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In plant sciences, curation and availability of interoperable phenotypic and genomic data is still in its infancy and represents an obstacle to rapid scientific discoveries in this field. To that end, supplementing the efforts being made to generate open access wheat genome, pan wheat genome and other bioinformatic resources, we present the GABI-WHEAT panel of elite European cultivars comprising 358 winter and 14 summer wheat varieties released between 1975 to 2007. The panel has been genotyped with SNP arrays of increasing density to investigate several important agronomic, quality and disease resistance traits. The robustness of investigated traits and interoperability of genomic and phenotypic data was assessed in the current publication with the aim to transform this panel into a public data resource for future genetic research in wheat. Consecutively, the phenotypic data was formatted to comply with FAIR principles and linked to online databases to substantiate panel origin information and quality. Thus, we were able to make a valuable resource available for plant science in a sustainable way., (© 2022. The Author(s).)

The resistance to cereal cyst nematode (Heterodera avenae Woll.) in wheat (Triticum aestivum L.) was studied using 114 doubled haploid lines from a novel ITMI mapping population. These lines were screened for nematode infestation in a controlled environment for two years. QTL-mapping analyses were performed across two years (Y1 and Y2) as well as combining two years (CY) data. On the 114 lines that were screened, a total of 2,736 data points (genotype, batch or years, and replication combinations) were acquired. For QTL analysis, 12,093 markers (11,678 SNPs and 415 SSRs markers) were used, after filtering the genotypic data, for the QTL mapping. Composite interval mapping, using Haley-Knott regression (hk) method in R/QTL, was used for QTL analysis. In total, 19 QTLs were detected out of which 13 were novel and six were found to be colocalized or nearby to previously reported Cre genes, QTLs or MTAs for H. avenae or H. filipjevi. Nine QTLs were detected across all three groups (Y1, Y2 and CY) including a significant QTL "QCcn.ha-2D" on chromosome 2D that explains 23% of the variance. This QTL colocalized with a previously identified Cre3 locus. Novel QTL, QCcn.ha-2A, detected in the present study could be the possible unreported homeoloci to QCcn.ha-2D, QCcn.ha-2B.1 and QCcn.ha-2B.2. Six significant digenic epistatic interactions were also observed. In addition, 26 candidate genes were also identified including genes known for their involvement in PPNs (plant parasitic nematodes) resistance in different plant species. In-silico expression of putative candidate genes showed differential expression in roots during specific developmental stages. Results obtained in the present study are useful for wheat breeding to generate resistant genetic resources against H. avenae., (© 2022. The Author(s).)

Tan spot, caused by the fungus Pyrenophora tritici-repentis ( Ptr ), is a severe foliar disease of wheat ( Triticum aestivum L.). Improving genetic resistance is a durable strategy to reduce Ptr -related losses. Here, we dissected Ptr -infection's genetic basis in 372 European wheat varieties via single sequence repeats (SSR) in addition to 35k and 90k single nucleotide polymorphism (SNP) marker platforms. In our phenotypic data analyses, Ptr infection showed a significant genotypic variance and a significant negative correlation with plant height. Genome-wide association studies revealed a highly quantitative nature of Ptr infection and identified two quantitative trait loci (QTL), viz., QTs.ipk-7A and QTs.ipk-7B , which imparted 21.23 and 5.84% of the genotypic variance, respectively. Besides, the Rht-D1 gene showed a strong allelic influence on the resistance scores. Due to the complex genetic nature of the Ptr infection, the potential of genome-wide prediction (GP) was assessed via three different genetic models on individual and combined marker platforms. The GP results indicated that the marker density and marker platforms do not considerably impact prediction accuracy (~40-42%) and that higher-order epistatic interactions may not be highly pervasive. Our results provide a further understanding of Ptr -infection's genetic nature, serve as a resource for marker-assisted breeding, and highlight the potential of genome-wide selection for improved Ptr resistance.

Total spikelet number per spike (TSN) is a major component of spike architecture in wheat (Triticum aestivum L.). A major and consistent quantitative trait locus (QTL) was discovered for TSN in a doubled haploid spring wheat population grown in the field over 4 years. The QTL on chromosome 7B explained up to 20.5% of phenotypic variance. In its physical interval (7B: 6.37-21.67 Mb), the gene FLOWERING LOCUS T (FT-B1) emerged as candidate for the observed effect. In one of the parental lines, FT-B1 carried a non-synonymous substitution on position 19 of the coding sequence. This mutation modifying an aspartic acid (D) into a histidine (H) occurred in a highly conserved position. The mutation was observed with a frequency of ca. 68% in a set of 135 hexaploid wheat varieties and landraces, while it was not found in other plant species. FT-B1 only showed a minor effect on heading and flowering time (FT) which were dominated by a major QTL on chromosome 5A caused by segregation of the vernalization gene VRN-A1. Individuals carrying the FT-B1 allele with amino acid histidine had, on average, a higher number of spikelets (15.1) than individuals with the aspartic acid allele (14.3) independent of their VRN-A1 allele. We show that the effect of TSN is not mainly related to flowering time; however, the duration of pre-anthesis phases may play a major role.

Hexaploid wheat (Triticum aestivum L.) is a natural allopolyploid and provides a usable model system to better understand the genetic mechanisms that underlie allopolyploid speciation through the hybrid genome doubling. Here we aimed to identify the contribution of chromosome 1D in the development and evolution of hexaploid wheat. We identified and mapped a novel DEFECTIVE ENDOSPERM-D1 (Dee-D1) locus on 1DL that is involved in the genetic control of endosperm development. The absence of Dee-D1 leads to non-viable grains in distant crosses and alters grain shape, which negatively affects grain number and thousand-grain weight. Dee-D1 can be classified as speciation locus with a positive effect on the function of genes which are involved in endosperm development in hybrid genomes. The presence of Dee-D1 is necessary for the normal development of endosperm, and thus play an important role in the evolution and improvement of grain yield in hexaploid wheat.

Grain quality traits determine the classification of registered wheat (Triticum aestivum L.) varieties. Although environmental factors and crop management practices exert a considerable influence on wheat quality traits, a significant proportion of the variance is attributed to the genetic factors. To identify the underlying genetic factors of wheat quality parameters viz., grain protein content (GPC), grain starch content (GSC), and grain hardness (GH), we evaluated 372 diverse European wheat varieties in replicated field trials in up to eight environments. We observed that all of the investigated traits hold a wide and significant genetic variation, and a significant negative correlation exists between GPC and GSC plus grain yield. Our association analyses based on 26,694 high-quality single nucleotide polymorphic markers revealed a strong quantitative genetic nature of GPC and GSC with associations on groups 2, 3, and 6 chromosomes. The identification of known Puroindoline-b gene for GH provided a positive analytic proof for our studies. We report that a locus QGpc.ipk-6A controls both GPC and GSC with opposite allelic effects. Based on wheat's reference and pan-genome sequences, the physical characterization of two loci viz., QGpc.ipk-2B and QGpc.ipk-6A facilitated the identification of the candidate genes for GPC. Furthermore, by exploiting additive and epistatic interactions of loci, we evaluated the prospects of predictive breeding for the investigated traits that suggested its efficient use in the breeding programs.

