Insights into breeding history, hotspot regions of selection, and untapped allelic diversity for bread wheat breeding.

Breeding has increasingly altered the genetics of crop plants since the domestication of their wild progenitors. It is postulated that the genetic diversity of elite wheat breeding pools is too narrow to cope with future challenges. In contrast, plant genetic resources (PGRs) of wheat stored in genebanks are valuable sources of unexploited genetic diversity. Therefore, to ensure breeding progress in the future, it is of prime importance to identify the useful allelic diversity available in PGRs and to transfer it into elite breeding pools. Here, a diverse collection consisting of modern winter wheat cultivars and genebank accessions was investigated based on reduced-representation genomic sequencing and an iSelect single nucleotide polymorphism (SNP) chip array. Analyses of these datasets provided detailed insights into population structure, levels of genetic diversity, sources of new allelic diversity, and genomic regions affected by breeding activities. We identified 57 regions representing genomic signatures of selection and 827 regions representing private alleles associated exclusively with genebank accessions. The presence of known functional wheat genes, quantitative trait loci, and large chromosomal modifications, i.e., introgressions from wheat wild relatives, provided initial evidence for putative traits associated within these identified regions. These findings were supported by the results of ontology enrichment analyses. The results reported here will stimulate further research and promote breeding in the future by allowing for the targeted introduction of novel allelic diversity into elite wheat breeding pools.

Genetics of mycorrhizal symbiosis in winter wheat (Triticum aestivum).

Bread wheat (Triticum aestivum) is a major staple food and therefore of prime importance for feeding the Earth's growing population. Mycorrhiza is known to improve plant growth, but although extensive knowledge concerning the interaction between mycorrhizal fungi and plants is available, genotypic differences concerning the ability of wheat to form mycorrhizal symbiosis and quantitative trait loci (QTLs) involved in mycorrhization are largely unknown. Therefore, a diverse set of 94 bread wheat genotypes was evaluated with regard to root colonization by arbuscular mycorrhizal fungi. In order to identify genomic regions involved in mycorrhization, these genotypes were analyzed using the wheat 90k iSelect chip, resulting in 17 823 polymorphic mapped markers, which were used in a genome-wide association study. Significant genotypic differences (P < 0.0001) were detected in the ability to form symbiosis and 30 significant markers associated with root colonization, representing six QTL regions, were detected on chromosomes 3A, 4A and 7A, and candidate genes located in these QTL regions were proposed. The results reported here provide key insights into the genetics of root colonization by mycorrhizal fungi in wheat.

Finding needles in a haystack: identification of inter-specific introgressions in wheat genebank collections using low-coverage sequencing data.

Recently, entire genebank collections of wheat have been extensively characterized with sequencing data. We have identified introgressions using these genotyping-by-sequencing and whole-genome sequencing data. On the basis of our results, we provide information about predicted introgressions at 1-Mb resolution for 9,172 wheat samples as a resource for breeders and scientists. We recommend that all plant genetic resources, including genebank collections, be characterized using a combination of variant calling and introgression prediction. This is necessary to identify potential duplicates in collections efficiently and reliably, and to select promising germplasms with potentially beneficial introgressions for further characterization and prospective breeding application.

Identification of Markers Associated with Wheat Dwarf Virus (WDV) Tolerance/Resistance in Barley (Hordeum vulgare ssp. vulgare) Using Genome-Wide Association Studies.

Wheat dwarf virus (WDV) causes an important vector transmitted virus disease, which leads to significant yield losses in barley production. Due to the fact that, at the moment, no plant protection products are approved to combat the vector Psammotettix alienus, and this disease cannot be controlled by chemical means, the use of WDV-resistant or -tolerant genotypes is the most efficient method to control and reduce the negative effects of WDV on barley growth and production. In this study, a set of 480 barley genotypes were screened to identify genotypic differences in response to WDV, and five traits were assessed under infected and noninfected conditions. In total, 32 genotypes showed resistance or tolerance to WDV. Subsequently, phenotypic data of 191 out of 480 genotypes combined with 34,408 single-nucleotide polymorphisms (SNPs) were used for a genome-wide association study to identify quantitative trait loci (QTLs) and markers linked to resistance/tolerance to WDV. Genomic regions significantly associated with WDV resistance/tolerance in barley were identified on chromosomes 3H, 4H, 5H, and 7H for traits such as relative virus titer, relative performance of total grain weight, plant height, number of ears per plant, and thousand grain weight.

