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Net form net blotch disease, caused by Pyrenophora teres f. teres, results in significant yield losses to barley industries. Up-to-date knowledge of the genetic diversity and structure of pathogen populations is critical for elucidating the disease epidemiology and unraveling pathogen survival and dispersal mechanisms. Thus, this study investigated long-distance dispersal and adaptation by analyzing the genetic structure of 250 P. teres f. teres isolates collected from Australia, Canada, Hungary, and Republic of South Africa (RSA), and historical isolates from Canada, Denmark, Japan, and Sweden. The population genetic structure detected by discriminant analysis of principal components, with the use of 5,890 Diversity Arrays Technology markers, revealed the presence of four clusters. Two of these contained isolates from all regions, and all isolates from RSA were grouped in these two. Australia and Hungary showed three clusters each. One of the Australian clusters contained only Australian isolates. One of the Hungarian clusters contained only Hungarian isolates and one Danish isolate. STRUCTURE analysis indicated that some isolates from Australia and Hungary shared recent ancestry with RSA, Canada, and historical isolates and were thus admixed. Subdivisions of the neighbor joining network indicated that isolates from distinct countries were closely related, suggesting that multiple introduction events conferred genetic heterogeneity in these countries. Through a neighbor joining analysis and amplification with form-specific DNA markers, we detected two hybrid isolates, CBS 281.31 from Japan and H-919 from Hungary, collected in 1931 and 2018, respectively. These results provide a foundation for exploring improved management of disease incursions and pathogen control through strategic deployment of resistance.

Phalaris aquatica L. (phalaris) is a cool-season perennial grass originating from the Mediterranean Basin, north-west Africa and Middle Eastern regions that is used for livestock agriculture mainly in temperate areas with dry summers. It has been the subject of breeding programs in Australia, South America, New Zealand and the USA. Increased knowledge of relationships between wild and cultivated germplasm through use of molecular markers has the potential to facilitate future breeding gains. For this purpose, we conducted an analysis of P. aquatica by using 3905 polymorphic DArTseq SNP markers. Genetic diversity as measured by expected heterozygosity was similar for wild (HE = 0.14; n = 57) and cultivated (HE = 0.13; n = 37) accessions. Diversity in wild germplasm was generally continuous in nature, largely related to geographical location, with a division at the broadest scale into eastern and western clades, with more admixture in the western than the eastern clade. Structure analysis of wild germplasm indicated six subpopulations consistent with country of origin, with some admixture among subpopulations likely resulting from natural and human influences. There were nine subpopulations among wild and cultivated accessions combined. This population structure should be considered if genomic selection is applied in P. aquatica. Analysis of molecular variance indicated that 71% of the genetic variation occurred within subpopulations and 29% among subpopulations. Genetic distances were low among cultivated germplasm from most countries except the USA, which was more distinct. Evaluation of material from the US pool by breeding programs in other countries, and additional material from the less utilised eastern clade, may be worthwhile.

Conservation genomics research often relies on accurate sex information to make inferences about species demography, dispersal, and population structure. However, field determined sex data are not always available and can be subject to human error, while laboratory sex assignment methods such as PCR assays can often be costly and challenging for non-model species. Conservation genomics programs increasingly use reduced-representation genome sequencing to assess neutral and functional genetic diversity, population structure, gene flow and pedigrees in threatened species. Here we demonstrate that sex can be determined from reduced-representation sequencing data produced by the increasingly popular Diversity Arrays Technology sequencing workflow (DArT-seq) using a program originally designed for application to shotgun data. This program—sexassign—compares the “dosage” of sequencing reads mapping to autosomes versus the X chromosome. In the present study, sexassign was used to identify the sex of 60 field-collected Greater Stick-Nest Rat (Leporillus conditor) samples, despite the absence of an annotated reference genome for the species. This “read-dosage” approach is not only more accurate and affordable than traditional sex assignment methods, but can be applied to any diploid organism with a heterogametic sex determination system—including non-model and understudied species of conservation importance—by using FASTQs generated by DArT.

The Coffee Berry Disease (CBD) epidemics destroy up to 100% of the crop on a susceptible variety. Resistance to CBD is conferred by the T-gene in Hibrido De Timor (HDT), R-gene in Rume Sudan (RS), and k-gene in K7, which were assembled in resistance varieties Ruiru 11(R11) and Batian. This study aimed to evaluate the genetic relationship between R11 and Batian with their parents' HDT, RS, SL8 and confirm the occurrence of T and R genes using DNA markers. Genome-wide single nucleotide polymorphism (SNP) markers were obtained through Diversity Arrays Technology sequencing (DArTseq). The genetic relationship was analyzed by Principal Component Analysis and hierarchical clustering. The Tgene was confirmed using Microsatellite primer, Sat 235 while the R gene was by marker sequence search within the DArTseq result files. The PC1 accounted for 42% of the total variation. Hierarchical clustering revealed less than 10% dissimilarity index apart from HDT that recorded above 20%. All the R11 and Batian genotypes carry the T gene. Eleven genotypes carry both T and R genes, therefore, with broad-based resistance to CBD. The study confirmed the narrow genetic relations within the Coffea arabica coffee varieties and further confirmed the occurrence of multiple gene resistance in R11 and Batian that will not break easily to new pathogen races. Key words: Coffee berry disease, diversity arrays technology sequencing, T gene, R gene, Ruiru 11, Batian, SL 28, codominant.

