While genetic manipulation for improvement of agricultural crops is not new, technologies such as gene editing that allow precise and targeted gene modifications are becoming more common. Highly specific, sensitive, and practical methods to detect such edits are desirable for a number of reasons. In this chapter, we provide readers with an overview of their application and what different technology platforms that can used to develop detection methods for edits. The platforms include PCR, digital PCR and sequencing and are discussed in the context of their strengths and weakness. This chapter also includes a discussion on challenges to differentiate a genetic change created through gene-editing technologies from same changes that might arise through other means such as conventional breeding or mutagenesis.

Genome editing (also known as gene editing) employs a range of tools such as Meganucleases, Zinc Finger Nucleases, TALENs, and more recently CRISPR to make defined changes in genes, regulatory sequences, untranslated regions, or intergenic regions. It is increasingly being applied in plant science research and to improve plant varieties. The benefits of having effective detection tools begin with optimization of the genome editing process itself and continue with selection and characterization of tissue cultures and/or regenerated plants. Detection tools are also used throughout the breeding process, and for preparation of regulatory dossiers when required, as well as for seed production, and may be necessary for monitoring products in the marketplace. Detection and identification of genome edits employs a wide range of analytical approaches including PCR, digital PCR, and sequencing methods. This article examines the applicability of each category of detection or identification approach, from the optimization of genome editing processes, through creation of edits, selection and characterization, and breeding. The challenges surrounding the detection of genome edits present at low levels in large seed, plant, or grain populations and of differentiating directed genome edits from conventional mutations are also explained.

Fusarium head blight (FHB) damage in durum wheat (Triticum turgidum L. var. durum Desf., turgidum) inflicted massive economic losses worldwide. Meanwhile, FHB resistant durum wheat germplasm is extremely limited. ‘Tunisian108’ is a newly identified tetraploid wheat with FHB resistance. However, genomic regions in ‘Tunisian108’ that significantly associated with FHB resistance are yet unclear. Therefore, a population of 171 backcross inbred lines (BC1F7) derived from a cross between ‘Tunisian108’ and a susceptible durum cultivar ‘Ben’ was characterized. Fusarium graminearum (R010, R1267, and R1322) was point inoculated (greenhouse) or spawn inoculated (field) in 2010 and 2011. Disease severity, Fusarium-damaged kernel (FDK) and mycotoxins were measured. Analysis of variance showed significant genotype and genotype by environment effect on all traits. Approximately 8% of the lines in field and 25% of the lines in greenhouse were more resistance than Tunisian108. A framework linkage map of 267 DArt plus 62 SSR markers was developed representing 239 unique loci and covering a total distance of 1887.6 cM. Composite interval mapping revealed nine QTL for FHB severity, four QTL for DON, and four QTL for FDK on seven chromosomes. Two novel QTL, Qfhb.ndsu-3BL and Qfhb.ndsu-2B, were identified for disease severity, explaining 11 and 6% of the phenotypic variation, respectively. Also, a QTL with large effect on severity and a QTL with negative effect on FDK on chromosome 5A were identified. Importantly, a novel region on chromosome 2B was identified with multiple FHB resistance. Validation on these QTL would facilitate the durum wheat resistance breeding.

Meiotic crossovers (COs) are not uniformly distributed across the genome. Factors affecting this phenomenon are not well understood. Although many species exhibit large differences in CO numbers between sexes, sex-specific aspects of CO landscape are particularly poorly elucidated. Here, we conduct high-resolution CO mapping in maize. Our results show that CO numbers as well as their overall distribution are similar in male and female meioses. There are, nevertheless, dissimilarities at local scale. Male and female COs differ in their locations relative to transcription start sites in gene promoters and chromatin marks, including nucleosome occupancy and tri-methylation of lysine 4 of histone H3 (H3K4me3). Our data suggest that sex-specific factors not only affect male–female CO number disparities but also cause fine differences in CO positions. Differences between male and female CO landscapes indicate that recombination has distinct implications for population structure and gene evolution in male and in female meioses.

