Enhancing thalassemia gene carrier identification in non-anemic populations using artificial intelligence erythrocyte morphology analysis and machine learning.

BACKGROUND: Non-anemic thalassemia trait (TT) accounted for a high proportion of TT cases in South China. OBJECTIVE: To use artificial intelligence (AI) analysis of erythrocyte morphology and machine learning (ML) to identify TT gene carriers in a non-anemic population. METHODS: Digital morphological data from 76 TT gene carriers and 97 controls were collected. The AI technology-based Mindray MC-100i was used to quantitatively analyze the percentage of abnormal erythrocytes. Further, ML was used to construct a prediction model. RESULTS: Non-anemic TT carriers accounted for over 60% of the TT cases. Random Forest was selected as the prediction model and named TT@Normal. The TT@Normal algorithm showed outstanding performance in the training, validation, and external validation sets and could efficiently identify TT carriers in the non-anemic population. The top three weights in the TT@Normal model were the target cells, microcytes, and teardrop cells. Elevated percentages of abnormal erythrocytes should raise a strong suspicion of being a TT gene carrier. TT@Normal could be promoted and used as a visualization and sharing tool. It is accessible through a URL link and can be used by medical staff online to predict the possibility of TT gene carriage in a non-anemic population. CONCLUSIONS: The ML-based model TT@Normal could efficiently identify TT carriers in non-anemic people. Elevated percentages of target cells, microcytes, and teardrop cells should raise a strong suspicion of being a TT gene carrier.

Identification of a Locus for High-Temperature Adult-Plant Resistance to Stripe Rust in the Wheat Yr8 Near-Isogenic Line through Mutagenesis and Molecular Mapping.

Aegilops species are wheat relatives that harbor valuable disease resistance genes for wheat breeding. The wheat Yr8 near-isogenic line, AvSYr8NIL, has long been believed to carry only Yr8 for race-specific all-stage resistance to stripe rust, caused by Puccinia striiformis f. sp. tritici, derived from Aegilops comosa. However, AvSYr8NIL has been found to have high-temperature adult-plant (HTAP) resistance in our field and greenhouse tests. To confirm both HTAP and Yr8 resistance, seeds from AvSYr8NIL were treated with ethyl methanesulfonate to generate mutant lines. The mutant lines with only Yr8 (M641) and only HTAP resistance (M488) were crossed with the susceptible recurrent parent, Avocet S (AvS). The F1 and F4 lines of AvS/M641 were phenotyped with Yr8-avirulent races in the seedling stage at the low-temperature (4-20oC) profile, while the F1, F2, F4, and F5 lines of AvS/M488 were phenotyped with Yr8-virulent races in the adult-plant stage at the high-temperature (10-30oC) profile. Both Yr8 and the HTAP resistance gene (YrM488) were recessive. The F4 populations of AvS/M641 and AvS/M488 were genotyped using polymorphic Kompetitive allele-specific PCR markers converted from SNPs. Yr8 was mapped to a 0.66 cM fragment and YrM488 to a 1.22 cM interval on chromosome 2D. The physical distance between the two resistance genes was estimated to be over 500 Mb, indicating their distinct loci. The mutant lines with separated resistance genes would be useful in enhancing our understanding of different types of resistance and in further studying the interactions between wheat and the stripe rust pathogen.

Genome-wide QTL mapping for stripe rust resistance in spring wheat line PI 660122 using the Wheat 15K SNP array.