Finger millet (Eleusine coracana(L.) Gaertn. subsp.coracana) is the most importantmillet in eastern Africa and perhaps the oldest domesticated cereal grain in Africa.One of the major factors limiting finger millet production is blast disease caused bythe fungusMagnaporthe grisea. Crop wild relatives and landraces present a poten-tial source of novel genes. This study investigated the response of cultivated and wildrelatives of finger millet to an isolate of blast disease from western Kenya. Previousgermplasm collections were purified through two generations of single-seed descentbefore screening alongside improved and farmer-preferred varieties (FPVs) under ascreen house across three seasons. Farmer-preferred varieties were identified throughparticipatory varietal selection (PVS). The plants were inoculated twice during eachgrowth period using hand-spraying method and data on disease incidence recorded atgrain-filling stage. Genotypic data was generated using diversity arrays technology(DArT) sequencing and data analysis done using Genstat 18.2 and TASSEL 5.2.58.We observed high heritability (81%), indicating that the variation observed was pre-dominantly genetic. Wild accessions were generally more resistant to the disease incomparison to the cultivated accessions. Preliminary genome-wide association study(GWAS) using general linear model with principal component analysis led to theidentification of 19 markers associated with blast disease that will be be developedinto assays for genotype quality control and trait introgression. Wild accessions andlandraces of finger millet present a good reservoir for novel genes that can be incor-porated into crop improvement programs.

Sorghum (Sorghum bicolor [L.] Moench) is the fifth most important cereal crop worldwide and second aftermaize (Zeamays L.) in Kenya. It is an important food security crop in arid and semi-arid lands, where its production potential is hampered by drought. Drought tolerance can be measured by a plant’s ability to resist premature senescence, often described as stay-green. This study was carried out with the objective of identifying novel stay-green trait among wild and landrace genotypes of sorghum. Forty-four sorghum genotypes that included 16 improved, nine landraces, and 17 wild relatives of sorghum alongside known stay-green sources, B35 and E36-1, were evaluated under well-watered and water-stressed conditions in an alpha-lattice design of three replications. Data was collected on plant height (PHT), flag leaf area (FLA), panicle weight (PWT), 100-seed weight (HSW), relative chlorophyll content (RCC), number of green leaves at maturity (GLAM), days to 50% flowering (DFL), and grain yield (YLD). Genetic diversity was determined using diversity arrays technology (DArT) sequencing and quality control (QC) markers were generated using a java script. Lodoka, a landrace, was the most drought-tolerant genotype, recorded the highest numbers of RCC and GLAM, and outperformed B35 and E36-1 in yield under water-stress and well-watered conditions. The RCC was highly correlated with GLAM (r = .71) and with yield-related traits, HSW (r = .85), PWT (r = .82), and YLD (r = .78). All traits revealed high heritability (broad-sense) ranging from 60.14 to 98.4% for RCC and DFL, respectively. These results confirm earlier reports that wild relatives and landraces are a good source of drought tolerance alleles.

Pearl millet hybrids biofortified with iron (Fe) and zinc (Zn) promise to be part of a long-term strategy to combat micronutrient malnutrition in the arid and semi-arid tropical (SAT) regions of the world. Biofortification through molecular breeding is the way forward to achieving a rapid trait-based breeding strategy. This genome-wide association study (GWAS) was conducted to identify significant marker-trait associations (MTAs) for Fe, Zn, and protein content (PC) for enhanced biofortification breeding. A diverse panel of 281 advanced inbred lines was evaluated for Fe, Zn, and PC over two seasons. Phenotypic evaluation revealed high variability (Fe: 32–120 mg kg−1, Zn: 19–87 mg kg−1, PC: 8–16%), heritability (hbs2 ≥ 90%) and significantly positive correlation among Fe, Zn and PC (P = 0.01), implying concurrent improvement. Based on the Diversity Arrays Technology (DArT) seq assay, 58,719 highly informative SNPs were filtered for association mapping. Population structure analysis showed six major genetic groups (K = 6). A total of 78 MTAs were identified, of which 18 were associated with Fe, 43 with Zn, and 17 with PC. Four SNPs viz., Pgl04_64673688, Pgl05_135500493, Pgl05_144482656, and Pgl07_101483782 located on chromosomes Pgl04 (1), Pgl05 (2) and Pgl07 (1), respectively were co-segregated for Fe and Zn. Promising genes, ‘Late embryogenesis abundant protein’, ‘Myb domain’, ‘pentatricopeptide repeat’, and ‘iron ion binding’ coded by 8 SNPs were identified. The SNPs/genes identified in the present study presents prospects for genomics assisted biofortification breeding in pearl millet.