Single nucleotide polymorphisms (SNPs) were genotyped using a high-density array and DNAs from individual plants of important onion ( Allium cepa L.) populations from major production regions and from the likely progenitor of onion, Allium vavilovii Popov et Vved. Genotypes at 1226 SNPs were used to estimate genetic relationships among these populations and revealed close associations among onions grown in Europe and those in North America, South America, and eastern Asia, supporting paths of introduction from Europe to the Americas and Asia. ‘Nasik Red’ is a population grown on the Indian subcontinent and was divergent from onions of European origin. Frequencies of SNPs among and within populations were used as a measure of informativeness, and 199 commonly polymorphic SNPs were identified distributed across the eight chromosomes of onion. These SNPs will be useful for estimations of relatedness among broader collections of onion populations, mapping of important phenotypes, fingerprinting of inbred lines and hybrids, and quality control of seed lots.

Agricultural Scientists has lifted the crop production many folds' overs last 100 years but nutritive quality of crop products has not been addressed accordingly as a result humans in many parts of the world are suffering from malnutrition. The efficient improvement of nutritive quality of important crop species like wheat is dependent on the understanding of the acquisition of micronutrients from soil environment and subsequent translocation and distribution into different tissues. The objectives of this work were to understand the effect of increased concentrations of Fe, Zn, and Cd 1) on overall mineral and metal concentrations, 2) on acquisition, translocation, and distribution of minerals among different tissues, and 3) on the inter-relationship of the minerals and metals as reflected in changing the relationship pattern in wheat. The application of increased concentrations of Fe and Zn resulted in three and 11 folds' increase of these micronutrients in wheat respectively and significantly increased seed Ca, P, and S contents however acquisition and translocation of 20 mineral elements varied from tissue to tissue. The improvement of major crop species for health-related micronutrient is important for combating world- wide malnutrition problem. The higher concentration of one micronutrient element may not always ensure higher concentration of that element in seed but increase concentration of Fe and Zn may ensure higher concentrations of others important minerals in wheat seed. The results from our research unveiled key aspects on interrelation among some minerals and metals due to higher concentration of Fe, Zn, and Cd application in wheat.

In eukaryotic organisms, nuclear and cytoplasmic genomes interact to drive cellular functions. These genomes have co-evolved to form speciic nuclear-cytoplasmic interactions that are essential to the origin, success, and evolution of diploid and polyploid species. Hundreds of genetic diseases in humans and phenotypic variations in plants are known to be the result of alterations affecting nuclear-mitochondrial (NM) communication. The genetic bottleneck in the nuclear genome of modern polyploid wheat species is mirrored by the homogeneity of cytoplasmic genomes in durum and bread wheat cultivars. This lack of variation is illustrated by our data indicating that the mitochondrial genome of durum wheat is almost identical to that of published bread wheat genome. The data by our group and others clearly illustrate that genes affecting NM interactions are directly or indirectly related to hybrid compatibility. Therefore, their manipulation and use would permit wider usage of alien germplasm and more eficient introgression. Thus, we have embarked on a series of studies to: 1) isolate, characterize and manipulate genes involved in NM interaction; 2) better understand the inluence of cytoplasmic genome by analyzing the vast collection of wheat alloplasmic lines; and 3) determine the extent of mitochondrial genome variability in Triticeae and Aegilops species in order to generate more cytoplasmically variable, and agronomically adapted cultivars. Utilizing traditional genetic mapping and radiation hybrid mapping, we located a gene in durum wheat (T. turgidum L. var. durum) involved in NM compat bility to a chromosome segment of a few hundred Kb in size. Isolation and characterization of this gene will provide us the ability to understand and manipulate regulatory mechanisms respons ble for a number of developmental processes in durum wheat, including embryo/seed development and plant vigor. In parallel, we have demonstrated that variation in the cytoplasmic genome can inluence plant-pathogen response such as the interaction with Pyrenophora tritici-repentis (tan spot) and Puccinia triticina (leaf rust). Sequencing the mitochondrial genome of an alloplasmic wheat line indicated a great amount of sequence and structural changes in the genome, and at a much higher frequency than is observed in evolutionarily distant species. Additionally, our data indicated paternal leakage, heteroplasmy and stoichiometric changes in the mitochondrial genomes. These results have important implications in terms of the potential to manipulate plant mitochondrial genomes and select for changes that are critical to plant development and adaptation. Since plants cannot escape from adverse environmental conditions, adaptation is paramount to species survival. Cytoplasmic genomes play a critical role in adaptation, and poss bly speciation. Therefore, manipulation of mitochondrial genomes and creation of new cytoplasmic variability may provide a further mechanism for stress tolerance.