Introduction: Stripe rust is a global disease of wheat. Identification of new resistance genes is key to developing and growing resistant varieties for control of the disease. Wheat line PI 660122 has exhibited a high level of stripe rust resistance for over a decade. However, the genetics of stripe rust resistance in this line has not been studied. A set of 239 recombinant inbred lines (RILs) was developed from a cross between PI 660122 and an elite Chinese cultivar Zhengmai 9023. Methods: The RIL population was phenotyped for stripe rust response in three field environments and genotyped with the Wheat 15K single-nucleotide polymorphism (SNP) array. Results: A total of nine quantitative trait loci (QTLs) for stripe rust resistance were mapped to chromosomes 1B (one QTL), 2B (one QTL), 4B (two QTLs), 4D (two QTLs), 6A (one QTL), 6D (one QTL), and 7D (one QTL), of which seven QTLs were stable and designated as QYrPI660122.swust-4BS, QYrPI660122.swust-4BL, QYrPI660122.swust-4DS, QYrPI660122.swust-4DL, QYrZM9023.swust-6AS, QYrZM9023.swust-6DS, and QYrPI660122.swust-7DS. QYrPI660122.swust-4DS was a major all-stage resistance QTL explaining the highest percentage (10.67%-20.97%) of the total phenotypic variation and was mapped to a 12.15-cM interval flanked by SNP markers AX-110046962 and AX-111093894 on chromosome 4DS. Discussion: The QTL and their linked SNP markers in this study can be used in wheat breeding to improve resistance to stripe rust. In addition, 26 lines were selected based on stripe rust resistance and agronomic traits in the field for further selection and release of new cultivars.

Mining the Australian Grains Gene Bank for Rust Resistance in Barley.

Global barley production is threatened by plant pathogens, especially the rusts. In this study we used a targeted genotype-by-sequencing (GBS) assisted GWAS approach to identify rust resistance alleles in a collection of 287 genetically distinct diverse barley landraces and historical cultivars available in the Australian Grains Genebank (AGG) and originally sourced from Eastern Europe. The accessions were challenged with seven US-derived cereal rust pathogen races including Puccinia hordei (Ph-leaf rust) race 17VA12C, P. coronata var. hordei (Pch-crown rust) race 91NE9305 and five pathogenically diverse races of P. striiformis f. sp. hordei (Psh-stripe rust) (PSH-33, PSH-48, PSH-54, PSH-72 and PSH-100) and phenotyped quantitatively at the seedling stage. Novel resistance factors were identified on chromosomes 1H, 2H, 4H and 5H in response to Pch, whereas a race-specific QTL on 7HS was identified that was effective only to Psh isolates PSH-72 and PSH-100. A major effect QTL on chromosome 5HL conferred resistance to all Psh races including PSH-72, which is virulent on all 12 stripe rust differential tester lines. The same major effect QTL was also identified in response to leaf rust (17VA12C) suggesting this locus contains several pathogen specific rust resistance genes or the same gene is responsible for both leaf rust and stripe rust resistance. Twelve accessions were highly resistant to both leaf and stripe rust diseases and also carried the 5HL QTL. We subsequently surveyed the physical region at the 5HL locus for across the barley pan genome variation in the presence of known resistance gene candidates and identified a rich source of high confidence protein kinase and antifungal genes in the QTL region.

Increasing catalytic efficiency of SceCPR by semi-rational engineering towards the asymmetric reduction of D-pantolactone.

D-pantolactone (D-PL) is one of the important chiral intermediates in the synthesis of D-pantothenic acid. Our previous study has revealed that ketopantolactone (KPL) reductase in Saccharomyces cerevisiae (SceCPR) could asymmetrically reduce KPL to D-PL with a relatively weak activity. In this study, engineering of SceCPR was performed using a semi-rational design to enhance its catalytic activity. Based on the computer-aided design including phylogenetic analysis and molecular dynamics simulation, Ser158, Asn159, Gln180, Tyr208, Tyr298 and Trp299 were identified as the potential sites. Semi-saturation, single and combined-site mutagenesis was performed on all six residues, and several mutants with improved enzymatic activities were obtained. Among them, the mutant SceCPRS158A/Y298H exhibited the highest catalytic efficiency in which the kcat/Km value is 2466.22 s-1·mM-1, 18.5 times higher than that of SceCPR. The 3D structural analysis showed that the mutant SceCPRS158A/Y298H had an expanded and increased hydrophilicity catalytic pocket, and an enhanced π-π interaction which could contribute to faster conversion efficiency and higher catalytic rate. The whole cell system containing SceCPRS158A/Y298H and glucose dehydrogenase (GDH), under the optimized condition, could reduce 490.21 mM D-PL with e.e.≧ 99%, conversion rate = 98%, and the space-time yield = 382.80 g·L-1·d-1, which is the highest level reported so far.