Synthetic hexaploid wheat (SHW), possesses numerous genes for drought that can help breeding for drought-tolerant wheat varieties. We evaluated 10 root traits at seedling stage in 111 F9 recombinant inbred lines derived from a F2 population of a SHW line (SHW-L1) and a common wheat line, under normal (NC) and polyethylene glycol-simulated drought stress conditions (DC). We mapped quantitative trait loci (QTLs) for root traits using an enriched high-density genetic map containing 120370 single nucleotide polymorphisms (SNPs), 733 diversity arrays technology markers (DArT) and 119 simple sequence repeats (SSRs). With four replicates per treatment, we identified 19 QTLs for root traits under NC and DC, and 12 of them could be consistently detected with three or four replicates. Two novel QTLs for root fresh weight and root diameter under NC explained 9 and 15.7% of the phenotypic variation respectively, and six novel QTLs for root fresh weight, the ratio of root water loss, total root surface area, number of root tips, and number of root forks under DC explained 8.5–14% of the phenotypic variation. Here seven of eight novel QTLs could be consistently detected with more than three replicates. Results provide essential information for fine-mapping QTLs related to drought tolerance that will facilitate breeding drought-tolerant wheat cultivars.

Systematic genetic profiling of the genetic pool is fundamental to select desirable parents for conventional and genomic-assisted breeding. The objective of this study was to assess genetic variati...

The durum wheat lines Heller#1 and Dunkler from the International Maize and Wheat Improvement Center Global Wheat Program showed moderate and stable adult plant resistance to leaf rust under high disease pressure over field environments in northwestern Mexico. Leaf rust phenotyping was performed on two recombinant inbred line (RIL) populations derived from crosses of Heller#1 and Dunkler with the susceptible parent Atred#2, conducted under artificially induced Puccinia triticina epidemics in 2013, 2014, 2015, and 2016. The Atred#2 × Heller#1 and Atred#2 × Dunkler populations were genotyped by single nucleotide polymorphism (SNP) platforms and diversity arrays technology markers, respectively. Four leaf rust resistance quantitative trait loci were detected simultaneously in the two RIL populations: Lr46, QLr.cim-2BC, QLr.cim-5BL, and QLr.cim-6BL based on phenotypic data across all four crop seasons. They explained 11.7 to 46.8%, 7.2 to 26.1%, 8.4 to 24.1%, and 12.4 to 28.5%, respectively, of the phenotypic variation for leaf rust resistance in Atred#2 × Heller#1 and 16.3 to 56.6%, 6.7 to 15.7%, 4.1 to 10.1%, and 5.1 to 20.2% of the variation in the Atred#2 × Dunkler population. Only the resistance allele of QLr.cim-2BC was from the susceptible parent Atred#2, and resistance alleles at other loci came from the resistant parents Heller#1 and Dunkler. The SNP markers closely linked to Lr46 and QLr.cim-2BC were converted to kompetitive allele specific PCR markers for use in marker-assisted selection to improve leaf rust resistance through crosses with Heller#1 and Dunkler sources.

Net form net blotch (NFNB), caused by the fungal pathogen Pyrenophora teres f. teres, is an important foliar disease present in all barley-producing regions of the world. This fungus is a hemibiotrophic and heterothallic ascomycete, where sexual recombination can lead to changes in disease expression in the host. Knowledge of the genetic architecture and genes involved in virulence is vital to increase the durability of NFNB resistance in barley cultivars. We used a genome-wide association mapping approach to characterize P. teres f. teres genomic regions associated with virulence in Australian barley cultivars. One hundred eighty-eight P. teres f. teres isolates collected across five Australian states were genotyped using Diversity Arrays Technology sequence markers and phenotyped across 20 different barley genotypes. Association mapping identified 14 different genomic regions associated with virulence, with the majority located on P. teres f. teres chromosomes 3 and 5 and one each present on chromosomes 1, 6, and 9. Four of the regions identified were confirmed by quantitative trait loci (QTL) mapping. The QTL regions are discussed in the context of their genomic architecture together with examination of their gene contents, which identified 20 predicted effectors. The number of QTL shown in this study at the population level clearly illustrates a complex genetic basis of P. teres f. teres virulence compared with pure necrotrophs, such as the wheat pathogens Parastagonospora nodorum and Parastagonospora tritici-repentis.