We dissected the genetic basis of total spikelet number (TSN) along with other traits, viz. spike length (SL) and flowering time (FT) in a panel of 518 elite European winter wheat varieties. Genome-wide association studies (GWAS) based on 39,908 SNP markers revealed highly significant quantitative trait loci (QTL) for TSN on chromosomes 2D, 7A, and 7B, for SL on 5A, and FT on 2D, with 2D-QTL being the functional marker for the gene Ppd-D1. The physical region of the 7A-QTL for TSN revealed the presence of a wheat ortholog (TaAPO-A1) to APO1-a rice gene that positively controls the spikelet number on the panicles. Interspecific analyses of the TaAPO-A1 orthologs showed that it is a highly conserved gene important for floral development and present in a wide range of terrestrial plants. Intraspecific studies of the TaAPO-A1 across wheat genotypes revealed a polymorphism in the conserved F-box domain, defining two haplotypes. A KASP marker developed on the polymorphic site showed a highly significant association of TaAPO-A1 with TSN, explaining 23.2% of the total genotypic variance. Also, the TaAPO-A1 alleles showed weak but significant differences for SL and grain yield. Our results demonstrate the importance of wheat sequence resources to identify candidate genes for important traits based on genetic analyses.

blotch (STB) caused by the fungus is a devastating foliar disease of wheat ( L.) that can lead to substantial yield losses. Quantitative genetic resistance has been proposed as a durable strategy for STB control. In this study, we dissected the genetic basis of STB infection in 371 European wheat varieties based on 35k and 90k single nucleotide polymorphism marker arrays. The phenotypic data analyses suggested that large genetic variance exists for STB infection with a broad-sense heritability of 0.78. Genome-wide association studies (GWAS) propose the highly quantitative nature of STB infection with potential associations on chromosomes 1A, 1B, 2D, 4A, 5A, 6A, 6D, 7A, and 7B. Increased marker density in GWAS by combining markers from both arrays helped to detect additional markers explaining increased genotypic variance. Linkage disequilibrium analyses revealed genes with a possible role in disease resistance. The potential of genomic prediction (GP) assessed via two models accounting for additive effects and additive plus epistatic interactions among the loci suggested the possibility of genomic selection for improved STB resistance. Genomic prediction results also indicated that the higher-order epistatic interactions are not abundant and that both marker platforms are equally suitable for GP of STB infection. Our results provide further understanding of the quantitative genetic nature of STB infection, serve as a resource for marker-assisted breeding, and highlight the potential of genomic selection for improved STB resistance., (Copyright © 2019 Crop Science Society of America.)

Malnutrition of iron (Fe) affects two billion people worldwide. Therefore, enhancing grain Fe concentration (GFeC) in wheat ( Triticum aestivum L.) is an important goal for breeding. Here we study the genetic factors underlying GFeC trait by genome-wide association studies (GWAS) and the prediction abilities using genomic prediction (GP) in a panel of 369 European elite wheat varieties which was genotyped with 15,523 mapped single-nucleotide polymorphism markers (SNP) and a subpanel of 183 genotypes with 44,233 SNP markers. The resulting means of GFeC from three field experiments ranged from 24.42 to 52.42 μg·g -1 with a broad-sense heritability ( H ²) equaling 0.59 over the years. GWAS revealed 41 and 137 significant SNPs in the whole and subpanel, respectively, including significant marker-trait associations (MTAs) for best linear unbiased estimates (BLUEs) of GFeC on chromosomes 2A, 3B and 5A. Putative candidate genes such as NAC transcription factors and transmembrane proteins were present on chromosome 2A (763,689,738⁻765,710,113 bp). The GP for a GFeC trait ranged from low to moderate values. The current study reported GWAS of GFeC for the first time in hexaploid wheat varieties. These findings confirm the utility of GWAS and GP to explore the genetic architecture of GFeC for breeding programs aiming at the improvement of wheat grain quality.

One of the primary objectives of wheat breeding was to increase grain yield. Floral abortion during the stem elongation phase (SEP) leads to a loss of more than 50% of the grain number potential. In this study, we quantified 75 plant growth-associated traits at seven stages during the SEP and mapped 15 696 single nucleotide polymorphism (SNP) markers in 210 accessions of wheat (Triticum aestivum). Our genomewide association study identified trait-associated SNPs that are shared among various stages of the SEP, as well as SNPs that are shared between plant growth traits and grain yield in the field. The genomic selection analysis shows variation among the prediction abilities of various traits and stages. Furthermore, we found that the allelic variants of Ppd-D1 (chromosome 2D) and Rht-D1 (chromosome 4D) loci affect some plant growth traits (e.g. leaf area and spike length). These results have identified a narrow time window within the SEP in which plant growth traits can be manipulated to alter grain yield. This suggests that there may be multiple ways to regulate plant growth during the SEP, to ultimately influence grain number in wheat., (© 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

In wheat (Triticum spp.), modifying inflorescence (spike) morphology can increase grain number and size and thus improve yield. Here, we demonstrated the potential for manipulating and predicting spike morphology, based on 44 traits. In 12 wheat cultivars, we observed that detillering (removal of branches), which alters photosynthate distribution, changed spike morphology. Our genome-wide association study detected close associations between carbon partitioning (e.g. tiller number, main shoot dry weight) and spike morphology (e.g. spike length, spikelet density) traits in 210 cultivars. Most carbon-partitioning traits (e.g. tiller dry weight, harvest index) demonstrated high prediction abilities (>0.5). For spike morphology, some traits (e.g. total and fertile spikelet number, spike length) displayed high prediction abilities (0.3-0.5), but others (e.g. spikelet fertility, spikelet density) exhibited low prediction abilities (<0.2). Grain size traits were closely correlated in field and greenhouse experiments. Stepwise regression analysis suggests that significantly associated traits in the greenhouse explain 35.35% of the variation in grain yield and 67.63% of the variation in thousand-kernel weight in the field. Therefore, the traits identified in this study affect spike morphology; these traits can be used to predict and improve plant architecture and thus increase yield.

Wheat ( Triticum aestivum L.) is one of the major staple food crops worldwide. Despite efforts in improving wheat quality, micronutrient levels are still below the optimal range for human nutrition. In particular, zinc (Zn) deficiency is a widespread problem in human nutrition in countries relying mainly on a cereal diet; hence improving Zn accumulation in grains is an imperative need. This study was designed to understand the genetic architecture of Zn grain concentrations in wheat grains. We performed a genome-wide association study (GWAS) for grain Zn concentrations in 369 European wheat genotypes, using field data from 3 years. The complete wheat panel was genotyped by high-density arrays of single nucleotide polymorphic (SNP) markers (90k iSELECT Infinium and 35k Affymetrix arrays) resulting in 15,523 polymorphic markers. Additionally, a subpanel of 183 genotypes was analyzed with a novel 135k Affymetrix marker array resulting in 28,710 polymorphic SNPs for high-resolution mapping of the potential genomic regions. The mean grain Zn concentration of the genotypes ranged from 25.05-52.67 μg g -1 dry weight across years with a moderate heritability value. Notably, 40 marker-trait associations (MTAs) were detected in the complete panel of varieties on chromosomes 2A, 3A, 3B, 4A, 4D, 5A, 5B, 5D, 6D, 7A, 7B, and 7D. The number of MTAs in the subpanel was increased to 161 MTAs whereas the most significant and consistent associations were located on chromosomes 3B (723,504,241-723,611,488 bp) and 5A (462,763,758-466,582,184 bp) having major effects. These genomic regions include newly identified putative candidate genes, which are related to Zn uptake and transport or represent bZIP and mitogen-activated protein kinase genes. These findings provide the basis for understanding the genetic background of Zn concentration in wheat grains that in turn may help breeders to select high Zn-containing genotypes to improve human health and grain quality.