SNP Diversity and Genetic Structure of "Rogosija", an Old Western Balkan Durum Wheat Collection.

Durum wheat landraces represent a genetic resource for the identification and isolation of new valuable genes and alleles, useful to increase the crop adaptability to climate change. Several durum wheat landraces, all denominated "Rogosija", were extensively cultivated in the Western Balkan Peninsula until the first half of the 20th century. Within the conservation program of the Montenegro Plant Gene Bank, these landraces were collected, but without being characterized. The main goal of this study was to estimate the genetic diversity of the "Rogosija collection" consisting of 89 durum accessions, using 17 morphological descriptors and the 25K Illumina single nucleotide polymorphism (SNP) array. The genetic structure analysis of the Rogosija collection showed two distinguished clusters localized in two different Montenegro eco-geographic micro-areas, characterized by continental Mediterranean climate and maritime Mediterranean climate. Data suggest that these clusters could be composed of two different Balkan durum landrace collections evolved in two different eco-geographic micro-areas. Moreover, the origin of Balkan durum landraces is discussed.

Detecting major introgressions in wheat and their putative origins using coverage analysis.

Introgressions from crop wild relatives (CWRs) have been used to introduce beneficial traits into cultivated plants. Introgressions have traditionally been detected using cytological methods. Recently, single nucleotide polymorphism (SNP)-based methods have been proposed to detect introgressions in crosses for which both parents are known. However, for unknown material, no method was available to detect introgressions and predict the putative donor species. Here, we present a method to detect introgressions and the putative donor species. We demonstrate the utility of this method using 10 publicly available wheat genome sequences and identify nine major introgressions. We show that the method can distinguish different introgressions at the same locus. We trace introgressions to early wheat cultivars and show that natural introgressions were utilised in early breeding history and still influence elite lines today. Finally, we provide evidence that these introgressions harbour resistance genes.

Genome wide association study of frost tolerance in wheat.

Winter wheat growing areas in the Northern hemisphere are regularly exposed to heavy frost. Due to the negative impact on yield, the identification of genetic factors controlling frost tolerance (FroT) and development of tools for breeding is of prime importance. Here, we detected QTL associated with FroT by genome wide association studies (GWAS) using a diverse panel of 276 winter wheat genotypes that was phenotyped at five locations in Germany and Russia in three years. The panel was genotyped using the 90 K iSelect array and SNPs in FroT candidate genes. In total, 17,566 SNPs were used for GWAS resulting in the identification of 53 markers significantly associated (LOD ≥ 4) to FroT, corresponding to 23 QTL regions located on 11 chromosomes (1A, 1B, 2A, 2B, 2D, 3A, 3D, 4A, 5A, 5B and 7D). The strongest QTL effect confirmed the importance of chromosome 5A for FroT. In addition, to our best knowledge, eight FroT QTLs were discovered for the first time in this study comprising one QTL on chromosomes 3A, 3D, 4A, 7D and two on chromosomes 1B and 2D. Identification of novel FroT candidate genes will help to better understand the FroT mechanism in wheat and to develop more effective combating strategies.

Which Factors Affect the Occurrence of Off-Target Effects Caused by the Use of CRISPR/Cas: A Systematic Review in Plants.