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is a serious disease in bread wheat (Triticum aestivum L.). Identification and use of adult plant resistance (APR) resources are important for stripe rust resistance breeding. Bread wheat line C33 is an exotic germplasm that has shown stable APR to stripe rust for more than 10 years in Sichuan Province of China. Here, 183 recombinant inbred lines (RILs) derived from the cross between C33 and a susceptible line X440 were genotyped with diversity arrays technology (DArT) markers to identify resistance quantitative trait locus (QTL). Field trials were conducted in five years at Chengdu and Xindu of Sichuan Province, using maximum disease severity (MDS) as stripe rust reaction phenotypes. A total of four quantitative trait loci (QTLs) were detected, respectively designed as QYr.saas-3AS, QYr.saas-5AL, QYr.saas-5BL, and QYr.saas-7DS, explaining 4.14–15.21% of the phenotypic variances. QYr.saas-5BL and QYr.saas-7DS were contributed by C33. However, the level for stripe rust resistance contributed by them was not strong as C33, suggesting the presence of other unidentified QTLs in C33. QYr.saas-7DS corresponded to Yr18 and QYr.saas-5BL remains to be formally named. The RIL lines carrying combinations QYr.saas-5AL, QYr.saas-5BL, and QYr.saas-7DS showed comparability resistance with C33. The present study provides resources to pyramid diverse genes into locally adapted elite germplasm to improve the stripe rust resistance of bread wheat.

Reproductive strategies are an essential component of invasion ecology that influence invasion success and rates of population growth. Burmese Pythons (Python bivittatus) are large constrictor snakes that were introduced to the Greater Everglades Ecosystem of southern Florida, USA, from Asia. Since their introduction, these giant constrictors have spread throughout wetlands of southern Florida while increasing in abundance and causing declines in the native species upon which they prey. Multiple paternity in reproduction could facilitate invasion success by increasing the genetic diversity produced within each reproductive event. We used Diversity Arrays Technology genome-wide genotyping to assess multiple paternity in the progeny of wild Burmese Pythons in Florida. We analyzed >4,000 single nucleotide polymorphisms from 153 neonates belonging to 4 clutches collected in southwestern Florida. Complementary hierarchical and K-means clustering analyses of the genetic distances within clutches revealed that three clutches were each fertilized by two sires, with a fourth fertilized by a single sire. The proportions of offspring attributable to each sire within multiple paternity clutches ranged from nearly even to highly skewed. Analysis of multivariate dispersion showed significantly increased genetic variability in the multiple paternity clutches. These results improve our understanding of the reproductive strategy and invasion potential of a giant constrictor with significant ecological impacts.

The barley net blotch diseases are caused by two fungal species of the Pyrenophora genus. Specifically, spot form net blotch is caused by P. teres f. sp. maculata (Ptm) whereas net form net blotch is caused by P. teres f. sp. teres (Ptt). Ptt and Ptm show high genetic diversity in the field due to intraspecific sexual recombination and hybridisation of the two species although the latter is considered rare. Here we present occurrence of a natural Ptt/Ptm hybrid with azole fungicides resistance and its implication to barley disease management in Australia. We collected and sequenced a hybrid, 3 Ptm and 10 Ptt isolates and performed recombination analyses in the intergenic and whole genome level. Eleven out of 12 chromosomes showed significant (P < 0.05) recombination events in the intergenic regions while variable recombination rate showed significant recombination across all the chromosomes. Locus specific analyses of Cyp51A1 gene showed at least four recombination breakpoints including a point mutation that alter target protein function. This point mutation did not found in Ptt and Ptm collected prior to 2013 and 2017, respectively. Further genotyping of fourteen Ptt, 48 HR Ptm, fifteen Ptm and two P. teres isolates from barley grass using Diversity Arrays Technology markers showed that all HR Ptm isolates were clonal and not clustered with Ptt or Ptm. The result confirms occurrence of natural recombination between Ptt and Ptm in Western Australia and the HR Ptm is likely acquired azole fungicide resistance through recombination and underwent recent rapid selective sweep likely within the last decade. The use of available fungicide resistance management tactics are essential to minimise and restrict further dissemination of these adaptive HR Ptm isolates.

The development of nutritionally enhanced wheat (Triticum aestivum L.) with higher levels of grain iron (Fe) and zinc (Zn) offers a sustainable solution to micronutrient deficiency among resource-poor wheat consumers. One hundred and ninety recombinant inbred lines (RILs) from ‘Kachu’ × ‘Zinc-Shakti’ cross were phenotyped for grain Fe and Zn concentrations and phenological and agronomically important traits at Ciudad Obregon, Mexico in the 2017–2018, 2018–2019, and 2019–2020 growing seasons and Diversity Arrays Technology (DArT) molecular marker data were used to determine genomic regions controlling grain micronutrients and agronomic traits. We identified seven new pleiotropic quantitative trait loci (QTL) for grain Zn and Fe on chromosomes 1B, 1D, 2B, 6A, and 7D. The stable pleiotropic QTL identified have expanded the diversity of QTL that could be used in breeding for wheat biofortification. Nine RILs with the best combination of pleiotropic QTL for Zn and Fe have been identified to be used in future crossing programs and to be screened in elite yield trials before releasing as biofortified varieties. In silico analysis revealed several candidate genes underlying QTL, including those belonging to the families of the transporters and kinases known to transport small peptides and minerals (thus assisting mineral uptake) and catalyzing phosphorylation processes, respectively.