Molecular Mapping of Yr85 and Comparison with Other Genes for Resistance to Stripe Rust on Wheat Chromosome 1B.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most serious plant diseases worldwide. Resistant cultivars are the most effective way to control the disease. YrTr1 is an important stripe rust resistance gene that has been used in wheat breeding programs and is represented in the host differential set to identify P. striiformis f. sp. tritici races in the United States. To map YrTr1, AvSYrTr1NIL was backcrossed to its recurrent parent Avocet S (AvS). Seedlings of BC7F2, BC7F3, and BC8F1 populations were tested with YrTr1-avirulent races under controlled conditions, and BC7F2 plants were genotyped using simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers. YrTr1 was mapped to the short arm of chromosome 1B using four SSR and seven SNP markers. The genetic distances of YrTr1 from the nearest flanking markers IWA2583 and IWA7480 were 1.8 and 1.3 centimorgans (cM), respectively. DNA amplification of a set of 21 Chinese Spring (CS) nulli-tetrasomic lines and seven CS 1B deletion lines with three SSR markers confirmed the chromosome arm location and further placed the gene in chromosomal bin region 1BS18 (0.5). The gene was determined to be about 7.4 cM proximal to Yr10. Based on multirace response array and chromosomal location, YrTr1 was determined to be different from other permanently named stripe rust resistance genes in chromosome arm 1BS and was named Yr85.

Characterization and Molecular Mapping of a Gene Conferring High-Temperature Adult-Plant Resistance to Stripe Rust Originally from Aegilops ventricosa.

Wheat near-isogenic line AvSYr17NIL carrying Yr17, originally from Aegilops ventricosa for all-stage resistance to Puccinia striiformis f. sp. tritici, also shows nonrace-specific, high-temperature adult-plant (HTAP) resistance to the stripe rust pathogen. To separate and identify the HTAP resistance gene, seeds of AvSYr17NIL were treated with ethyl methanesulfonate. Mutant lines with only HTAP resistance were obtained, and one of the lines, M1225, was crossed with the susceptible recurrent parent Avocet S (AvS). Field responses of the F2 plants and F3 lines, together with the parents, were recorded at the adult-plant stage in Pullman and Mount Vernon, WA under natural P. striiformis f. sp. tritici infection. The parents and the F4 population were phenotyped with a Yr17-virulent P. striiformis f. sp. tritici race in the adult-plant stage under the high-temperature profile in the greenhouse. The phenotypic results were confirmed by testing the F5 population in the field under natural P. striiformis f. sp. tritici infection. The F2 data indicated a single recessive gene, temporarily named YrM1225, for HTAP resistance. The F4 lines were genotyped with Kompetitive allele-specific PCR markers converted from single-nucleotide polymorphism markers polymorphic between M1225 and AvS. The HTAP resistance gene was mapped on the short arm of chromosome 2A in an interval of 7.5 centimorgans using both linkage and quantitative trait locus mapping approaches. The separation of the HTAP resistance gene from Yr17 should improve the understanding and utilization of the different types of resistance.

Genome-wide association study of common resistance to rust species in tetraploid wheat.