Flowering time is an important factor affecting grain yield in wheat. In this study, we divided reproductive spike development into eight sub-phases. These sub-phases have the potential to be delicately manipulated to increase grain yield. We measured 36 traits with regard to sub-phase durations, determined three grain yield-related traits in eight field environments and mapped 15 696 single nucleotide polymorphism (SNP, based on 90k Infinium chip and 35k Affymetrix chip) markers in 210 wheat genotypes. Phenotypic and genetic associations between grain yield traits and sub-phase durations showed significant consistency (Mantel test; r = 0.5377, P < 0.001). The shared quantitative trait loci (QTLs) revealed by the genome-wide association study suggested a close association between grain yield and sub-phase duration, which may be attributed to effects on spikelet initiation/spikelet number (double ridge to terminal spikelet stage, DR-TS) and assimilate accumulation (green anther to anthesis stage, GA-AN). Moreover, we observed that the photoperiod-sensitivity allele at the Ppd-D1 locus on chromosome 2D markedly extended all sub-phase durations, which may contribute to its positive effects on grain yield traits. The dwarfing allele at the Rht-D1 (chromosome 4D) locus altered the sub-phase duration and displayed positive effects on grain yield traits. Data for 30 selected genotypes (from among the original 210 genotypes) in the field displayed a close association with that from the greenhouse. Most importantly, this study demonstrated specific connections to grain yield in narrower time windows (i.e. the eight sub-phases), rather than the entire stem elongation phase as a whole., (© 2018 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.)

The production and cultivation of hybrid wheat is a possible strategy to close the yield gap in wheat. Efficient hybrid wheat seed production largely depends on high rates of cross-pollination which can be ensured through high anther extrusion (AE) by male parental lines. Here, we report the AE capacity and elucidate its genetics in 514 elite European winter wheat varieties via genome-wide association studies (GWAS). We observed a wide range of variation among genotypes and a high heritability (0.80) for AE. The whole panel was genotyped with the 35k Affymetrix and 90k iSELECT single nucleotide polymorphism (SNP) arrays plus Ppd-D1, Rht-B1 and Rht-D1 candidate markers. GWAS revealed 51 marker-trait associations (MTAs) on chromosomes 1A, 1B, 2A, 4D and 5B, with Rht-D1 (4D) being the most significant marker. Division of whole panel according to the Rht-D1 genotype resulted in 212 and 294 varieties harboring Rht-D1a and Rht-D1b allele, respectively. The presence of Rht-D1a compared to Rht-D1b (mutant) allele had a large phenotypic influence on AE resulting in its ~17% increase. GWAS performed on the sub-panels detected novel MTAs on chromosomes 2D, 3B and 6A with increased phenotypic variance imparted by individual markers. Our study shows that AE is a highly quantitative trait and wild type Rht-D1a allele greatly improves AE. Moreover, demarcating the quantitative trait loci regions based on intra-chromosomal linkage disequilibrium revealed AE's candidate genes/genomic regions. Understanding the genetics of AE in elite European wheat and utilizing the linked markers in breeding programs can help to enhance cross-pollination for better exploitation of heterosis.

Grain yield (GY) of bread wheat (Triticum aestivum L.) is quantitatively inherited. Correlated GY-syndrome traits such as plant height (PH), heading date (HD), thousand grain weight (TGW), test weight (TW), grains per ear (GPE), and ear weight (EW) influence GY. Most quantitative genetics studies assessed the multiple-trait (MT) complex of GY-syndrome using single-trait approaches, and little is known about its underlying pleiotropic architecture. We investigated the pleiotropic architecture of wheat GY-syndrome through MT association mapping (MT-GWAS) using 372 varieties phenotyped in up to eight environments and genotyped with 18 832 single nucleotide polymorphisms plus 24 polymorphic functional markers. MT-GWAS revealed a total of 345 significant markers spread genome wide, representing 8, 40, 11, 40, 34, and 35 effective GY-PH, GY-HD, GY-TGW, GY-TW, GY-GPE, and GY-EW associations, respectively. Among them, pleiotropic roles of Rht-B1 and TaGW2-6B loci were corroborated. Only one marker presented simultaneous associations for three traits (i.e. GY-TGW-TW). Close linkage was difficult to differentiate from pleiotropy; thus, the pleiotropic architecture of GY-syndrome was dissected more as a cause of pleiotropy rather than close linkage. Simulations showed that minor allele frequencies, along with sizes and distances between quantitative trait loci for two traits, influenced the ability to distinguish close linkage from pleiotropy., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Increasing grain yield is still the main target of wheat breeding; yet today's wheat plants utilize less than half of their yield potential. Owing to the difficulty of determining grain yield potential in a large population, few genetic factors regulating floret fertility (i.e. the difference between grain yield potential and grain number) have been reported to date. In this study, we conducted a genome-wide association study (GWAS) by quantifying 54 traits (16 floret fertility traits and 38 traits for assimilate partitioning and spike morphology) in 210 European winter wheat accessions. The results of this GWAS experiment suggested potential associations between floret fertility, assimilate partitioning and spike morphology revealed by shared quantitative trait loci (QTLs). Several candidate genes involved in carbohydrate metabolism, phytohormones or floral development colocalized with such QTLs, thereby providing potential targets for selection. Based on our GWAS results we propose a genetic network underlying floret fertility and related traits, nominating determinants for improved yield performance., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

The Capsicum genus (Pepper) is a part of the Solanacae family. It has been important in many cultures worldwide for its key nutritional components and uses as spices, medicines, ornamentals and vegetables. Worldwide population growth is associated with demand for more nutritionally valuable vegetables while contending with decreasing resources and available land. These conditions require increased efficiency in pepper breeding to deal with these imminent challenges. Through resequencing of inbred lines we have completed a valuable haplotype map (HapMap) for the pepper genome based on single-nucleotide polymorphisms (SNP). The identified SNPs were annotated and classified based on their gene annotation in the pepper draft genome sequence and phenotype of the sequenced inbred lines. A selection of one marker per gene model was utilized to create the PepperSNP16K array, which simultaneously genotyped 16 405 SNPs, of which 90.7% were found to be informative. A set of 84 inbred and hybrid lines and a mapping population of 90 interspecific F2 individuals were utilized to validate the array. Diversity analysis of the inbred lines shows a distinct separation of bell versus chile/hot pepper types and separates them into five distinct germplasm groups. The interspecific population created between Tabasco (C. frutescens chile type) and P4 (C. annuum blocky type) produced a linkage map with 5546 markers separated into 1361 bins on twelve 12 linkage groups representing 1392.3 cM. This publically available genotyping platform can be used to rapidly assess a large number of markers in a reproducible high-throughput manner for pepper. As a standardized tool for genetic analyses, the PepperSNP16K can be used worldwide to share findings and analyze QTLs for important traits leading to continued improvement of pepper for consumers. Data and information on the array are available through the Solanaceae Genomics Network.

