Einkorn genomics sheds light on history of the oldest domesticated wheat.

Einkorn (Triticum monococcum) was the first domesticated wheat species, and was central to the birth of agriculture and the Neolithic Revolution in the Fertile Crescent around 10,000 years ago1,2. Here we generate and analyse 5.2-Gb genome assemblies for wild and domesticated einkorn, including completely assembled centromeres. Einkorn centromeres are highly dynamic, showing evidence of ancient and recent centromere shifts caused by structural rearrangements. Whole-genome sequencing analysis of a diversity panel uncovered the population structure and evolutionary history of einkorn, revealing complex patterns of hybridizations and introgressions after the dispersal of domesticated einkorn from the Fertile Crescent. We also show that around 1% of the modern bread wheat (Triticum aestivum) A subgenome originates from einkorn. These resources and findings highlight the history of einkorn evolution and provide a basis to accelerate the genomics-assisted improvement of einkorn and bread wheat.

Multiple wheat genomes reveal global variation in modern breeding.

Advances in genomics have expedited the improvement of several agriculturally important crops but similar efforts in wheat (Triticum spp.) have been more challenging. This is largely owing to the size and complexity of the wheat genome1, and the lack of genome-assembly data for multiple wheat lines2,3. Here we generated ten chromosome pseudomolecule and five scaffold assemblies of hexaploid wheat to explore the genomic diversity among wheat lines from global breeding programs. Comparative analysis revealed extensive structural rearrangements, introgressions from wild relatives and differences in gene content resulting from complex breeding histories aimed at improving adaptation to diverse environments, grain yield and quality, and resistance to stresses4,5. We provide examples outlining the utility of these genomes, including a detailed multi-genome-derived nucleotide-binding leucine-rich repeat protein repertoire involved in disease resistance and the characterization of Sm16, a gene associated with insect resistance. These genome assemblies will provide a basis for functional gene discovery and breeding to deliver the next generation of modern wheat cultivars.

Extrachromosomal circular DNA-mediated spread of herbicide resistance in interspecific hybrids of pigweed.

Extrachromosomal circular DNAs (eccDNAs) are found in many eukaryotic organisms. EccDNA-powered copy number variation plays diverse roles, from oncogenesis in humans to herbicide resistance in crop weeds. Here, we report interspecific eccDNA flow and its dynamic behavior in soma cells of natural populations and F1 hybrids of Amaranthus sp. The glyphosate-resistance (GR) trait is controlled by eccDNA-based amplification harboring the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (eccDNA replicon), the molecular target of glyphosate. We documented pollen-mediated transfer of eccDNA in experimental hybrids between glyphosate-susceptible Amaranthus tuberculatus and GR Amaranthus palmeri. Experimental hybridization and fluorescence in situ hybridization (FISH) analysis revealed that the eccDNA replicon in Amaranthus spinosus derived from GR A. palmeri by natural hybridization. FISH analysis also revealed random chromosome anchoring and massive eccDNA replicon copy number variation in soma cells of weedy hybrids. The results suggest that eccDNAs are inheritable across compatible species, contributing to genome plasticity and rapid adaptive evolution.

Genetic characterization and curation of diploid A-genome wheat species.

A-genome diploid wheats represent the earliest domesticated and cultivated wheat species in the Fertile Crescent and include the donor of the wheat A sub-genome. The A-genome species encompass the cultivated einkorn (Triticum monococcum L. subsp. monococcum), wild einkorn (T. monococcum L. subsp. aegilopoides (Link) Thell.), and Triticum urartu. We evaluated the collection of 930 accessions in the Wheat Genetics Resource Center (WGRC) using genotyping by sequencing and identified 13,860 curated single-nucleotide polymorphisms. Genomic analysis detected misclassified and genetically identical (>99%) accessions, with most of the identical accessions originating from the same or nearby locations. About 56% (n = 520) of the WGRC A-genome species collections were genetically identical, supporting the need for genomic characterization for effective curation and maintenance of these collections. Population structure analysis confirmed the morphology-based classifications of the accessions and reflected the species geographic distributions. We also showed that T. urartu is the closest A-genome diploid to the A-subgenome in common wheat (Triticum aestivum L.) through phylogenetic analysis. Population analysis within the wild einkorn group showed three genetically distinct clusters, which corresponded with wild einkorn races α, ß, and γ described previously. The T. monococcum genome-wide FST scan identified candidate genomic regions harboring a domestication selection signature at the Non-brittle rachis 1 (Btr1) locus on the short arm of chromosome 3Am at ∼70 Mb. We established an A-genome core set (79 accessions) based on allelic diversity, geographical distribution, and available phenotypic data. The individual species core set maintained at least 79% of allelic variants in the A-genome collection and constituted a valuable genetic resource to improve wheat and domesticated einkorn in breeding programs.