Rusts of the genus Puccinia are wheat pathogens. Stem (black; Sr), leaf (brown; Lr), and stripe (yellow; Yr) rust, caused by Puccinia graminis f. sp. tritici (Pgt), Puccinia triticina (Pt), and Puccinia striiformis f. sp. tritici (Pst), can occur singularly or in mixed infections and pose a threat to wheat production globally in terms of the wide dispersal of their urediniospores. The development of durable resistant cultivars is the most sustainable method for controlling them. Many resistance genes have been identified, characterized, genetically mapped, and cloned; several quantitative trait loci (QTLs) for resistance have also been described. However, few studies have considered resistance to all three rust pathogens in a given germplasm. A genome-wide association study (GWAS) was carried out to identify loci associated with resistance to the three rusts in a collection of 230 inbred lines of tetraploid wheat (128 of which were Triticum turgidum ssp. durum) genotyped with SNPs. The wheat panel was phenotyped in the field and subjected to growth chamber experiments across different countries (USA, Mexico, Morocco, Italy, and Spain); then, a mixed linear model (MLM) GWAS was performed. In total, 9, 34, and 5 QTLs were identified in the A and B genomes for resistance to Pgt, Pt, and Pst, respectively, at both the seedling and adult plant stages. Only one QTL on chromosome 4A was found to be effective against all three rusts at the seedling stage. Six QTLs conferring resistance to two rust species at the adult plant stage were mapped: three on chromosome 1B and one each on 5B, 7A, and 7B. Fifteen QTLs conferring seedling resistance to two rusts were mapped: five on chromosome 2B, three on 7B, two each on 5B and 6A, and one each on 1B, 2A, and 7A. Most of the QTLs identified were specific for a single rust species or race of a species. Candidate genes were identified within the confidence intervals of a QTL conferring resistance against at least two rust species by using the annotations of the durum (cv. 'Svevo') and wild emmer wheat ('Zavitan') reference genomes. The 22 identified loci conferring resistance to two or three rust species may be useful for breeding new and potentially durable resistant wheat cultivars.

Folding Features and Dynamics of 3D Genome Architecture in Plant Fungal Pathogens.

The folding and dynamics of three-dimensional (3D) genome organization are fundamental for eukaryotes executing genome functions but have been largely unexplored in nonmodel fungi. Using high-throughput sequencing coupled with chromosome conformation capture (Hi-C) data, we generated two chromosome-level assemblies for Puccinia striiformis f. sp. tritici, a fungus causing stripe rust disease on wheat, for studying 3D genome architectures of plant pathogenic fungi. The chromatin organization of the fungus followed a combination of the fractal globule model and the equilibrium globule model. Surprisingly, chromosome compartmentalization was not detected. Dynamics of 3D genome organization during two developmental stages of P. striiformis f. sp. tritici indicated that regulation of gene activities might be independent of the changes of genome organization. In addition, chromatin conformation conservation was found to be independent of genome sequence synteny conservation among different fungi. These results highlighted the distinct folding principles of fungal 3D genomes. Our findings should be an important step toward a holistic understanding of the principles and functions of genome architecture across different eukaryotic kingdoms. IMPORTANCE Previously, our understanding of 3D genome architecture has mainly come from model mammals, insects, and plants. However, the organization and regulatory functions of 3D genomes in fungi are largely unknown. In this study, we comprehensively investigated P. striiformis f. sp. tritici, a plant fungal pathogen, and revealed distinct features of the 3D genome, comparing it with the universal folding feature of 3D genomes in higher eukaryotic organisms. We further suggested that there might be distinct regulatory mechanisms of gene expression that are independent of chromatin organization changes during the developmental stages of this rust fungus. Moreover, we showed that the evolutionary pattern of 3D genomes in this fungus is also different from the cases in mammalian genomes. In addition, the genome assembly pipeline and the generated two chromosome-level genomes will be valuable resources. These results highlighted the unexplored distinct features of 3D genome organization in fungi. Therefore, our study provided complementary knowledge to holistically understand the organization and functions of 3D genomes across different eukaryotes.

Current Status and Future Perspectives of Genomics Research in the Rust Fungi.