Grain weight, an essential yield component, is under strong genetic control and at the same time markedly influenced by the environment. Genetic analysis of the thousand grain weight (TGW) by genome-wide association study (GWAS) was performed with a panel of 358 European winter wheat (Triticum aestivum L.) varieties and 14 spring wheat varieties using phenotypic data of field tests in eight environments. Wide phenotypic variations were indicated for the TGW with BLUEs (best linear unbiased estimations) values ranging from 35.9 to 58.2 g with a mean value of 45.4 g and a heritability of H(2) = 0.89. A total of 12 candidate genes for plant height, photoperiodism and grain weight were genotyped on all varieties. Only three candidates, the photoperiodism gene Ppd-D1, dwarfing gene Rht-B1and the TaGW-6A gene were significant explaining up to 14.4, 2.3, and 3.4% of phenotypic variation, respectively. For a comprehensive genome-wide analysis of TGW-QTL genotyping data from 732 microsatellite markers and a set of 7769 mapped SNP-markers genotyped with the 90k iSELECT array were analyzed. In total, 342 significant (-log10 (P-value) ≥ 3.0) marker trait associations (MTAs) were detected for SSR-markers and 1195 MTAs (-log10(P-value) ≥ 3.0) for SNP-markers in all single environments plus the BLUEs. After Bonferroni correction, 28 MTAs remained significant for SSR-markers (-log10 (P-value) ≥ 4.82) and 58 MTAs for SNP-markers (-log10 (P-value) ≥ 5.89). Apart from chromosomes 4B and 6B for SSR-markers and chromosomes 4D and 5D for SNP-markers, MTAs were detected on all chromosomes. The highest number of significant SNP-markers was found on chromosomes 3B and 1B, while for the SSRs most markers were significant on chromosomes 6D and 3D. Overall, TGW was determined by many markers with small effects. Only three SNP-markers had R(2) values above 6%.

High-throughput genotyping arrays provide a standardized resource for plant breeding communities that are useful for a breadth of applications including high-density genetic mapping, genome-wide association studies (GWAS), genomic selection (GS), complex trait dissection, and studying patterns of genomic diversity among cultivars and wild accessions. We have developed the CottonSNP63K, an Illumina Infinium array containing assays for 45,104 putative intraspecific single nucleotide polymorphism (SNP) markers for use within the cultivated cotton species Gossypium hirsutum L. and 17,954 putative interspecific SNP markers for use with crosses of other cotton species with G. hirsutum. The SNPs on the array were developed from 13 different discovery sets that represent a diverse range of G. hirsutum germplasm and five other species: G. barbadense L., G. tomentosum Nuttal × Seemann, G. mustelinum Miers × Watt, G. armourianum Kearny, and G. longicalyx J.B. Hutchinson and Lee. The array was validated with 1,156 samples to generate cluster positions to facilitate automated analysis of 38,822 polymorphic markers. Two high-density genetic maps containing a total of 22,829 SNPs were generated for two F2 mapping populations, one intraspecific and one interspecific, and 3,533 SNP markers were co-occurring in both maps. The produced intraspecific genetic map is the first saturated map that associates into 26 linkage groups corresponding to the number of cotton chromosomes for a cross between two G. hirsutum lines. The linkage maps were shown to have high levels of collinearity to the JGI G. raimondii Ulbrich reference genome sequence. The CottonSNP63K array, cluster file and associated marker sequences constitute a major new resource for the global cotton research community., (Copyright © 2015 Hulse-Kemp et al.)

Genome-wide mapping approaches in diverse populations are powerful tools to unravel the genetic architecture of complex traits. The main goals of our study were to investigate the potential and limits to unravel the genetic architecture and to identify the factors determining the accuracy of prediction of the genotypic variation of Fusarium head blight (FHB) resistance in wheat (Triticum aestivum L.) based on data collected with a diverse panel of 372 European varieties. The wheat lines were phenotyped in multi-location field trials for FHB resistance and genotyped with 782 simple sequence repeat (SSR) markers, and 9k and 90k single-nucleotide polymorphism (SNP) arrays. We applied genome-wide association mapping in combination with fivefold cross-validations and observed surprisingly high accuracies of prediction for marker-assisted selection based on the detected quantitative trait loci (QTLs). Using a random sample of markers not selected for marker-trait associations revealed only a slight decrease in prediction accuracy compared with marker-based selection exploiting the QTL information. The same picture was confirmed in a simulation study, suggesting that relatedness is a main driver of the accuracy of prediction in marker-assisted selection of FHB resistance. When the accuracy of prediction of three genomic selection models was contrasted for the three marker data sets, no significant differences in accuracies among marker platforms and genomic selection models were observed. Marker density impacted the accuracy of prediction only marginally. Consequently, genomic selection of FHB resistance can be implemented most cost-efficiently based on low- to medium-density SNP arrays.

The genetic architecture of plant height was investigated in a set of 358 recent European winter wheat varieties plus 14 spring wheat varieties based on field data in eight environments. Genotyping of diagnostic markers revealed the Rht-D1b mutant allele in 58% of the investigated varieties, while the Rht-B1b mutant was only present in 7% of the varieties. Rht-D1 was significantly associated with plant height by using a mixed linear model and employing a kinship matrix to correct for population stratification. Further genotyping data included 732 microsatellite markers, resulting in 770 loci, of which 635 markers were placed on the ITMI map plus a set of 7769 mapped SNP markers genotyped with the 90 k iSELECT chip. When Bonferroni correction was applied, a total of 153 significant marker-trait associations (MTAs) were observed for plant height and the SSR markers (-log10 (P-value) ≥ 4.82) and 280 (-log10 (P-value) ≥ 5.89) for the SNPs. Linear regression between the most effective markers and the BLUEs for plant height indicated additive effects for the MTAs of different chromosomal regions. Analysis of syntenic regions in the rice genome revealed closely linked rice genes related to gibberellin acid (GA) metabolism and perception, i.e. GA20 and GA2 oxidases orthologous to wheat chromosomes 1A, 2A, 3A, 3B, 5B, 5D and 7B, ent-kaurenoic acid oxidase orthologous to wheat chromosome 7A, ent-kaurene synthase on wheat chromosome 2B, as well as GA-receptors like DELLA genes orthologous to wheat chromosomes 4B, 4D and 7A and genes of the GID family orthologous to chromosomes 2B and 5B. The data indicated that besides the widely used GA-insensitive dwarfing genes Rht-B1 and Rht-D1 there is a wide spectrum of loci available that could be used for modulating plant height in variety development.

A genome-wide association study (GWAS) for heading date (HD) was performed with a panel of 358 European winter wheat (Triticum aestivum L.) varieties and 14 spring wheat varieties through the phenotypic evaluation of HD in field tests in eight environments. Genotyping data consisted of 770 mapped microsatellite loci and 7934 mapped SNP markers derived from the 90K iSelect wheat chip. Best linear unbiased estimations (BLUEs) were calculated across all trials and ranged from 142.5 to 159.6 days after the 1st of January with an average value of 151.4 days. Considering only associations with a -log10 (P-value) ≥ 3.0, a total of 340 SSR and 2983 SNP marker-trait associations (MTAs) were detected. After Bonferroni correction for multiple testing, a total of 72 SSR and 438 SNP marker-trait associations remained significant. Highly significant MTAs were detected for the photoperiodism gene Ppd-D1, which was genotyped in all varieties. Consistent associations were found on all chromosomes with the highest number of MTAs on chromosome 5B. Linear regression showed a clear dependence of the HD score BLUEs on the number of favorable alleles (decreasing HD) and unfavorable alleles (increasing HD) per variety meaning that genotypes with a higher number of favorable or a low number of unfavorable alleles showed lower HD and therefore flowered earlier. For the vernalization gene Vrn-A2 co-locating MTAs on chromosome 5A, as well as for the photoperiodism genes Ppd-A1 and Ppd-B1 on chromosomes 2A and 2B were detected. After the construction of an integrated map of the SSR and SNP markers and by exploiting the synteny to sequenced species, such as rice and Brachypodium distachyon, we were able to demonstrate that a marker locus on wheat chromosome 5BL with homology to the rice photoperiodism gene Hd6 played a significant role in the determination of the heading date in wheat.