A panel of 387 durum wheat genotypes including Mediterranean landraces and modern cultivars was characterized with 46,161 diversity arrays technology (DArTseq) markers. Analysis of population structure uncovered the existence of five subpopulations (SP) related to the pattern of migration of durum wheat from the domestication area to the west of the Mediterranean basin (SPs 1, 2, and 3) and further improved germplasm (SPs 4 and 5). The total genetic diversity (HT) was 0.40 with a genetic differentiation (GST) of 0.08 and a mean gene flow among SPs of 6.02. The lowest gene flow was detected between SP 1 (presumably the ancient genetic pool of the panel) and SPs 4 and 5. However, gene flow from SP 2 to modern cultivars was much higher. The highest gene flow was detected between SP 3 (western Mediterranean germplasm) and SP 5 (North American and European cultivars). A genome wide association study (GWAS) approach using the top ten eigenvectors as phenotypic data revealed the presence of 89 selective sweeps, represented as quantitative trait loci (QTL) hotspots, widely distributed across the durum wheat genome. A principal component analysis (PCoA) using 147 markers with −log10p &gt, 5 identified three regions located on chromosomes 2A, 2B and 3A as the main drivers for differentiation of Mediterranean landraces. Gene flow between SPs offers clues regarding the putative use of Mediterranean old durum germplasm by the breeding programs represented in the structure analysis. EigenGWAS identified selective sweeps among landraces and modern cultivars. The analysis of the corresponding genomic regions in the ‘Zavitan’, ‘Svevo’ and ‘Chinese Spring’ genomes discovered the presence of important functional genes including Ppd, Vrn, Rht, and gene models involved in important biological processes including LRR-RLK, MADS-box, NAC, and F-box.

This chapter reviews the applications of data science methodologies for analysis of omics data in agricultural sciences. In view of omics observational Big Data, a focus on validation of model-based inference and causality is emphasized. The review is also focused on artificial intelligence random decision tree forest modelling with propensity score method for sample adjustments to reduce bias due to cofounding covariates. Special attention is paid to wheat genome wide association studies (GWAS) data sets obtained by diversity arrays technology of 30 000 markers and 2000 landraces, for eight traits, cultivated in four environments. Random forest decision tree models are applied for trait predictions and propensity score classification. Bootstrap simulation of the data sets provided estimation of 13% relative root mean square error (RMSE) for predictions with untrained data sets. The evaluation of causalities of single nucleotide polymorphism (SNP) markers for prediction of grain yield data depending on environment is also emphasized and causalities of individual SNPs are evaluated as average treatment exposures (ATE). Upregulations and downregulations of SNPs are identified as positive and negative ATE values in the range of [−0.2, 0.2] depending on environmental conditions. Intensities of effects of SNPs on traits are estimated as odd ratios (OR) by relative odds data of the subsets with presence and absence of corresponding SNPs. Maximal upregulation OR are in the range [3, 5] while for downregulation the range is [0.2, 0.4]. SNPs causalities show strong dependency on environment.

Conservation genomics research often relies on accurate sex information to make inferences about species demography, dispersal, and population structure. However, field determined sex data are not always available and can be subject to human error, while laboratory sex determination is costly and often challenging for non-model species. Conservation genomics programs increasingly use reduced-representation genome sequencing to assess neutral and functional genetic diversity, population structure, gene flow and pedigrees in threatened species. Here we demonstrate that sex can be determined from reduced-representation sequencing data produced by the increasingly popular Diversity Arrays Technology sequencing workflow (DArT-seq) using a program originally designed for application to shotgun data. This program – sexassign – compares the “dosage” of sequencing reads mapping to autosomes versus the X chromosome. In the present study, sexassign accurately determined the sex of 60 field-collected Greater Stick-Nest Rat (Leporillus conditor) samples, despite the absence of an annotated reference genome for the species. This “read-dosage” approach is not only more accurate and affordable than traditional sex determination methods, but can be applied to any diploid organism with a heterogametic sex determination system – including non-model and understudied species of conservation importance – by using FASTQs generated by DArT.