Rust fungi in Pucciniales have caused destructive plant epidemics, have become more aggressive with new virulence, rapidly adapt to new environments, and continually threaten global agriculture. With the rapid advancement of genome sequencing technologies and data analysis tools, genomics research on many of the devastating rust fungi has generated unprecedented insights into various aspects of rust biology. In this review, we first present a summary of the main findings in the genomics of rust fungi related to variations in genome size and gene composition between and within species. Then we show how the genomics of rust fungi has promoted our understanding of the pathogen virulence and population dynamics. Even with great progress, many questions still need to be answered. Therefore, we introduce important perspectives with emphasis on the genome evolution and host adaptation of rust fungi. We believe that the comparative genomics and population genomics of rust fungi will provide a further understanding of the rapid evolution of virulence and will contribute to monitoring the population dynamics for disease management.

A transcriptomic-guided strategy used in identification of a wheat rust pathogen target and modification of the target enhanced host resistance to rust pathogens.

Transcriptional reprogramming is an essential feature of plant immunity and is governed by transcription factors (TFs) and co-regulatory proteins associated with discrete transcriptional complexes. On the other hand, effector proteins from pathogens have been shown to hijack these vast repertoires of plant TFs. Our current knowledge of host genes' role (including TFs) involved in pathogen colonization is based on research employing model plants such as Arabidopsis and rice with minimal efforts in wheat rust interactions. In this study, we begun the research by identifying wheat genes that benefit rust pathogens during infection and editing those genes to provide wheat with passive resistance to rust. We identified the wheat MYC4 transcription factor (TF) located on chromosome 1B (TaMYC4-1B) as a rust pathogen target. The gene was upregulated only in susceptible lines in the presence of the pathogens. Down-regulation of TaMYC4-1B using barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) in the susceptible cultivar Chinese Spring enhanced its resistance to the stem rust pathogen. Knockout of the TaMYC4-1BL in Cadenza rendered new resistance to races of stem, leaf, and stripe rust pathogens. We developed new germplasm in wheat via modifications of the wheat TaMYC4-1BL transcription factor.

Changes of Barley Stripe Rust Populations in the United States from 1993 to 2017.

Barley stripe rust is a relatively new disease in the United States. The pathogen, Puccinia striiformis f. sp. hordei (Psh), was first observed in Texas in 1991 and has spread north and westwards and mainly caused epidemics in the western United States. A total of 447 isolates collected from 1993 to 2017 were identified as 382 multilocus genotypes (MLGs) using 14 simple sequence repeat markers. The MLGs were clustered into six molecular groups (MGs) using the discriminant analysis of principal components and the hierarchical cluster analysis, and the MGs had significant differences in frequency in different years. MG1 was present in the population prior to the year 2000. MG2, MG3, and MG4 became predominate after 2000. MG5 was detected in all 24 years but more frequent from 2010 to 2017. MG6 was the most recent group detected mainly from 2011 to 2017 and had the highest correlation coefficient with the virulence phenotypes among the MGs. The heterozygosity and genotypic diversity of the Psh populations increased from 2000 to 2017, even more from 2010 to 2017. The results indicate rapid genetic changes from year to year, with major molecular group changes around 2000 and 2010. The possible mechanisms underlying the population changes are discussed.

Determination of three sites involved in the divergence of L-aspartate-α-decarboxylase self-cleavage in bacteria.

L-aspartate-α-decarboxylase (PanD) is an essential enzyme catalysing the decarboxylation of L-aspartate to ß-alanine in organisms. To perform the catalytic functions, PanD pro-proteins need to be self-cleaved to form two subunits: active α-subunit and ß-subunit. However, the processes of self-cleavage have diverged in different organisms for unknown reasons. To reveal the possible divergence mechanisms, the molecular evolution, selection pressures and site-directed mutagenesis of the panD gene family were explored in this study. The evolution analysis revealed that the panD genes in bacteria have diverged into three clades: Class I, Class II and Class III. Furthermore, 9 positive selection sites (A13, T14, V23, L32, V44, N49, L55, L78, and V85 in BsupanD) were detected. As shown by SDS-PAGE assay and catalytic activity determination in the mutants of BsupanD and EcoPanD, three of those sites (T14, V44 and V85) affect the PanD activities and are involved in the divergence of panD self-cleavage, while the other 6 sites only influenced the enzymatic activities of PanD. Furthermore, the structure analysis indicated that the structural mechanisms of the 9 sites affecting the catalysis were various. In all, three sites contributing to the divergence of PanD self-cleavage were revealed, and the results also provide foundation for the industrial application of PanD in ß-alanine synthesis.