Deciphering the Mechanism of Glyphosate Resistance in Amaranthus palmeri by Cytogenomics.

In agriculture, various chemicals are used to control the weeds. Out of which, glyphosate is an important herbicide invariably used in the cultivation of glyphosate-resistant crops to control weeds. Overuse of glyphosate results in the evolution of glyphosate-resistant weeds. Evolution of glyphosate resistance (GR) in Amaranthus palmeri (AP) is a serious concern in the USA. Investigation of the mechanism of GR in AP identified different resistance mechanisms of which 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene amplification is predominant. Molecular analysis of GR AP identified the presence of a 5- to >160-fold increase in copies of the EPSPS gene than in a glyphosate-susceptible (GS) population. This increased copy number of the EPSPS gene increased the genome size ranging from 3.5 to 11.8%, depending on the copy number compared to the genome size of GS AP. FISH analysis using a 399-kb EPSPS cassette derived from bacterial artificial chromosomes (BACs) as probes identified that amplified EPSPS copies in GR AP exist in extrachromosomal circular DNA (eccDNA) in addition to the native copy in the chromosome. The EPSPS gene-containing eccDNA having a size of ∼400 kb is termed EPSPS-eccDNA and showed somatic mosacism in size and copy number. EPSPS-eccDNA has a genetic mechanism to tether randomly to mitotic or meiotic chromosomes during cell division or gamete formation and is inherited to daughter cells or progeny generating copy number variation. These eccDNAs are stable genetic elements that can replicate and exist independently. The genomic characterization of the EPSPS locus, along with the flanking regions, identified the presence of a complex array of repeats and mobile genetic elements. The cytogenomics approach in understanding the biology of EPSPS-eccDNA sheds light on various characteristics of EPSPS-eccDNA that favor GR in AP.

An approach for high-resolution genetic mapping of distant wild relatives of bread wheat: example of fine mapping of Lr57 and Yr40 genes.

KEY MESSAGE: The article reports a powerful but simple approach for high-resolution mapping and eventual map-based cloning of agronomically important genes from distant relatives of wheat, using the already existing germplasm resources. Wild relatives of wheat are a rich reservoir of genetic diversity for its improvement. The effective utilization of distant wild relatives in isolation of agronomically important genes is hindered by the lack of recombination between the homoeologous chromosomes. In this study, we propose a simple yet powerful approach that can be applied for high-resolution mapping of a targeted gene from wheat's distant gene pool members. A wheat-Aegilops geniculata translocation line TA5602 with a small terminal segment from chromosome 5 Mg of Ae. geniculata translocated to 5D of wheat contains genes Lr57 and Yr40 for leaf rust and stripe rust resistance, respectively. To map these genes, TA5602 was crossed with a susceptible Ae. geniculata 5 Mg addition line. Chromosome pairing between the 5 Mg chromosomes of susceptible and resistant parents resulted in the development of a high-resolution mapping panel for the targeted genes. Next-generation-sequencing data from flow-sorted 5 Mg chromosome of Ae. geniculata allowed us to generate 5 Mg-specific markers. These markers were used to delineate Lr57 and Yr40 genes each to distinct ~ 1.5 Mb physical intervals flanked by gene markers on 5 Mg. The method presented here will allow researchers worldwide to utilize existing germplasm resources in genebanks and seed repositories toward routinely performing map-based cloning of important genes from tertiary gene pools of wheat.

The Aegilops ventricosa 2NvS segment in bread wheat: cytology, genomics and breeding.