CRISPR/Cas enables a targeted modification of DNA sequences. Despite their ease and efficient use, one limitation is the potential occurrence of associated off-target effects. This systematic review aims to answer the following research question: Which factors affect the occurrence of off-target effects caused by the use of CRISPR/Cas in plants? Literature published until March 2019 was considered for this review. Articles were screened for relevance based on pre-defined inclusion criteria. Relevant studies were subject to critical appraisal. All studies included in the systematic review were synthesized in a narrative report, but studies rated as high and medium/high validity were reported separately from studies rated as low and medium/low or unclear validity. In addition, we ran a binary logistic regression analysis to verify five factors that may affect the occurrence of off-target effects: (1) Number of mismatches (2) Position of mismatches (3) GC-content of the targeting sequence (4) Altered nuclease variants (5) Delivery methods. In total, 180 relevant articles were included in this review containing 468 studies therein. Seventy nine percentage of these studies were rated as having high or medium/high validity. Within these studies, 6,416 potential off-target sequences were assessed for the occurrence of off-target effects. Results clearly indicate that an increased number of mismatches between the on-target and potential off-target sequence steeply decreases the likelihood of off-target effects. The observed rate of off-target effects decreased from 59% when there is one mismatch between the on-target and off-target sequences toward 0% when four or more mismatches exist. In addition, mismatch/es located within the first eight nucleotides proximal to the PAM significantly decreased the occurrence of off-target effects. There is no evidence that the GC-content significantly affects off-target effects. The database regarding the impact of the nuclease variant and the delivery method is very poor as the majority of studies applied the standard nuclease SpCas9 and the CRISPR/Cas system was stably delivered in the genome. Hence, a general significant impact of these two factors on the occurrence of off-target effects cannot be proved. This identified evidence gap needs to be filled by systematic studies exploring these individual factors in sufficient numbers.

Comparison Between Core Set Selection Methods Using Different Illumina Marker Platforms: A Case Study of Assessment of Diversity in Wheat.

Collections of plant genetic resources stored in genebanks are an important source of genetic diversity for improvement in plant breeding programs and for conservation of natural variation. The establishment of reduced representative collections from a large set of genotypes is a valuable tool that provides cost-effective access to the diversity present in the whole set. Software like Core Hunter 3 is available to generate high quality core sets. In addition, general clustering approaches, e.g., k-medoids, are available to subdivide a large data set into small groups with maximum genetic diversity between groups. Illumina genotyping platforms are a very efficient tool for the assessment of genetic diversity of plant genetic resources. The accumulation of genotyping data over time using commercial genotyping platforms raises the question of how such huge amount of information can be efficiently used for creating core collections. In the present study, after developing a 15K wheat Infinium array with 12,908 SNPs and genotyping a set of 479 hexaploid winter wheat lines (Triticum aestivum), a larger data set was created by merging 411 lines previously genotyped with the 90K iSelect array. Overlaying the markers from the 15K and 90K arrays enabled the identification of a common set of 12,806 markers, suggesting that the 15K array is a valuable and cost-effective resource for plant breeding programs. Finally, we selected genetically diverse core sets out of these 890 wheat genotypes derived from five collections based on the common markers from the 15K and 90K SNP arrays. Two different approaches, k-medoids and Core Hunter 3 were compared,and k-medoids was identified as an efficient method for selecting small core sets out of a large collection of genotypes while retaining the genetic diversity of the original population.

On genetic diversity in caraway: Genotyping of a large germplasm collection.

Caraway (Carum carvi) is a widespread and frequently used spice and medicinal plant with a long history of cultivation. However, due to ongoing climatic changes, the cultivation is becoming increasingly risky. To secure caraway cultivation in future, timely breeding efforts to develop adapted material are necessary. Analysis of genetic diversity can accompany this process, for instance, by revealing untapped gene pools. Here, we analyzed 137 accessions using genotyping by sequencing (GBS). Hence, we can report a broad overview of population structure and genetic diversity of caraway. Population structure was determined using a principal coordinate analysis, a Bayesian clustering analysis, phylogenetic trees and a neighbor network based on 13,155 SNPs. Genotypic data indicate a clear separation of accessions into two subpopulations, which correlates with the flowering type (annual vs. biennial). Four winter-annual accessions were closer related to biennial accessions. In an analysis of molecular variance, genetic variation between the two subpopulations was 7.84%. In addition, we estimated the genome size for 35 accessions by flow cytometry. An average genome size of 4.282 pg/2C (± 0.0096 S.E.) was estimated. Therefore, we suggest a significantly smaller genome size than stated in literature.

Detecting Large Chromosomal Modifications Using Short Read Data From Genotyping-by-Sequencing.