Identification of Secreted Protein Gene-Based SNP Markers Associated with Virulence Phenotypes of Puccinia striiformis f. sp. tritici, the Wheat Stripe Rust Pathogen.

Stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is a destructive disease that occurs throughout the major wheat-growing regions of the world. This pathogen is highly variable due to the capacity of virulent races to undergo rapid changes in order to circumvent resistance in wheat cultivars and genotypes and to adapt to different environments. Intensive efforts have been made to study the genetics of wheat resistance to this disease; however, no known avirulence genes have been molecularly identified in Pst so far. To identify molecular markers for avirulence genes, a Pst panel of 157 selected isolates representing 126 races with diverse virulence spectra was genotyped using 209 secreted protein gene-based single nucleotide polymorphism (SP-SNP) markers via association analysis. Nineteen SP-SNP markers were identified for significant associations with 12 avirulence genes: AvYr1, AvYr6, AvYr7, AvYr9, AvYr10, AvYr24, AvYr27, AvYr32, AvYr43, AvYr44, AvYrSP, and AvYr76. Some SP-SNPs were associated with two or more avirulence genes. These results further confirmed that association analysis in combination with SP-SNP markers is a powerful tool for identifying markers for avirulence genes. This study provides genomic resources for further studies on the cloning of avirulence genes, understanding the mechanisms of host-pathogen interactions, and developing functional markers for tagging specific virulence genes and race groups.

Race Characterization of Puccinia striiformis f. sp. tritici in the United States from 2013 to 2017.

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is an important disease of wheat. In this study, 1,567 isolates collected from the United States from 2013 to 2017 were tested for virulence on 18 wheat Yr single-gene lines to differentiate races. In total, 72 races, including 20 new, were identified, and their frequencies in different years and different epidemiological regions were determined and compared. The 20 new races had low frequencies, and 7 of them each were detected from only one sample and 10 only in a single year. Frequencies of virulence to Yr10, Yr24, and Yr32 were low (<10%); to Yr1, Yr76, YrTr1, and YrSP were moderate (10 to 40%); and to Yr6, Yr7, Yr8, Yr9, Yr17, Yr27, Yr43, Yr44, and Exp2 were high (>70%), although they varied from year to year and from region to region. No virulence was detected to either Yr5 or Yr15, indicating that these genes were still effective against the pathogen in the United States. Based on the virulence data, the diversity of the U.S. P. striiformis f. sp. tritici population was the highest in 2016 and lowest in 2015, and the diversity of the regional population was the highest in region 1 and lowest in region 11. The yearly populations between consecutive years were closer than nonconsecutive years, and the eastern populations were closer to each other than those among the western populations. The findings are useful for understanding the pathogen evolution and for developing resistant cultivars for control of the disease.