A total of 358 recent European winter wheat varieties plus 14 spring wheat varieties were evaluated for resistance to Fusarium head blight (FHB) caused by Fusarium graminearum and Fusarium culmorum in four separate environments. The FHB scores based on FHB incidence (Type I resistance)×FHB severity (Type II resistance) indicated a wide phenotypic variation of the varieties with BLUE (best linear unbiased estimation) values ranging from 0.07 to 33.67. Genotyping with 732 microsatellite markers resulted in 782 loci of which 620 were placed on the ITMI map. The resulting average marker distance of 6.8 cM allowed genome wide association mapping employing a mixed model. Though no clear population structure was discovered, a kinship matrix was used for stratification. A total of 794 significant (-log10(p)-value≥3.0) associations between SSR-loci and environment-specific FHB scores or BLUE values were detected, which included 323 SSR alleles. For FHB incidence and FHB severity a total of 861 and 877 individual marker-trait associations (MTA) were detected, respectively. Associations for both traits co-located with FHB score in most cases. Consistent associations detected in three or more environments were found on all chromosomes except chromosome 6B, and with the highest number of MTA on chromosome 5B. The dependence of the number of favourable and unfavourable alleles within a variety to the respective FHB scores indicated an additive effect of favourable and unfavourable alleles, i.e. genotypes with more favourable or less unfavourable alleles tended to show greater resistance to FHB. Assessment of a marker specific for the dwarfing gene Rht-D1 resulted in strong effects. The results provide a prerequisite for designing genome wide breeding strategies for FHB resistance.

Genotyping with large numbers of molecular markers is now an indispensable tool within plant genetics and breeding. Especially through the identification of large numbers of single nucleotide polymorphism (SNP) markers using the novel high-throughput sequencing technologies, it is now possible to reliably identify many thousands of SNPs at many different loci in a given plant genome. For a number of important crop plants, SNP markers are now being used to design genotyping arrays containing thousands of markers spread over the entire genome and to analyse large numbers of samples. In this article, we discuss aspects that should be considered during the design of such large genotyping arrays and the analysis of individuals. The fact that crop plants are also often autopolyploid or allopolyploid is given due consideration. Furthermore, we outline some potential applications of large genotyping arrays including high-density genetic mapping, characterization (fingerprinting) of genetic material and breeding-related aspects such as association studies and genomic selection.

The concurrent development of high-throughput genotyping platforms and next generation sequencing (NGS) has increased the number and density of genetic markers, the efficiency of constructing detailed linkage maps, and our ability to overlay recombination and physical maps of the genome. We developed an array for tomato with 8,784 Single Nucleotide Polymorphisms (SNPs) mainly discovered based on NGS-derived transcriptome sequences. Of the SNPs, 7,720 (88%) passed manufacturing quality control and could be scored in tomato germplasm. The array was used to generate high-density linkage maps for three interspecific F(2) populations: EXPEN 2000 (Solanum lycopersicum LA0925 x S. pennellii LA0716, 79 individuals), EXPEN 2012 (S. lycopersicum Moneymaker x S. pennellii LA0716, 160 individuals), and EXPIM 2012 (S. lycopersicum Moneymaker x S. pimpinellifolium LA0121, 183 individuals). The EXPEN 2000-SNP and EXPEN 2012 maps consisted of 3,503 and 3,687 markers representing 1,076 and 1,229 unique map positions (genetic bins), respectively. The EXPEN 2000-SNP map had an average marker bin interval of 1.6 cM, while the EXPEN 2012 map had an average bin interval of 0.9 cM. The EXPIM 2012 map was constructed with 4,491 markers (1,358 bins) and an average bin interval of 0.8 cM. All three linkage maps revealed an uneven distribution of markers across the genome. The dense EXPEN 2012 and EXPIM 2012 maps showed high levels of colinearity across all 12 chromosomes, and also revealed evidence of small inversions between LA0716 and LA0121. Physical positions of 7,666 SNPs were identified relative to the tomato genome sequence. The genetic and physical positions were mostly consistent. Exceptions were observed for chromosomes 3, 10 and 12. Comparing genetic positions relative to physical positions revealed that genomic regions with high recombination rates were consistent with the known distribution of euchromatin across the 12 chromosomes, while very low recombination rates were observed in the heterochromatic regions.

The effects of selection on genome variation were investigated and visualized in tomato using a high-density single nucleotide polymorphism (SNP) array. 7,720 SNPs were genotyped on a collection of 426 tomato accessions (410 inbreds and 16 hybrids) and over 97% of the markers were polymorphic in the entire collection. Principal component analysis (PCA) and pairwise estimates of F(st) supported that the inbred accessions represented seven sub-populations including processing, large-fruited fresh market, large-fruited vintage, cultivated cherry, landrace, wild cherry, and S. pimpinellifolium. Further divisions were found within both the contemporary processing and fresh market sub-populations. These sub-populations showed higher levels of genetic diversity relative to the vintage sub-population. The array provided a large number of polymorphic SNP markers across each sub-population, ranging from 3,159 in the vintage accessions to 6,234 in the cultivated cherry accessions. Visualization of minor allele frequency revealed regions of the genome that distinguished three representative sub-populations of cultivated tomato (processing, fresh market, and vintage), particularly on chromosomes 2, 4, 5, 6, and 11. The PCA loadings and F(st) outlier analysis between these three sub-populations identified a large number of candidate loci under positive selection on chromosomes 4, 5, and 11. The extent of linkage disequilibrium (LD) was examined within each chromosome for these sub-populations. LD decay varied between chromosomes and sub-populations, with large differences reflective of breeding history. For example, on chromosome 11, decay occurred over 0.8 cM for processing accessions and over 19.7 cM for fresh market accessions. The observed SNP variation and LD decay suggest that different patterns of genetic variation in cultivated tomato are due to introgression from wild species and selection for market specialization.

SNP genotyping arrays have been useful for many applications that require a large number of molecular markers such as high-density genetic mapping, genome-wide association studies (GWAS), and genomic selection. We report the establishment of a large maize SNP array and its use for diversity analysis and high density linkage mapping. The markers, taken from more than 800,000 SNPs, were selected to be preferentially located in genes and evenly distributed across the genome. The array was tested with a set of maize germplasm including North American and European inbred lines, parent/F1 combinations, and distantly related teosinte material. A total of 49,585 markers, including 33,417 within 17,520 different genes and 16,168 outside genes, were of good quality for genotyping, with an average failure rate of 4% and rates up to 8% in specific germplasm. To demonstrate this array's use in genetic mapping and for the independent validation of the B73 sequence assembly, two intermated maize recombinant inbred line populations - IBM (B73×Mo17) and LHRF (F2×F252) - were genotyped to establish two high density linkage maps with 20,913 and 14,524 markers respectively. 172 mapped markers were absent in the current B73 assembly and their placement can be used for future improvements of the B73 reference sequence. Colinearity of the genetic and physical maps was mostly conserved with some exceptions that suggest errors in the B73 assembly. Five major regions containing non-colinearities were identified on chromosomes 2, 3, 6, 7 and 9, and are supported by both independent genetic maps. Four additional non-colinear regions were found on the LHRF map only; they may be due to a lower density of IBM markers in those regions or to true structural rearrangements between lines. Given the array's high quality, it will be a valuable resource for maize genetics and many aspects of maize breeding.