The nuclear-encoded species cytoplasm specific (scs) genes control nuclear-cytoplasmic compatibility in wheat (genus Triticum). Alloplasmic cells, which have nucleus and cytoplasm derived from different species, produce vigorous and vital organisms only when the correct version of scs is present in their nucleus. In this study, bulks of invivo radiation hybrids segregating for the scs phenotype have been genotyped by sequencing with over 1.9million markers. The high marker saturation obtained for a critical region of chromosome 1D allowed identification of 3318 reads that mapped in close proximity of the scs. A novel in silico approach was deployed to extend these short reads to sequences of up to 70Kb in length and identify candidate open reading frames (ORFs). Markers were developed to anchor the short contigs containing ORFs to a radiation hybrid map of 650 individuals with resolution of 288Kb. The region containing the scs locus was narrowed to a single Bacterial Artificial Chromosome (BAC) contig of Aegilops tauschii. Its sequencing and assembly by nano-mapping allowed rapid identification of a rhomboid gene as the only ORF existing within the refined scs locus. Resequencing of this gene from multiple germplasm sources identified a single nucleotide mutation, which gives rise to a functional amino acid change. Gene expression characterization revealed that an active copy of this rhomboid exists on all homoeologous chromosomes of wheat, and depending on the specific cytoplasm each copy is preferentially expressed. Therefore, a new methodology was applied to unique genetic stocks to rapidly identify a strong candidate gene for the control of nuclear-cytoplasmic compatibility in wheat.

Mapping and map-based cloning of genes that control agriculturally and economically important traits remain great challenges for plants with complex highly repetitive genomes such as those within the grass tribe, Triticeae. Mapping limitations in the Triticeae are primarily due to low frequencies of polymorphic gene markers and poor genetic recombination in certain genetic regions. Although the abundance of repetitive sequence may pose common problems in genome analysis and sequence assembly of large and complex genomes, they provide repeat junction markers with random and unbiased distribution throughout chromosomes. Hence, development of a high-throughput mapping technology that combine both gene-based and repeat junction-based markers is needed to generate maps that have better coverage of the entire genome.

Mutation and chromosomal rearrangements are the two main forces of increasing genetic diversity for natural selection to act upon, and ultimately drive the evolutionary process. Although genome evolution is a function of both forces, simultaneously, the ratio of each can be varied among different genomes and genomic regions. It is believed that in plant mitochondrial genome, rearrangements play a more important role than point mutations, but relatively few studies have directly addressed this phenomenon. To address this issue, we isolated and sequenced the ATP6-1 and ATP6-2 genes from 46 different euplasmic and alloplasmic wheat lines. Four different ATP6-1 orthologs were detected, two of them reported for the first time. Expression analysis revealed that all four orthologs are transcriptionally active. Results also indicated that both point mutation and genomic rearrangement are involved in the evolution of ATP6. However, rearrangement is the predominant force that triggers drastic variation. Data also indicated that speciation of domesticated wheat cultivars were simultaneous with the duplication of this gene. These results directly support the notion that rearrangement plays a significant role in driving plant mitochondrial genome evolution.