Molecular Characterization of Wheat Stripe Rust Pathogen (Puccinia striiformis f. sp. tritici) Collections from Nine Countries.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most important diseases of wheat worldwide. To understand the worldwide distribution of its molecular groups, as well as the diversity, differentiation, and migration of the Pst populations, 567 isolates collected from nine countries (China, Pakistan, Italy, Egypt, Ethiopia, Canada, Mexico, Ecuador, and the U.S.) in 2010-2018 were genotyped using 14 codominant simple sequence repeat markers. A total of 433, including 333 new multi-locus genotypes (MLGs), were identified, which were clustered into ten molecular groups (MGs). The MGs and country-wise populations differed in genetic diversity, heterozygosity, and correlation coefficient between the marker and virulence data. Many isolates from different countries, especially the isolates from Mexico, Ecuador, and the U.S., were found to be identical or closely related MLGs, and some of the MGs were present in all countries, indicating Pst migrations among different countries. The analysis of molecular variance revealed 78% variation among isolates, 12% variation among countries, and 10% variation within countries. Only low levels of differentiation were found by the pairwise comparisons of country populations. Of the 10 MGs, 5 were found to be involved in sexual and/or somatic recombination. Identical and closely related MLGs identified from different countries indicated international migrations. The study provides information on the distributions of various Pst genetic groups in different countries and evidence for the global migrations, which should be useful in understanding the pathogen evolution and in stressing the need for continual monitoring of the disease and pathogen populations at the global scale.

Population Diversity, Dynamics, and Differentiation of Wheat Stripe Rust Pathogen Puccinia striiformis f. sp. tritici From 2010 to 2017 and Comparison With 1968 to 2009 in the United States.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a serious disease on wheat in the United States, especially after 2000. In the present study, 2,247 Pst isolates collected over all stripe rust epidemiological regions in the United States from 2010 to 2017 were genotyped at 14 simple sequence repeat (SSR) loci to investigate the population diversity, dynamics, and differentiation. A total of 1,454 multilocus genotypes (MLGs) were detected. In general, the populations in the west (regions 1-6) had more MLGs and higher diversities than the populations in the east (regions 7-12). The populations of 2010 and 2011 were more different from the other years. Genetic variation was higher among years than among regions, indicating the fast changes of the population. The divergence (Gst) was bigger between the west population and east population than among regions within either the west or east population. Gene flow was stronger among the regional populations in the east than in the west. Clustering analyses revealed 3 major molecular groups (MGs) and 10 sub-MGs by combining the genotypic data of 2010-2017 isolates with those of 1968-2009. MG1 contained both 1968-2009 isolates (23.1%) and 2010-2017 isolates (76.9%). MG2 had 99.4% of isolates from 1968-2009. MG3, which was the most recent and distinct group, had 99.1% of isolates from 2010-2017. Of the 10 sub-MGs, 5 (MG1-3, MG1-5, MG3-2, MG3-3, and MG3-4) were detected only from 2011 to 2017. The SSR genotypes had a moderate, but significant correlation (r = 0.325; p < 0.0001) with the virulence phenotype data. The standard index values of association (rbarD = 0.11) based on either regional or yearly populations suggest clonal reproduction. This study indicated high diversity, fast dynamics, and various levels of differentiation of the Pst population over the years and among epidemiological regions, and the results should be useful for managing wheat stripe rust.

Comparative transcriptome analysis during seeds development between two soybean cultivars.

Soybean is one of the important economic crops, which supplies a great deal of vegetable oil and proteins for human. The content of nutrients in different soybean seeds is different, which is related to the expression of multiple genes, but the mechanisms are complicated and still largely uncertain. In this study, to reveal the possible causes of the nutrients difference in soybeans A7 (containing low oil and high protein) and A35 (containing high oil and low protein), RNA-seq technology was performed to compare and identify the potential differential expressed genes (DEGs) at different seed developmental stages. The results showed that DEGs mainly presented at the early stages of seeds development and more DEGs were up-regulated at the early stage than the late stages. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis showed that the DEGs have diverged in A7 and A35. In A7, the DEGs were mainly involved in cell cycle and stresses, while in A35 were the fatty acids and sugar metabolism. Specifically, when the DEGs contributing to oil and protein metabolic pathways were analyzed, the differences between A7 and A35 mainly presented in fatty acids metabolism and seeds storage proteins (SSPs) synthesis. Furthermore, the enzymes, fatty acid dehydrogenase 2, 3-ketoacyl-CoA synthase and 9S-lipoxygenase, in the synthesis and elongation pathways of fatty acids, were revealed probably to be involved in the oil content difference between A7 and A35, the SSPs content might be due to the transcription factors: Leafy Cotyledon 2 and Abscisic acid-intensitive 3, while the sugar transporter, SWEET10a, might contribute to both oil and protein content differences. Finally, six DEGs were selected to analyze their expression using qRT-PCR, and the results were consistent with the RNA-seq results. Generally, the study provided a comprehensive and dynamic expression trends for the seed development processes, and uncovered the potential DEGs for the differences of oil in A7 and A35.