Oilseed rape (Brassica napus) is an allotetraploid species consisting of two genomes, derived from B. rapa (A genome) and B. oleracea (C genome). The presence of these two genomes makes single nucleotide polymorphism (SNP) marker identification and SNP analysis more challenging than in diploid species, as for a given locus usually two versions of a DNA sequence (based on the two ancestral genomes) have to be analyzed simultaneously during SNP identification and analysis. One hundred amplicons derived from expressed sequence tag (ESTs) were analyzed to identify SNPs in a panel of oilseed rape varieties and within two sister species representing the ancestral genomes. A total of 604 SNPs were identified, averaging one SNP in every 42 bp. It was possible to clearly discriminate SNPs that are polymorphic between different plant varieties from SNPs differentiating the two ancestral genomes. To validate the identified SNPs for their use in genetic analysis, we have developed Illumina GoldenGate assays for some of the identified SNPs. Through the analysis of a number of oilseed rape varieties and mapping populations with GoldenGate assays, we were able to identify a number of different segregation patterns in allotetraploid oilseed rape. The majority of the identified SNP markers can be readily used for genetic mapping, showing that amplicon sequencing and Illumina GoldenGate assays can be used to reliably identify SNP markers in tetraploid oilseed rape and to convert them into successful SNP assays that can be used for genetic analysis.

Hordeum vulgare subsp. spontaneum, the wild progenitor of barley, is a potential source of useful genetic variation for barley breeding programs. The objective of this study was to map quantitative trait loci (QTLs) in an advanced backcross population of barley. A total of 207 BC3 lines were developed using the 2-rowed German spring cultivar Hordeum vulgare subsp. vulgare 'Brenda' as a recurrent parent and the H. vulgare subsp. spontaneum accession HS584 as a donor parent. The lines were genotyped by 108 simple-sequence repeat (SSR) markers and evaluated in field tests for the measurement of grain yield and its components, such as ear length, spikelet number per spike, grain number per spike, spike number, and 1000-grain mass, as well as heading date and plant height. A total of 100 QTLs were detected. Ten QTLs with increasing effects were found for ear length, spikelet number, and grain number per spike. Three QTLs contributed by HS584 were found to significantly decrease days to heading across all years at 2 locations. In addition, 2 QTLs from HS584 on chromosomes 2H and 3H were associated with resistance to leaf rust. Based on genotypic data obtained from this population, 55 introgression lines carrying 1 or 2 donor segments were selected to develop a set of doubled-haploid lines, which will be used to reconfirm and investigate the effects of 100 QTLs for future genetic studies.

Background: The goal of our study was a systematic survey of the molecular diversity in barley genetic resources. To this end 953 cultivated barley accessions originating from all inhabited continents except Australia were genotyped with 48 SSR markers. Molecular diversity was evaluated with routine statistics (allelic richness, gene diversity, allele frequency, heterozygosity and unique alleles), Principal Coordinate Analysis (PCoA), and analysis of genome-wide linkage disequilibrium., Results: A genotyping database for 953 cultivated barley accessions profiled with 48 SSR markers was established. The PCoA revealed structuring of the barley population with regard to (i) geographical regions and (ii) agronomic traits. Geographic origin contributed most to the observed molecular diversity. Genome-wide linkage disequilibrium (LD) was estimated as squared correlation of allele frequencies (r2). The values of LD for barley were comparable to other plant species (conifers, poplar, maize). The pattern of intrachromosomal LD with distances between the genomic loci ranging from 1 to 150 cM revealed that in barley LD extended up to distances as long as 50 cM with r2 > 0.05, or up to 10 cM with r2 > 0.2. Few loci mapping to different chromosomes showed significant LD with r2 > 0.05. The number of loci in significant LD as well as the pattern of LD were clearly dependent on the population structure. The LD in the homogenous group of 207 European 2-rowed spring barleys compared to the highly structured worldwide barley population was increased in the number of loci pairs with r2 > 0.05 and had higher values of r2, although the percentage of intrachromosomal loci pairs in significant LD based on P < 0.001 was 100% in the whole set of varieties, but only 45% in the subgroup of European 2-rowed spring barleys. The value of LD also varied depending on the polymorphism of the loci selected for genotyping. The 17 most polymorphic loci (PIC > 0.80) provided higher LD values as compared to 19 low polymorphic loci (PIC < 0.73) in both structured (all accessions) and non-structured (European 2-rowed spring varieties) barley populations., Conclusion: A global population of cultivated barley accessions was highly structured. Clustering highlighted the accessions with the same geographic origin, as well as accessions possessing similar agronomic characters. LD in barley extended up to 50 cM, and was strongly dependent on the population structure. The data on LD were summarized as a genome-wide LD map for barley.

Genetic analysis of the wound response pathway in tomato indicates that systemin and its precursor protein, prosystemin, are upstream components of a defensive signaling cascade that involves the synthesis and subsequent action of the octadecatrienoic acid (18:3)-derived plant hormone jasmonic acid (JA). The suppressor of prosystemin-mediated responses2 (spr2) mutation, which was isolated previously as a suppressor of (pro)systemin-mediated signaling, impairs wound-induced JA biosynthesis and the production of a long-distance signal for the expression of defensive Proteinase inhibitor genes. Using a map-based cloning approach, we demonstrate here that Spr2 encodes a chloroplast fatty acid desaturase involved in JA biosynthesis. Loss of Spr2 function reduced the 18:3 content of leaves to <10% of wild-type levels, abolished the accumulation of hexadecatrienoic acid, and caused a corresponding increase in the level of dienoic fatty acids. The effect of spr2 on the fatty acyl content of various classes of glycerolipids indicated that the Spr2 gene product catalyzes most, if not all, omega3 fatty acid desaturation within the "prokaryotic pathway" for lipid synthesis in tomato leaves. Despite the reduced levels of trienoic fatty acids, spr2 plants exhibited normal growth, development, and reproduction. However, the mutant was compromised in defense against attack by tobacco hornworm larvae. These results indicate that jasmonate synthesis from chloroplast pools of 18:3 is required for wound- and systemin-induced defense responses and support a role for systemin in the production of a transmissible signal that is derived from the octadecanoid pathway.

The Hero gene of tomato is a broad spectrum resistance gene that confers a high level of resistance to all pathotypes of the potato cyst nematodes Globodera rostochiensis and partial resistance to G. pallida. The gene was identified by map-based cloning, sequencing and complementation analysis of two susceptible tomato lines with an array of 13 overlapping cosmids spanning a total distance of 135 kb. Hero encodes a protein with a nucleotide-binding site (NBS) and a leucine-rich-repeat (LRR) domain and is a member of a gene family of 14 highly homologous genes, which are clustered within a continuous 118-kb region. The isolated Hero gene displayed resistance to various G. rostochiensis pathotypes and partial resistance to G. pallida pathotype Pa2/3 in transgenic tomato lines. None of the Hero homologues conferred resistance to G. rostochiensis pathotypes. Hero can be distinguished from its homologues by the length of a compound hexanucleotide microsatellite, which codes for a charged and repetitive amino acid domain within the LRR. We propose that the expansion of this microsatellite may be involved in the evolution of the Hero resistance gene.