Markers linked to agronomic traits are of the prerequisite for molecular breeding. Genotyping-by-sequencing (GBS) data enables to detect small polymorphisms including single nucleotide polymorphisms (SNPs) and short insertions or deletions (InDels) that can be used, for instance, for marker-assisted selection, population genetics, and genome-wide association studies (GWAS). Here, we aim at detecting large chromosomal modifications in barley and wheat based on GBS data. These modifications could be duplications, deletions, substitutions including introgressions as well as alterations of DNA methylation. We demonstrate that GBS coverage analysis is capable to detect Hordeum vulgare/Hordeum bulbosum introgression lines. Furthermore, we identify large chromosomal modifications in barley and wheat collections. Hence, large chromosomal modifications, including introgressions and copy number variations (CNV), can be detected easily and can be used as markers in research and breeding without additional wet-lab experiments.

Genome-Wide Association Studies Reveal Genomic Regions Associated With the Response of Wheat (Triticum aestivum L.) to Mycorrhizae Under Drought Stress Conditions.

In the majority of wheat growing areas worldwide, the incidence of drought stress has increased significantly resulting in a negative impact on plant development and grain yield. Arbuscular mycorrhizal symbiosis is known to improve drought stress tolerance of wheat. However, quantitative trait loci (QTL) involved in the response to drought stress conditions in the presence of mycorrhizae are largely unknown. Therefore, a diverse set consisting of 94 bread wheat genotypes was phenotyped under drought stress and well watered conditions in the presence and absence of mycorrhizae. Grain yield and yield components, drought stress related traits as well as response to mycorrhizae were assessed. In parallel, wheat accessions were genotyped by using the 90k iSelect chip, resulting in a set of 15511 polymorphic and mapped SNP markers, which were used for genome-wide association studies (GWAS). In general, drought stress tolerance of wheat was significantly increased in the presence of mycorrhizae compared to drought stress tolerance in the absence of mycorrhizae. However, genotypes differed in their response to mycorrhizae under drought stress conditions. Several QTL regions on different chromosomes were detected associated with grain yield and yield components under drought stress conditions. Furthermore, two genome regions on chromosomes 3D and 7D were found to be significantly associated with the response to mycorrhizae under drought stress conditions. Overall, the results reveal that inoculation of wheat with mycorrhizal fungi significantly improves drought stress tolerance and that QTL regions associated with the response to mycorrhizae under drought stress conditions exist in wheat. Further research is necessary to validate detected QTL regions. However, this study may be the starting point for the identification of candidate genes associated with drought stress tolerance and response to mycorrhizae under drought stress conditions. Maybe in future, these initial results will help to contribute to use mycorrhizal fungi effectively in agriculture and combine new approaches i.e., use of genotypic variation in response to mycorrhizae under drought stress conditions with existing drought tolerance breeding programs to develop new drought stress tolerant genotypes.

The Terpene Synthase Gene Family of Carrot (Daucus carota L.): Identification of QTLs and Candidate Genes Associated with Terpenoid Volatile Compounds.

Terpenes are an important group of secondary metabolites in carrots influencing taste and flavor, and some of them might also play a role as bioactive substances with an impact on human physiology and health. Understanding the genetic and molecular basis of terpene synthases (TPS) involved in the biosynthesis of volatile terpenoids will provide insights for improving breeding strategies aimed at quality traits and for developing specific carrot chemotypes possibly useful for pharmaceutical applications. Hence, a combination of terpene metabolite profiling, genotyping-by-sequencing (GBS), and genome-wide association study (GWAS) was used in this work to get insights into the genetic control of terpene biosynthesis in carrots and to identify several TPS candidate genes that might be involved in the production of specific monoterpenes. In a panel of 85 carrot cultivars and accessions, metabolite profiling was used to identify 31 terpenoid volatile organic compounds (VOCs) in carrot leaves and roots, and a GBS approach was used to provide dense genome-wide marker coverage (>168,000 SNPs). Based on this data, a total of 30 quantitative trait loci (QTLs) was identified for 15 terpenoid volatiles. Most QTLs were detected for the monoterpene compounds ocimene, sabinene, ß-pinene, borneol and bornyl acetate. We identified four genomic regions on three different carrot chromosomes by GWAS which are both associated with high significance (LOD ≥ 5.91) to distinct monoterpenes and to TPS candidate genes, which have been identified by homology-based gene prediction utilizing RNA-seq data. In total, 65 TPS candidate gene models in carrot were identified and assigned to known plant TPS subfamilies with the exception of TPS-d and TPS-h. TPS-b was identified as largest subfamily with 32 TPS candidate genes.