Sunflower (Helianthus annuus L.) is largely consumed in Iran as an oleaginous and confectionery product. In this study, the total phenolic and flavonoid contents, antioxidant activity and phenolic composition in different parts of the seeds from six sunflower genotypes were investigated. The antioxidant activity of extract was evaluated by different assays. The total phenolic and flavonoid contents in kernel were 6.8 and 4.1 times higher than those in shell. Antioxidant activity of kernel extract was significantly higher than shell. It can be attributed to higher total phenolic and flavonoid content. In kernel extracts, a positive correlation coefficient was observed between total phenolic content and FRAP (r = 0.636) and Nitric oxide radical inhibition (r= 0. 721) assays. The amount of identified phenolic compounds varied in different extracts and ranged from 0.29 µg/g (Vanillic acid, shell of S5 genotype) to 433.6 µg/g (Syringic acid, kernel of S2 genotype). Therefore, the result indicated that kernel sunflower seeds can be used as potent natural antioxidants in diet.

Background: Wheat is an excellent plant species for nuclear mitochondrial interaction studies due to availability of large collection of alloplasmic lines. These lines exhibit different vegetative and physiological properties than their parents. To investigate the level of sequence changes introduced into the mitochondrial genome under the alloplasmic condition, three mitochondrial genomes of the Triticum-Aegilops species were sequenced: 1) durum alloplasmic line with the Ae. longissima cytoplasm that carries the T. turgidum nucleus designated as (lo) durum, 2) the cytoplasmic donor line, and 3) the nuclear donor line.Results: The mitochondrial genome of the T. turgidum was 451,678 bp in length with high structural and nucleotide identity to the previously characterized T. aestivum genome. The assembled mitochondrial genome of the (lo) durum and the Ae. longissima were 431,959 bp and 399,005 bp in size, respectively. The high sequence coverage for all three genomes allowed analysis of heteroplasmy within each genome. The mitochondrial genome structure in the alloplasmic line was genetically distant from both maternal and paternal genomes. The alloplasmic durum and the Ae. longissima carry the same versions of atp6, nad6, rps19-p, cob and cox2 exon 2 which are different from the T. turgidum parent. Evidence of paternal leakage was also observed by analyzing nad9 and orf359 among all three lines. Nucleotide search identified a number of open reading frames, of which 27 were specific to the (lo) durum line.Conclusions: Several heteroplasmic regions were observed within genes and intergenic regions of the mitochondrial genomes of all three lines. The number of rearrangements and nucleotide changes in the mitochondrial genome of the alloplasmic line that have occurred in less than half a century was significant considering the high sequence conservation between the T. turgidum and the T. aestivum that diverged from each other 10,000 years ago. We showed that the changes in genes were not limited to paternal leakage but were sufficiently significant to suggest that other mechanisms, such as recombination and mutation, were responsible. The newly formed ORFs, differences in gene sequences and copy numbers, heteroplasmy, and substoichiometric changes show the potential of the alloplasmic condition to accelerate evolution towards forming new mitochondrial genomes.

Aeluropus lagopoides is a salt and drought tolerant grass from Poaceae family, distributed widely in arid regions. There is almost no information about the genetics or genome of this close relative of wheat that stands harsh conditions of deserts. Differential Display Amplified fragment length polymorphism (DD-AFLP) led to the improvement of a non-radioactive method for which many parameters were optimized. Having screened approximately 1600 transcript-derived fragments, 1.4 percent of them showed varied expression levels in response to high salt or drought treatments. The relative abundance of twenty one selected differentially expressed fragments was inspected by reverse northern blotting that affirmed the potential of this applied method. Sequence comparisons revealed that some of the isolated genes are involved in osmotic adjustment, regulation of transcription, cation transportation and stress responses. These data clearly show that the modified DD-AFLP method was a successful and reliable approach for the isolation of differentially expressed genes.Background: Aeluropus lagopoides is a salt and drought tolerant grass from Poaceae family, distributed widely in arid regions. There is almost no information about the genetics or genome of this close relative of wheat that stands harsh conditions of deserts. Objectives: The main aim of this research was to isolation and characterization of salt and drought inducible genes from A. lagopoides by Differential Display Amplified fragment length polymorphism (DD-AFLP) method. Material and Methods: In this research A. lagopoides was grown under salt or drought conditions and after modifying the DD-AFLP method several fragments were isolated and after nomination their induction was studied by reverse northern blotting. Results: DD-AFLP led to the improvement of a non-radioactive method for which many parameters were optimized. Having screened approximately 1600 transcript-derived fragments, 1.4 percent of them showed varied expression levels in response to high salt or drought treatments. The relative abundance of twenty one selected differentially expressed fragments was inspected by reverse northern blotting that affirmed the potential of this applied method. Sequence comparisons revealed that some of the isolated genes are involved in osmotic adjustment, regulation of transcription, cation transportation and stress responses. These data clearly show that the modified DD-AFLP method was a successful and reliable approach for the isolation of differentially expressed genes.