Tolerance to metribuzin, a broad-spectrum herbicide, is an important trait for weed control in wheat breeding. However, the genetics of metribuzin tolerance in relation to the underlying quantitative trait loci (QTL) and genes is limited. This study developed F8 recombinant inbred lines (RILs) from a cross between a highly resistant genotype (Chuan Mai 25) and highly susceptible genotype (Ritchie), which were used for QTL mapping of metribuzin tolerance. Genotyping was done using a diversity arrays technology sequencing (DArTseq) platform, and phenotyping was done in controlled environments. Herbicide tolerance was measured using three traits, visual score (VS), reduction of chlorophyll content (RCC), and mean value of chlorophyll content for metribuzin-treated plants (MCC). A high-density genetic linkage map was constructed using 2,129 DArTseq markers. Inclusive composite interval mapping (ICIM) identified seven QTL, one each on chromosomes 2A, 2D, 3A, 3B, 4A, 5A, and 6A. Three major QTL—Qrcc.uwa.2AS, Qrcc.uwa.5AL, and Qrcc.uwa.6AL—explained 11.39%, 11.06%, and 11.45% of the phenotypic variation, respectively. The 5A QTL was further validated using kompetitive allele-specific PCR (KASP) assays in an F3 validation population developed from Chuan Mai 25 × Dagger. Blasting the single-nucleotide polymorphisms (SNPs) flanking the QTL in the wheat reference genome RefV1.0 revealed SNP markers within or very close to annotated genes which could be candidate genes responsible for metribuzin tolerance. Most of the candidate genes were related to metabolic detoxification, especially those of P450 pathway and xenobiotic transmembrane transporter activity, which are reportedly key molecules responsible for herbicide tolerance. This study is the first to use specially developed populations to conduct QTL mapping on the metribuzin tolerance trait. The three major QTL and candidate genes identified in this study could facilitate marker-assisted metribuzin breeding in wheat. The QTL could be fine-mapped to locate the genes responsible for metribuzin tolerance, which could be introgressed into elite wheat cultivars.

Background Transcription factor (TF) GAMYB, belonging to MYB family (named after the gene of the avian myeloblastosis virus) is a master gibberellin (GA)-induced regulatory protein that is crucial for development and germination of cereal grain and involved in anther formation. It activates many genes including high-molecular-weight glutenin and α-amylase gene families. This study presents the first attempt to characterize the rye gene encoding GAMYB in relation to its sequence, polymorphisms, and phenotypic effects. Results ScGAMYB was mapped on rye chromosome 3R using high-density Diversity Arrays Technology (DArT)/DArTseq-based maps developed in three mapping populations. The ScGAMYB sequences were identified in RNA-seq libraries of four rye inbred lines. The transcriptome used for the search contained almost 151,000 transcripts with a median contig length of 500 nt. The average amount of total base raw data was approximately 9 GB. Comparative analysis of the ScGAMYB sequence revealed its high level of homology to wheat and barley orthologues. Single nucleotide polymorphisms (SNPs) detected among rye inbred lines allowed the development of allele specific-PCR (AS-PCR) markers for ScGAMYB that might be used to detect this gene in wide genetic stocks of rye and triticale. Segregation of the ScGAMYB alleles showed significant relationship with α-amylase activity (AMY). Conclusions The research showed the strong similarity of rye GAMYB sequence to its orthologues in other Graminae and confirmed the position in the genome consistent with the collinearity rule of cereal genomes. Concurrently, the ScGAMYB coding sequence (cds) showed stronger variability (24 SNPs) compared to the analogous region of wheat (5 SNPs) and barley (7 SNPs). The moderate regulatory effect of ScGAMYB on AMY was confirmed, therefore, ScGAMYB was identified as a candidate gene for partial control of α-amylase production in rye grain. The predicted structural protein change in the aa region 362–372, caused by a single SNP (C/G) at the 1100 position in ScGAMYB cds and single aa sequence change (S/C) at the 367 position, is the likely cause of the differences in the effectiveness of ScGAMYB regulatory function associated with AMY. The development of sequence-based, allele-specific (AS) PCR markers could be useful in research and application.

Elucidation of the process of sex chromosome differentiation is necessary to understand the dynamics of evolutionary mechanisms in organisms. The W sex chromosome of the Siamese cobra (Naja kaouthia) contains a large number of repeats and shares amniote sex chromosomal linkages. Diversity Arrays Technology provides an effective approach to identify sex-specific loci that are epoch-making, to understand the dynamics of molecular transitions between the Z and W sex chromosomes in a snake lineage. From a total of 543 sex-specific loci, 90 showed partial homology with sex chromosomes of several amniotes and 89 loci were homologous to transposable elements. Two loci were confirmed as W-specific nucleotides after PCR amplification. These loci might result from a sex chromosome differentiation process and involve putative sex-determination regions in the Siamese cobra. Sex-specific loci shared linkage homologies among amniote sex chromosomes, supporting an ancestral super-sex chromosome.

Yam (Dioscorea spp.) is a nutritional and medicinal staple tuber crop grown in the tropics and sub-tropics. Among the food yam species, water yam (Dioscorea alata L.) is the most widely distributed and cultivated species worldwide. Tuber dry matter content (DMC) and oxidative browning (OxB) are important quality attributes that determine cultivar acceptability in water yam. This study used a single nucleotide polymorphism (SNP) assay from a diversity arrays technology (DArT) platform for a genome-wide association study (GWAS) of the two quality traits in a panel of 100 water yam clones grown in three environments. The marker&ndash, trait association analysis identified significant SNPs associated with tuber DMC on chromosomes 6 and 19 and with OxB on chromosome 5. The significant SNPs cumulatively explained 45.87 and 12.74% of the total phenotypic variation for the tuber DMC and OxB, respectively. Gene annotation for the significant SNP loci identified important genes associated in the process of the proteolytic modification of carbohydrates in the dry matter accumulation pathway as well as fatty acid &beta, oxidation in peroxisome for enzymatic oxidation. Additional putative genes were also identified in the peak SNP sites for both tuber dry matter and enzymatic oxidation with unknown functions. The results of this study provide valuable insight for further dissection of the genetic architecture of tuber dry matter and enzymatic oxidation in water yam. They also highlight SNP variants and genes useful for genomics-informed selection decisions in the breeding process for improving food quality traits in water yam.

Stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is one of the most devastating foliar diseases in wheat. Host resistance is the most effective strategy for the management of the disease. To screen for accessions with stable resistance and identify effective stripe rust resistance loci, a genome-wide association study (GWAS) was conducted using a panel of 140 Chinese wheat landraces. The panel was evaluated for stripe rust response at the adult-plant stage at six field-year environments with mixed races and at the seedling stage with two separate predominant races of the pathogen, and genotyped with the genome-wide Diversity Arrays Technology markers. The panel displayed abundant phenotypic variation in stripe rust responses, with 9 landraces showing stable resistance to the mixture of Pst races at the adult-plant stage in the field and 10 landraces showing resistance to individual races at the seedling stage in the greenhouse. GWAS identified 12 quantitative trait loci (QTL) significantly (P ≤ 0.001) associated to stripe rust resistance using the field data of at least two environments and 18 QTL using the seedling data with two races. Among these QTL, 10 were presumably novel, including 4 for adult-plant resistance mapped to chromosomes 1B (QYrcl.sicau-1B.3), 4A (QYrcl.sicau-4A.3), 6A (QYrcl.sicau-6A.2) and 7B (QYrcl.sicau-7B.2) and 6 for all-stage resistance mapped to chromosomes 2D (QYrcl.sicau-2D.1), 3B (QYrcl.sicau-3B.3), 3D (QYrcl.sicau-3D), 4B (QYrcl.sicau-4B), 6A (QYrcl.sicau-6A.1) and 6D (QYrcl.sicau-6D). The landraces with stable resistance can be used for developing wheat cultivars with effective resistance to stripe rust.

An investigation of sex-specific loci may provide important insights into fish sex determination strategies. This may be useful for biotechnological purposes, for example, to produce all-male or all-female fish for commercial breeding. The North African catfish species, Clarias gariepinus, has been widely adopted for aquaculture because its superior growth and disease resistance render the species suitable for hybridization with other catfish to improve the productivity and quality of fish meat. This species has either a ZZ/ZW or XX/XY sex determination system. Here, we investigate and characterize these systems using high-throughput genome complexity reduction sequencing as Diversity Arrays Technology. This approach was effective in identifying moderately sex-linked loci with both single-nucleotide polymorphisms (SNPs) and restriction fragment presence/absence (PA) markers in 30 perfectly sexed individuals of C. gariepinus. However, SNPs based markers were not found in this study. In total, 41 loci met the criteria for being moderately male-linked (with male vs. female ratios 80:20 and 70:30), while 25 loci were found to be moderately linked to female sex. No strictly male- or female-linked loci were detected. Seven moderately male-linked loci were partially homologous to some classes of transposable elements and three moderately male-linked loci were partially homologous to functional genes. Our data showed that the male heterogametic XX/XY sex determination system should co-exist with the ZZ/ZW system in C. gariepinus. Our finding of the co-existence of XX/XY and ZZ/ZW systems can be applied to benefit commercial breeding of this species in Thailand. This approach using moderately sex-linked loci provides a solid baseline for revealing sex determination mechanisms and identify potential sex determination regions in catfish, allowing further investigation of genetic improvements in breeding programs.

Background This study demonstrates the use of reduced-representation genotyping to provide preliminary identifications for thermophilic bacterial isolates. The approach combines restriction enzyme digestion and PCR with next-generation sequencing to provide thousands of short-read sequences from across the bacterial genomes. Isolates were obtained from compost, hot water systems, and artesian bores of the Great Artesian Basin. Genomic DNA was double-digested with two combinations of restriction enzymes followed by PCR amplification, using a commercial provider of DArTseq™, Diversity Arrays Technology Pty Ltd. (Canberra, Australia). The resulting fragments which formed a reduced-representation of approximately 2.3% of the genome were sequenced. The sequence tags obtained were aligned against all available RefSeq bacterial genome assemblies by BLASTn to identify the nearest reference genome. Results Based on the preliminary identifications, a total of 99 bacterial isolates were identified to species level, from which 8 isolates were selected for whole-genome sequencing to assess the identification results. Novel species and strains were discovered within this set of isolates. The preliminary identifications obtained by reduced-representation genotyping, as well as identifications obtained by BLASTn alignment of the 16S rRNA gene sequence, were compared with those derived from the whole-genome sequence data, using the same RefSeq sequence database for the three methods. Identifications obtained with reduced-representation sequencing agreed with the identifications provided by whole-genome sequencing in 100% of cases. The identifications produced by BLASTn alignment of 16S rRNA gene sequence to the same database differed from those provided by whole-genome sequencing in 37.5% of cases, and produced ambiguous identifications in 50% of cases. Conclusions Previously, this method has been successfully demonstrated for use in bacterial identification for medical microbiology. This study demonstrates the first successful use of DArTseq™ for preliminary identification of thermophilic bacterial isolates, providing results in complete agreement with those obtained from whole-genome sequencing of the same isolates. The growing database of bacterial genome sequences provides an excellent resource for alignment of reduced-representation sequence data for identification purposes, and as the available sequenced genomes continue to grow, the technique will become more effective.