The Rh2 resistance gene of barley (Hordeum vulgare) confers resistance against the scald pathogen (Rhynchosporium secalis). A high-resolution genetic map of the Rh2 region on chromosome I (7H) was established by the use of molecular markers. Tightly linked markers from this region were used to screen existing and a newly constructed yeast artificial chromosome (YAC) library of barley cv. Franka composed of 45,000 clones representing approximately two genome equivalents. Corresponding YAC clones were identified for most markers, indicating that the combined YAC library has good representation of the barley genome. The contiguous sets of YAC clones with the most tightly linked molecular markers represent entry points for map-based cloning of this resistance gene.

The potential of Aegilops tauschii, the diploid progenitor of the D genome of wheat, as a source of microsatellite markers for hexaploid bread wheat was investigated. By screening lambda phage and plasmid libraries of Ae. tauschii genomic DNA, dinucleotide microsatellites containing GA and GT motifs were isolated and a total of 65 functional microsatellite markers were developed. All primer pairs that were functional in Ae. tauschii amplified well in hexaploid wheat. Fifty-five loci amplified by 48 primer sets were placed onto a genetic framework map of the reference population of the International Triticeae Mapping Initiative (ITMI) 'Opata 85' x 'W7984'. The majority of microsatellite markers could be assigned to the chromosomes of the D genome of wheat. The distribution of the markers along the chromosomes is random. Chromosomal location of 22 loci nonpolymorphic in the reference population was determined using nullitetrasomic lines of Triticum aestivum 'Chinese Spring'. The results of this study demonstrate the value of microsatellite markers isolated from Ae. tauschii for the study of bread wheat. The microsatellite markers developed improve the existing wheat microsatellite map and can be used in a wide range of genetic studies and breeding programs.

The uptake of iron in plants is a highly regulated process that is induced on iron starvation. In tomato, the mutant chloronerva exhibits constitutive expression of iron uptake responses and intercostal chlorosis. Biochemically, chloronerva is an auxotroph for nicotianamine, a key polyamine in plant iron uptake metabolism. The chloronerva gene has been fine-mapped onto the long arm of chromosome 1 in a large segregating tomato population and yeast artificial chromosome clones encompassing the region were isolated by using flanking markers. A cosmid contig containing the chloronerva gene was established, and complementing cosmids were identified by transformation into the mutant. The chloronerva transcript was identified by cDNA isolation using the complementing cosmids. The gene encodes a unique protein of 35 kDa. The mutant harbors a single base change compared with the wild type. Based on enzyme activity and sequence similarity to the coding DNA sequence of the purified barley enzyme the chloronerva gene encodes the enzyme nicotianamine synthase.

Microsatellites as genetic markers are used in many crop plants. Major criteria for their usability as molecular markers include that they are highly polymorphic and evenly spread throughout a genome. In tomato, it has been reported that long arrays of tetranucleotide microsatellites containing the motif GATA are highly clustered around the centromeres of all chromosomes. In this study, we have isolated tomato microsatellites containing long arrays (> 20 repeats) of the dinucleotide motifs GA, GT, AT, as well as GATA, assessed their variability within Lycopersicon esculentum varieties and mapped them onto a genetic map of tomato. The investigated microsatellite markers exhibited between 1 and 5 alleles in a diverse set of L. esculentum lines. Mapping of the microsatellites onto the genetic map of tomato demonstrates that, as previously shown, GATA microsatellites are highly clustered in the regions of the tomato centromeres. Interestingly, the same centromeric location was now found for long dinucleotide microsatellite markers. Because of this uneven distribution, genetic mapping of the entire tomato genome using long dinucleotide microsatellites will be very difficult to achieve and microsatellite markers with shorter arrays of microsatellites could be more suitable for mapping experiments albeit their lower level of polymorphism. Some microsatellite markers described in this study might provide a useful tool to study the molecular structure of tomato centromeric regions and for variety identification.

Hexaploid bread wheat (Triticum aestivum L. em. Thell) is one of the world's most important crop plants and displays a very low level of intraspecific polymorphism. We report the development of highly polymorphic microsatellite markers using procedures optimized for the large wheat genome. The isolation of microsatellite-containing clones from hypomethylated regions of the wheat genome increased the proportion of useful markers almost twofold. The majority (80%) of primer sets developed are genome-specific and detect only a single locus in one of the three genomes of bread wheat (A, B, or D). Only 20% of the markers detect more than one locus. A total of 279 loci amplified by 230 primer sets were placed onto a genetic framework map composed of RFLPs previously mapped in the reference population of the International Triticeae Mapping Initiative (ITMI) Opata 85 x W7984. Sixty-five microsatellites were mapped at a LOD >2.5, and 214 microsatellites were assigned to the most likely intervals. Ninety-three loci were mapped to the A genome, 115 to the B genome, and 71 to the D genome. The markers are randomly distributed along the linkage map, with clustering in several centromeric regions.

The dense RFLP linkage map of tomato (Lycopersicon esculentum) contains >300 anonymous cDNA clones. Of those clones, 272 were partially or completely sequenced. The sequences were compared at the DNA and protein level to known genes in databases. For 57% of the clones, a significant match to previously described genes was found. The information will permit the conversion of those markers to STS markers and allow their use in PCR-based mapping experiments. Furthermore, it will facilitate the comparative mapping of genes across distantly related plant species by direct comparison of DNA sequences and map positions. [cDNA sequence data reported in this paper have been submitted to the EMBL database under accession nos. AA824695-AA825005 and the dbEST&#95;Id database under accession nos. 1546519-1546862.]

The rapid identification of sex in the dioecious hop (Humulus lupulus) is important for the breeding of this cultivated plant because only unfertilized flowers of the female plants are used as an ingredient in the production of beer. It is thought that a sex-chromosome mechanism controls the development of male or female plants. We have compared pools of male and female plants derived from a hop cross to identify molecular markers associated with the Y or male-specific chromosome. Of 900 functional RAPD primers, 32 revealed fragments specific for male plants that were absent in female plants of this cross. Subsequently, the 32 positive primers were tested on unrelated male and female plants. Three of these 32 primers were specific for the Y chromosome in all lines. The Y-specific product derived from one of these primers (OPJ9) was of low copy in hybridization experiments and predominantly present in male plants. Primers developed from the DNA sequence of this product provide a marker for rapid sex identification in crosses of hop by means of PCR.

In situ localization of short low- or single-copy sequences is still difficult in plants. One solution to this problem could be the use of large yeast artificial chromosomes (YACs) for fluorescence in situ hybridization. Two YACs specific for a single copy marker on the long arm of the NOR-chromosome 2 of tomato (Lycopersicon esculentum) were selected. Both probes hybridized exclusively to this chromosome, although one produced a slightly dispersed hybridization signal. Hybridization of these YACs onto potato chromosome showed a clear single locus on the homoeologous potato chromosome in both cases.