Chloride is an essential micronutrient in tobacco (Nicotiana tabaccum L.) cultivation. However, large amounts of it have many adverse effects on burning quality of tobacco leaves. The objective of this study was to evaluate the genetic variability among 70 oriental-type tobacco genotypes and determine the genomic regions associated with chloride accumulation rate using mixed linear model (MLM) procedure. A total number of 66 alleles were detected by 26 simple sequence repeat (SSR) loci with an average of 2.53 alleles per locus. A model-based Bayesian approach subdivided 70 tobacco genotypes into the three subgroups. Almost 5.85% of the 325 marker pairs showed a significant level of linkage disequilibrium (P <= 0.01). Using MLM procedure, 1 SSR locus (pt30027) from linkage group 13 was identified to be associated with the gene(s) controlling low chloride accumulation in oriental tobacco genotypes. Identified markers could be of great interest in marker-assisted selection in tobacco breeding programs.

Starch and cellulose are among the best known renewable reinforcing components. Scientists are continuously looking for various renewable sources such as flax, hemp, jute, and corn hulls with polymer matrixes to form composite materials and make structural biocomposites a reality. Wheat is a major cereal grain in the US and the world. During wheat milling, a large amount of wheat bran, a by-product, is disposed off as waste. The high percentage of water-insoluble fiber in wheat bran could be advantageous for reinforcing industrial material. However, the utilization of cellulosic fibers derived from wheat byproduct has not been explored in processing of biocomposites. Therefore, the objectives of this study were to characterize wheat bran fiber compositions including dry matter (DM), ash, crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), cellulose (Cell), hemicellulose (Hemi), calcium (Ca), fat, starch, and acid detergent lignin (ADL); identify the interrelationship between the fiber composition traits and the influence of the environment and genotype on these traits. The experiment included six diverse and popular hard red spring wheat (HRSW) cultivars commonly grown in spring wheat region of the Northern Plains of USA. The experiment was installed in three different environments in the Dakotas States, USA. Results from this study showed that the DM, ash, Ca, Cell, starch, and ADL contents were influenced mainly by environments. However, CP along with fat, ash and Ca contents were influenced by genotypes in addition to environment. All bran components were influenced by the genotype × environment (G × E) interactions. We observed significant negative correlation of Cell with CP and ADL which make wheat bran a suitable reinforcing industrial material. However surface treatment of bran fiber would make it even more efficient. These preliminary results indicate the potential use of wheat bran components as biocomposite, but further studies to elucidate more these finding are warranted.

