifer genome assemblies using BUSCO v5.0.0 in genome mode, on OrthoDB Embryophyta v10 , which dhttps://www.ncbi.nlm.nih.gov/bioproject/] under accession number PRJNA704616). We determined gene loci by transcript assembly alignments and EXONERATE v2.4.0  Iso-Seq, full-length cDNAs were synthesized from total RNA. We then generated transcript assemblies from 1.4 billion 2 × 150 and 50 million 2 × 100 stranded paired-end Illumina RNA-seq reads using PERTRAN , 18 millE v2.4.0  alignmenr v4.0.8  with up r v4.0.8 , FGENESHr v4.0.8  assemblyWe estimated completeness of the WRC primary transcript gene set and other conifer gene sets using BUSCO v5.0.0 in protein mode on OrthoDB Embryophyta v10. We also assessed coverage of 59 complete WRC sequences found on NCBI and 33 WRC TPS sequences . SequencDNA was isolated from lyophilized tissue with a modified protocol of Supplemental Methods.Putative SNPs were identified using FreeBayes v1.2.0  in 150 bD , using the following formula:HO is the observed heterozygosity of the locus, HE is the expected heterozygosity of the locus under HWE, and the factor of (1–0.17) accounts for the expected equilibrium fixation index of 0.17 in WRC based on the average outcrossing rate of 0.7 .Variant sites were filtered using VCFtools v0.1.17  with theD  >5 and <e of 0.7 , 2004. Ie of 0.7 . Relatioe of 0.7 . For ther2) was estimated in the RWP using PLINK v1.9 =[r2 threshold of 0.1 using a 2.17-Mb window; following the outcome of our LD analysis, a MAC threshold of three was used to remove singletons, and only SCO and intergenic SNPs were retained (n = 4765). For all other genetic differentiation analyses, all SCO and intergenic SNPs were used . The population structure of the RWP was estimated using STRUCTURE v2.3.4 and a DAPC using the adegenet package (v2.1.1) in R (FST test as implemented in the hierfstat package (v0.04-22) in R  in R . A hiera22) in R  was used22) in R  to assesSTRUCTURE software was run using 10,000 MCMC repetitions with 10,000 repetitions of burn-in and 10 iterations of each K. Results were analyzed using methods of n = 13,427) and all invariant sites were used to estimate π and dXY for n = 10,631 SCO genes and 10-kb windows in the RWP using pixy v1.2.7.beta1 .To avoid downward bias owing to missing data, SCO and intergenic variant sites . For seven SLs in which only the S3 generation had not been sequenced, we imputed the genotypes for the S3 generation at each locus for SNPs where no other genotype was possible and marked the rest as missing.Genotype correction in continuous SLs used two criteria: Individuals with homozygous calls in at least two consecutive generations were considered to be homozygous for that allele in all subsequent generations; and individuals with heterozygous calls in at least two consecutive generations were considered to be heterozygous for all preceding generations, up to and including the FS generation. SNPs that could not be corrected following these criteria were removed. We manually corrected genotypes for SLs that had been completely genotyped from either S1–S4 or S1–S5  were used to calculate the observed and expected changes in heterozygosity and inbreeding coefficients over time in the SLs, and observed heterozygosity in the RWP. Observed heterozygosity was calculated at each SNP locus for each generation across all SLs using the adegenet package in R. Expected heterozygosities in the S1–S5 generations were calculated for each SNP locus as half the observed heterozygosity of the previous generation. We calculated inbreeding coefficients in PLINK using the ‐‐ibc flag to obtain a measure for inbreeding based on the correlation between uniting gametes (FUNI). This metric is defined by ith SNP and each jth individual asx is the number of copies of the reference allele, and P is the population-wide allele frequency at that locus. Calculations of FUNI do not consider LD; thus, we used SNPs filtered for LD and MAC (n = 6123). In a diploid population, F should increase as F to increase by a factor of F was calculated using corrected genotypes in SLs and uncorrected genotypes in the RWP.Corrected genotypes for all filtered SNPs  under accession number PRJNA704616. The SNP data generated in this study have been submitted to Zenodo (https://zenodo.org/record/6562381#.Yz2fvS1h2v4) and are available in the Supplemental Data Sets. Supplemental Code and raw data used for generating data files and figures, including all filtered SNP sets for each step of the study, are available as Supplemental Code files, and have been uploaded together with copies of the genome annotation and Supplemental Data Set files to GitHub . Summaries of Supplemental Code are available in the Supplemental Information.The genome sequence reads, assembly and annotation, and transcriptomes used in annotation generated in this study have been submitted to the NCBI BioProject database (