The socio-economic impact of Fusarium odoratissimum, which is colloquially called tropical race 4 (TR4), is escalating as this fungal pathogen spreads to new banana-growing areas. Hence, the development of simple, reliable and rapid detection technologies is indispensable for implementing quarantine measures. Here, a versatile loop-mediated isothermal amplification (LAMP) assay has been developed that is applicable under field and laboratory conditions. DNA markers unique to TR4 isolates were obtained by diversity arrays technology sequencing (DArTseq), a genotyping by sequencing technology that was conducted on 27 genotypes, comprising 24 previously reported vegetative compatibility groups (VCGs) and three TR4 isolates. The developed LAMP TR4 assay was successfully tested using 22 TR4 isolates and 45 non-target fungal and bacterial isolates, as well as on infected plants under greenhouse and field conditions. The detection limit was 1 pg µL−1 pure TR4 DNA or 102 copies plasmid-localized TR4 unique sequence (SeqA) per reaction, which was not affected by background DNA in complex samples. The LAMP TR4 assay offers a powerful tool for the routine and unambiguous identification of banana plants infected with TR4, contributing to advanced diagnosis in field situations and monitoring of fusarium wilt.

A low level of available potassium (K) in soils is a major factor restricting crop growth and production. In earlier studies we found that Tibetan wild barley has a wider variation in low K stress tolerance than cultivated barley. In this study, we performed a genome-wide association analysis of 179 Tibetan wild barley (Hordeum vulgare L.) accessions exposed to low K stress and investigated the associations of K uptake rate (KUR), K translocation rate (KTR) and other K metabolism traits with 11,013 diversity arrays technology markers. The results showed that there were significant differences in tissue K concentration and content, KUR and KTR among the wild barley accessions analyzed. Scores for all traits showed a normal distribution. Three significant quantitative trait loci (QTLs) for KUR and KTR were identified and located on 6H and 1H. These QTLs were found to be associated with the cation/H(+) antiporter and cyclic nucleotide gated channel. A number of unique candidate genes associated with K-related signals, including K channels, K transporters and ethylene-related genes, were also identified. The K channels and K transporters involved in K uptake and translocation, respectively, were closely associated with KUR in response to low K stress. The present study highlights the potential of Tibetan wild barley to provide elite candidate genes for the improvement of K use efficiency of barley.

Fructans (fructo-oligosaccharides) is one of the most important carbohydrates in wheat vegetative tissues and plays a vital role in wheat grain development. One hundred and forty 'Avocet'× 'Chilero' wheat (Triticum aestivum L.) recombinant inbred lines (RILs) were phenotyped for grain fructans at Luoyang and Langzhou field sites in the 2015-2016 and 2016-2017 growing seasons and 2090 Diversity Arrays Technology (DArT) molecular marker data were used to determine genomic regions controlling fructan content. Eleven significant quantitative trait loci (QTL) for wheat fructan content were identified on chromosomes 1B, 2A, 2B, 2D, 3D, 4B, 6B, 6D, 7A, and 7B using the inclusive composite interval mapping (ICIM) method, explaining 4.08% to 27.40% of the phenotypic variance, with seven exceeding 10%. The single environment analysis identified QF.haust-6D.1 and QF.haust-7B in Luoyang, QF.haust-1B, QF.haust-2D, QF.haust-3D, and QF.haust-7B in Lanzhou, and QF.haust-1B, QF.haust-7A, and QF.haust-7B in average phenotypic performance. The multi-environment analysis identified QF.haust-2B and QF.haust-7B, which explained 21.01% and 17.39% of the phenotypic variance, respectively. The positive allele of QF.haust-2B and QF.haust-7B were all originated from Chilero. QF.haust-2B was flanked by the 3957031 and SNP1203021 markers; candidate genes for QF.haust-2B were identified by anchoring marker sequences in the IGWSC wheat genome sequence, involving a variety of biological processes, such as the regulation of ribosomes metabolism in wheat. We also constructed the physical map for wheat grain fructan QF.haust-2B and QF.haust-7B, QF.haust-7B with a physical length of 11.47 Mb between marker SNP3026392 and 3954877 and 103 genes within the segment, in which ten were related to glucose metabolism. This study provides a theoretical basis and technical support for wheat grain fructan content-related genes and molecular marker-assisted breeding.