The species cytoplasm specific (scs) genes affect nuclear-cytoplasmic interactions in interspecific hybrids. A radiation hybrid (RH) mapping population of 188 individuals was employed to refine the location of the scs (ae) locus on Triticum aestivum chromosome 1D. "Wheat Zapper," a comparative genomics tool, was used to predict synteny between wheat chromosome 1D, Oryza sativa, Brachypodium distachyon, and Sorghum bicolor. A total of 57 markers were developed based on synteny or literature and genotyped to produce a RH map spanning 205.2 cR. A test-cross methodology was devised for phenotyping of RH progenies, and through forward genetic, the scs (ae) locus was pinpointed to a 1.1 Mb-segment containing eight genes. Further, the high resolution provided by RH mapping, combined with chromosome-wise synteny analysis, located the ancestral point of fusion between the telomeric and centromeric repeats of two paleochromosomes that originated chromosome 1D. Also, it indicated that the centromere of this chromosome is likely the result of a neocentromerization event, rather than the conservation of an ancestral centromere as previously believed. Interestingly, location of scs locus in the vicinity of paleofusion is not associated with the expected disruption of synteny, but rather with a good degree of conservation across grass species. Indeed, these observations advocate the evolutionary importance of this locus as suggested by "Maan's scs hypothesis.".

Septoria tritici blotch (STB) caused by Mycosphaerella graminicola, is one of the most destructive foliar diseases of wheat (Triticum aestivum L.) especially in temperate and humid regions across the world. The susceptibility of recently released varieties, evolution of resistance to fungicides and increasing incidence of STB disease emphasizes the need to understand the genetics of resistance to this disease and to incorporate host resistance into adapted cultivars. This study aimed to decipher the genetics and map the resistance to STB using a recombinant inbred line (RIL) mapping population derived from ‘Steele-ND’ (susceptible parent) and ‘ND 735’ (resistant parent). The RILs were evaluated in three greenhouse experiments, using a North Dakota (ND) isolate of STB pathogen. The mean disease severity of parental genotypes, ‘ND 735’ (11.96%) and ‘Steele-ND’ (66.67%) showed significant differences (p < 0.05). The population segregated for STB and the frequency distribution of RILs indicated quantitative inheritance for resistance. The mean disease severity in RILs ranged from 0 to 71.55% with a mean of 21.98%. The genome map of this population was developed using diversity array technology (DArT) and simple sequence repeat (SSR) markers. The framework linkage map of this population was developed using 469 molecular markers. This map spanned a total distance of 1,789.3 cM and consisted of 17 linkage groups. QTL mapping using phenotypic data and the framework linkage maps detected three QTL through composite interval mapping. One QTL was consistently detected in all experiments on the long arm of chromosome 5B, and explained up to 10.2% phenotypic variation. The other two QTLs, detected in single environments, were mapped to 1D and 7A and explain 13% and 5.5% of the phenotypic variation, respectively. The map position of the consistent QTL on 5BL coincides with the map position of durable resistance gene Stb1 suggesting the importance of this region of ‘ND 735’ as a source of durable STB resistance for the wheat germplasm.

ABSTRACTFusarium head blight (FHB) caused by Fusarium graminearum Schwabe causes extensive losses in durum wheat [Triticum turgidum subsp. durum (Desf.) Husn.]. The only verified resistance contributed by a tetraploid wheat species comes from T. turgidum L. subsp. dicoccoides (Körn. ex Asch. & Graebn.) Thell. This research was conducted to determine whether five Tunisian durum lines exhibiting Type II FHB resistance carry the same resistance alleles as the hexaploid wheat ‘Frontana’, ‘Sumai 3’, and ‘Wangshuibai’ and subsp. dicoccoides genotype (Israel A). One hundred thirty‐one polymerase chain reaction (PCR)‐based markers were used to determine the genetic similarity among 20 genotypes including resistant and susceptible Tunisian lines and adapted tetraploid and hexaploid cultivars. Phenotypic evaluation was conducted in two greenhouse seasons. Cluster analysis of the Tunisian lines based on the genetic markers showed they are genetically distant from known sources of resistance. Tunisian 7, the most resistant line in this study, was distinctly placed on a separate branch from all the other Tunisian lines, indicating the presence of some genetic diversity. Nineteen of the genotypes were haplotyped with PCR‐based markers for nine loci on six chromosomes associated with known Type II FHB resistance. The Tunisian lines amplified different fragment sizes from the known resistant hexaploid wheat cultivars and subsp. dicoccoides. Results from this study indicate that these Tunisian lines may contain a novel source of Type II FHB resistance.