Genotyping Puccinia striiformis f. sp. tritici Isolates with SSR and SP-SNP Markers Reveals Dynamics of the Wheat Stripe Rust Pathogen in the United States from 1968 to 2009 and Identifies Avirulence-Associated Markers.

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is a devastating disease of wheat (Triticum aestivum) in the United States. The fungal pathogen can rapidly evolve, producing new virulent races infecting previously resistant cultivars and genotypes adapting to different environments. The objective of this study was to investigate the long-term population dynamics of P. striiformis f. sp. tritici in the United States. Through genotyping 1,083 isolates taken from 1968 to 2009, using 14 simple sequence repeat (SSR) markers and 92 secreted protein single nucleotide polymorphism (SP-SNP) markers, 614 and 945 genotypes were detected, respectively. In general, the two types of markers produced consistent genetic relationships among the P. striiformis f. sp. tritici populations over the 40-year period. The prior-to-2000 and the 2000-to-2009 populations were significantly different, with the latter showing higher genotypic diversity and higher heterozygosity than the earlier populations. Clustering analyses using genotypes of either SSR or SP-SNP markers revealed three molecular groups (MGs), MG1, MG2, and MG3. The prior-to-2000 and the 2000-to-2009 groups both had evidence of MG1 and MG2; however, MG3 was only found in the 2000-to-2009 population. Some of the isolates in the period of 2000 to 2009 formed individual clusters, suggesting exotic incursions. Other isolates of the same period were clustered with prior-to-2000 isolates, indicating that they were developed from the previously established populations. The data suggest the coexistence of newly introduced populations alongside established populations in the United States. Twenty SP-SNP markers were significantly associated to individual avirulence genes. These results are useful for developing more accurate monitoring systems and provide guidance for disease management.

Mapping Quantitative Trait Loci for High-Temperature Adult-Plant Resistance to Stripe Rust in Spring Wheat PI 197734 Using a Doubled Haploid Population and Genotyping by Multiplexed Sequencing.

Stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is a global concern for wheat production. Spring wheat cultivar PI 197734, of Sweden origin, has shown high-temperature adult-plant resistance (APR) to stripe rust for many years. To map resistance quantitative trait loci (QTL), 178 doubled haploid lines were developed from a cross of PI 197734 with susceptible AvS. The DH lines and parents were tested in fields in 2017 and 2018 under natural infection of Pst and genotyped with genotyping by multiplexed sequencing (GMS). Kompetitive allele specific PCR (KASP) and simple sequence repeat (SSR) markers from specific chromosomal regions were also used to genotype the population to validate and saturate resistance QTL regions. Two major QTL on chromosomes 1AL and 3BL and one minor QTL on 2AL were identified. The two major QTL, QYrPI197734.wgp-1A and QYrPI197734.wgp-3B, were detected in all tested environments explaining up to 20.7 and 46.8% phenotypic variation, respectively. An awnletted gene mapped to the expected distal end of chromosome 5AL indicated the accuracy of linkage mapping. The KASP markers converted from the GMS-SNPs in the 1A and 3B QTL regions were used to genotype 95 US spring wheat cultivars and breeding lines, and they individually showed different percentages of polymorphisms. The haplotypes of the three markers for the 1A QTL and four markers for the 3B QTL identified 37.9 and 21.1% of the wheat cultivar/breeding lines possibly carrying these two QTL, indicating their usefulness in marker-assisted selection (MAS) for incorporating the two major QTL into new wheat cultivars.