| Barley(Hordeum vulgare L.)is the fourth most important cereal crop after wheat,rice and maize,with both edible and industrial brewing benefits.Barley is confronting the challenges of frequent harsh weather,excessive salinity and other adversities as a result of global warming,and there is an urgent need to breed new better cultivars with synergistic improvements in production,quality and stress resistance.Due to the genetic bottleneck effect,the genetic base of farmed barley narrows and becomes progressively homogenized over time,limiting additional breeding breakthroughs.Wild barley,the progenitor species of current cultivated barley,has been an essential high genetic diversity,resistance and other excellent characteristics,has been an germplasm and gene pool for barley genetic development due to its high genetic variety,resistance,and other excellent qualities.Therefore,it is critical to strengthen the identification of excellent wild barley germplasm,explore and utilize the excellent genes of wild barley to extend the genetic base of cultivated barley,innovate barley germplasm,and breed new breakthrough barley varieties.In this study,118 wild barley samples collected from 13 regions in Israel were systematically investigated and identified for seedling root traits,field agronomic traits and grain traits,and the excellent germplasm was chosen.The candidate loci and candidate genes were identified and analyzed using genomewide association analysis.The following are the key findings:1.8 seedling root traits(number of root tips,total root length,maximum root length,total root surface area,total root volume,average root diameter,fresh root weight and dry root weight),8 field agronomic traits(plant height,spike length,grain number per spike,under spike section length,spike base width,flag leaf length,flag leaf width,flag leaf area)and 8grain traits(thousand grain weight,grain length,grain width,grain length-width ratio,grain area,grain perimeter,grain diameter and grain roundness).The coefficients of variation for root traits were found to ranged from 16.71% to 46.34%,for field agronomic traits from 8.12%to 14.96%,and for grain traits from 4.53% to 18.19%,indicating that phenotypic diversity is considerable in wild barley.Each trait’s mean or at two pilot BLUPs values were properly distributed.ANOVA revealed that both genetic regulation and environment influenced 5 field agronomic traits: plant height,spike length,grain number per spike,under spike section length,and stem base width.Both genetic and environmental factors influenced the 6 grain attributes of grain weight,grain length,grain width,grain area,grain perimeter,and grain diameter..2.Correlation analysis revealed that among the agronomic traits in the field under different environments,flag leaf length and width were highly significantly and favorably correlated with stem base width,spike length,and grain number per spike;under spike section length,flag leaf length and width,flag leaf area,and all of these variables were positively correlated with plant height;spike length was significantly and positively correlated with grain number per spike.With the exception of the negative correlation with the grain length-width ratio,grain traits in various environments showed significant or highly significant positive correlations between thousand grain weight and other grain traits,Grain length and grain width also showed strong positive correlations with grain area,grain diameter,and grain perimeter.The analysis of root characteristics at the seedling stage revealed that average root diameter was negatively and significantly correlated with maximum root length and number of root tips,whereas it was positively and significantly correlated with root fresh weight,dry weight,total root volume,and total root surface area.Maximum root length was also negatively and significantly correlated with maximum root length,number of root tips,and total root length.According to the analysis of agronomic traits and grain traits,plant height,under spike section length,and stem base width were all highly significant and positively correlated with thousand grain weight,grain width,grain area,and grain diameter.This finding suggests that the 3agronomic traits of under spike section length,stem base width,and plant height may each have an impact on the plant’s thousand grain weight by influencing the shape of the grain,such as grain length and width.3.118 barleys were genotyped using Barley 40 K,and after quality control and filtering,a total of 173,999 high-confidence SNP loci were obtained for downstream analysis;population structure analysis allowed them to be divided into 2 populations,with subpopulation I having the ideal population size.Genome-wide association studies were conducted for wild barley root traits,field agronomic traits,and grain traits using the phenotypes and genotypes mentioned above.The results showed that 13 SNP loci were strongly linked to field agronomic characteristics,and a total of 12 candidate genes were identified with gene annotation,of which 5 genes were associated with flag leaf area,3 genes with grain number per spike,and 4 genes with plant height.2 SNP loci were detected to be significantly associated with seedling root traits.These genes are involved in biological activities such amino acid synthesis,ubiquitin protease,and glycosyl transfer.For example,HORVU5Hr1G012780 encoding a product of homocysteine S-methyltransferase(HMT),which may be involved in barley growth and development affecting flag leaf traits.The gene HORVU5Hr1G012790,which encodes a small ubiquitin-like modifier protein(SUMO),so we speculate that this gene may regulate flag leaf size via controlling the phytohormone signaling pathway.The gene HORVU7Hr1G046270 regulates the cell cycle protein-dependent protein serine/threonine kinase activity,which is involved in signaling and other effects on spike number.4.80 SNP loci were detected to be significantly associated with grain traits,and a total of 84 candidate genes were identified in combination with gene annotation;among them,1gene were associated with thousand grain weight,9 genes were associated with grain area,17 genes were associated with grain diameter,25 genes were associated with grain length,and 32 genes were associated with grain perimeter.Among them,14 SNP loci were associated with four traits: grain length,grain area,grain perimeter and grain diameter,and 14 candidate genes were identified in these loci;1 SNP locus was associated with three traits: grain length,grain perimeter and grain diameter,and 9 candidate genes were identified in the vicinity of the locus;3 SNP loci were found to be associated with two traits: grain length and grain perimeter,and2 candidate genes were identified in the vicinity of the locus.These genes are involved in biological processes such as phytohormone signaling pathways,defense responses and ubiquitin proteins: for example,HORVU3Hr1G049670 and HORVU3Hr1G049680 encode E3 ubiquitin protein ligases,which are involved in ubiquitin-dependent proteolytic metabolism;HORVU7Hr1G046400 involved in growth hormone-activated signaling pathway,responding to water deficiency and cytokinin,etc.It can be seen that grain traits are associated with a variety of complex biological processes.In this study,we systematically investigated and compared the root,agronomic and grain traits of wild barley collected from its origin in Israel,and analyzed the loci and candidate genes associated with these traits in combination with genome-wide association analysis,which initially clarified the genetic basis for the formation of important agronomic traits in wild barley,and provided the basis for further fine localization and candidate gene cloning identification,as well as material and technical support for barley genetic improvement. |
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