The genomic organization of two different types of satellite DNA sequences was analysed by means of fluorescence in situ hybridization (FISH) and pulsed-field gel electrophoresis (PFGE) in barley. Satellite HvT01 was detected at all chromosome ends except the long arms of chromosomes 2 and 7. The unrelated satellite pAS1 was found at all chromosome ends except the long arm of chromosome 7 and at two interstitial sites, both located on the long arm of chromosome 4 on the standard karyotype. Southern and in situ hybridization further indicate that pAS1 also occurs interspersed in the barley genome. For most chromosome ends, the linear order of HvT01 and pAS1 could not be determined by in situ hybridization except at the short arms of chromosomes 2 and 6, where HvT01 is more distal than pAS1. This is confirmed by PFGE analysis, HvT01 being frequently associated with the telomeric repeat but not pAS1. Furthermore, we found that HvT01 occurred in clusters up to 1000 kb in size, whereas the pAS1 cluster had a maximum size of 500 kb. Sequence comparison revealed that both satellites are completely unrelated and differ considerably in their G + C contents.

The Pto gene in tomato confers resistance to races of Pseudomonas syringae pv. tomato that carry the avirulence gene avrPto. A yeast artificial chromosome clone that spans the Pto region was identified and used to probe a leaf complementary DNA (cDNA) library. A cDNA clone was isolated that represents a gene family, at least six members of which genetically cosegregate with Pto. When susceptible tomato plants were transformed with a cDNA from this family, they were resistant to the pathogen. Analysis of the amino acid sequence revealed similarity to serine-threonine protein kinases, suggesting a role for Pto in a signal transduction pathway.

High density molecular linkage maps, comprised of more than 1000 markers with an average spacing between markers of approximately 1.2 cM (ca. 900 kb), have been constructed for the tomato and potato genomes. As the two maps are based on a common set of probes, it was possible to determine, with a high degree of precision, the breakpoints corresponding to 5 chromosomal inversions that differentiate the tomato and potato genomes. All of the inversions appear to have resulted from single breakpoints at or near the centromeres of the affected chromosomes, the result being the inversion of entire chromosome arms. While the crossing over rate among chromosomes appears to be uniformly distributed with respect to chromosome size, there is tremendous heterogeneity of crossing over within chromosomes. Regions of the map corresponding to centromeres and centromeric heterochromatin, and in some instances telomeres, experience up to 10-fold less recombination than other areas of the genome. Overall, 28% of the mapped loci reside in areas of putatively suppressed recombination. This includes loci corresponding to both random, single copy genomic clones and transcribed genes (detected with cDNA probes). The extreme heterogeneity of crossing over within chromosomes has both practical and evolutionary implications. Currently tomato and potato are among the most thoroughly mapped eukaryotic species and the availability of high density molecular linkage maps should facilitate chromosome walking, quantitative trait mapping, marker-assisted breeding and evolutionary studies in these two important and well studied crop species.

A telomere-associated tandemly repeated DNA sequence of tomato, TGR I, has been used to map telomeres on the tomato RFLP linkage map. Mapping was performed by monitoring the segregation of entire arrays of TGR I from a segregating F2 population using pulsed-field gel electrophoresis (PFGE). With this strategy, four telomeres have been mapped to the ends of the short arm of chromosomes 9 and 12 and the long arms of chromosomes 5 and 11, using a saturated RFLP map of tomato containing approximately 1000 RFLP markers. In all four cases, the TGR I locus maps to the end of the chromosome, and the distance between the most distal single-copy RFLP marker and the telomeric TGR I locus was between 1.6 and 9.6 cM. This indicates that the region close to the telomeres does not show an excessive rate of recombination compared to other regions of the genome and that the RFLP map of tomato is essentially complete and covers the entire genome for all practical purposes. Additionally, the mapping technique presented here should be generally applicable to the mapping of other tandemly repeated DNA sequences.

Tomato telomeres are composed of a terminal 7-base-pair tandem repeat and a closely liked 162-base-pair subtelomeric repeat (TGRI). Together, these repeats account for 2% of the total chromosomal DNA. Pulsed-field gel electrophoresis has been used to examine the long-range physical structure of these arrays in closely related varieties, and we report here that these arrays are undergoing heritable changes at a frequency as great as 2% per generation. Moreover, comparisons with other known hypervariable probes (e.g., human minisatellites and M13 sequences) revealed that telomeric sites are more variable than any other known region of the plant genome and can be used to distinguish closely related plant varieties (tomato and melon) that are otherwise very similar at the DNA level. The fact that the polymorphisms are inherited in a mendelian fashion suggests applications in genetic mapping of telomeres and identification of varieties.

We present a general method for isolating molecular markers specific to any region of a chromosome using existing mapping populations. Two pools of DNA from individuals homozygous for opposing alleles for a targeted chromosomal interval, defined by two or more linked RFLP markers, are constructed from members of an existing mapping population. The DNA pools are then screened for polymorphism using random oligonucleotide primers and PCR (1). Polymorphic DNA bands should represent DNA sequences within or adjacent to the selected interval. We tested this method in tomato using two genomic intervals containing genes responsible for regulating pedicle abscission (jointless) and fruit ripening (non-ripening). DNA pools containing 7 to 14 F2 individuals for each interval were screened with 200 random primers. Three polymorphic markers were thus identified, two of which were subsequently shown to be tightly linked to the selected intervals. The third marker mapped to the same chromosome (11) but 45 cM away from the selected interval. A particularly attractive attribute of this method is that a single mapping population can be used to target any interval in the genome. Although this method has been demonstrated in tomato, it should be applicable to any sexually reproducing organism for which segregating populations are being used to construct genetic linkage maps.

The macrostructure of the tomato telomeres has been investigated by in situ hybridization, genomic sequencing, and pulsed-field gel electrophoresis. In situ hybridizations with a cloned telomeric sequence from Arabidopsis thaliana indicated that the telomeric repeat of tomato cross-hybridizes with that of Arabidopsis and is located at all telomeres. Bal31 digestion kinetics confirmed that the tomato telomeric repeat represents the outermost DNA sequence of each tomato chromosome. Genomic sequencing of enriched tomato telomeric sequences, using primers derived from the Arabidopsis sequence, revealed that the consensus sequence of the tomato telomeric repeat is TT(T/A)AGGG compared with the Arabidopsis consensus sequence of TTTAGGG. Furthermore, as shown by pulsed-field gel electrophoresis, the telomeric repeat of tomato is separated by not more than a few hundred kilobases from a previously described 162-base pair satellite DNA repeat of tomato (TGR I) at 20 of the 24 telomeres. Together, these sequences are found in the heterochromatic terminal knob observed in pachytene chromosomes. Therefore, these two repeats determine the structure of 20 of the 24 tomato chromosome ends over approximately 2% of the total chromosome length.

The Tm-2a gene of tomato confers resistance to the viral pathogen, tobacco mosaic virus. Like many economically important plant genes, Tm-2a has been characterized phenotypically and by classical linkage analysis, yet nothing is known about its gene product. We report here the isolation of two DNA clones which are very tightly linked to the Tm-2a gene. These clones were identified by testing 122 genomic clones as hybridization probes against Southern blots consisting of DNA from pairs of nearly isogenic lines with or without the Tm-2a gene. Screening such a large number of clones in a short period of time was facilitated by co-labeling and simultaneous probing of sets of up to 10 random genomic clones. Tightly linked clones were distinguished by the fact that they exhibited one or more restriction fragment length polymorphisms between the nearly isogenic lines. Tight linkage of the clones with Tm-2a was verified in a segregating F(2) population. Both mapped to the same locus 0.4 +/- 0.4 centimorgans away from Tm-2a and may provide starting points for a genomic ;;walk'' to this gene. Due to the availability of isogenic lines in many plant species, the strategy outlined in this paper should be widely applicable for selecting DNA clones tightly linked to genes of interest.