KEY MESSAGE: The first cytological characterization of the 2NvS segment in hexaploid wheat; complete de novo assembly and annotation of 2NvS segment; 2NvS frequency is increasing 2NvS and is associated with higher yield. The Aegilops ventricosa 2NvS translocation segment has been utilized in breeding disease-resistant wheat crops since the early 1990s. This segment is known to possess several important resistance genes against multiple wheat diseases including root knot nematode, stripe rust, leaf rust and stem rust. More recently, this segment has been associated with resistance to wheat blast, an emerging and devastating wheat disease in South America and Asia. To date, full characterization of the segment including its size, gene content and its association with grain yield is lacking. Here, we present a complete cytological and physical characterization of this agronomically important translocation in bread wheat. We de novo assembled the 2NvS segment in two wheat varieties, 'Jagger' and 'CDC Stanley,' and delineated the segment to be approximately 33 Mb. A total of 535 high-confidence genes were annotated within the 2NvS region, with > 10% belonging to the nucleotide-binding leucine-rich repeat (NLR) gene families. Identification of groups of NLR genes that are potentially N genome-specific and expressed in specific tissues can fast-track testing of candidate genes playing roles in various disease resistances. We also show the increasing frequency of 2NvS among spring and winter wheat breeding programs over two and a half decades, and the positive impact of 2NvS on wheat grain yield based on historical datasets. The significance of the 2NvS segment in wheat breeding due to resistance to multiple diseases and a positive impact on yield highlights the importance of understanding and characterizing the wheat pan-genome for better insights into molecular breeding for wheat improvement.

Extrachromosomal circular DNA-based amplification and transmission of herbicide resistance in crop weed Amaranthus palmeri.

Gene amplification has been observed in many bacteria and eukaryotes as a response to various selective pressures, such as antibiotics, cytotoxic drugs, pesticides, herbicides, and other stressful environmental conditions. An increase in gene copy number is often found as extrachromosomal elements that usually contain autonomously replicating extrachromosomal circular DNA molecules (eccDNAs). Amaranthus palmeri, a crop weed, can develop herbicide resistance to glyphosate [N-(phosphonomethyl) glycine] by amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, the molecular target of glyphosate. However, biological questions regarding the source of the amplified EPSPS, the nature of the amplified DNA structures, and mechanisms responsible for maintaining this gene amplification in cells and their inheritance remain unknown. Here, we report that amplified EPSPS copies in glyphosate-resistant (GR) A. palmeri are present in the form of eccDNAs with various conformations. The eccDNAs are transmitted during cell division in mitosis and meiosis to the soma and germ cells and the progeny by an as yet unknown mechanism of tethering to mitotic and meiotic chromosomes. We propose that eccDNAs are one of the components of McClintock's postulated innate systems [McClintock B (1978) Stadler Genetics Symposium] that can rapidly produce soma variation, amplify EPSPS genes in the sporophyte that are transmitted to germ cells, and modulate rapid glyphosate resistance through genome plasticity and adaptive evolution.

A spontaneous wheat-Aegilops longissima translocation carrying Pm66 confers resistance to powdery mildew.

KEY MESSAGE: A spontaneous Robertsonian T4SlS·4BL translocation chromosome carrying Pm66 for powdery mildew resistance was discovered and confirmed by RNA-seq, molecular marker, and in situ hybridization analyses. Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a severe disease of bread wheat worldwide. Discovery and utilization of resistance genes to powdery mildew from wild relatives of wheat have played important roles in wheat improvement. Aegilops longissima, one of the S-genome diploid wild relatives of wheat, is a valuable source of disease and pest resistance for wheat. Chromosome 4Sl from Ae. longissima confers moderate resistance to powdery mildew. In this study, we conducted RNA-seq on a putative Chinese Spring (CS)-Ae. longissima 4Sl(4B) disomic substitution line (TA3465) to develop 4Sl-specific markers to assist the transfer of a Bgt resistance gene from 4Sl by induced homoeologous recombination. A pairwise comparison of genes between CS and TA3465 demonstrated that a number of genes on chromosome 4BS in CS were not expressed in TA3465. Analysis of 4B- and 4Sl-specific molecular markers showed that 4BS and 4SlL were both missing in TA3465, whereas 4BL and 4SlS were present. Further characterization by genomic and fluorescent in situ hybridization confirmed that TA3465 carried a spontaneous Robertsonian T4SlS·4BL translocation. Powdery mildew tests showed that TA3465 was resistant to 10 of 16 Bgt isolates collected from different regions of China, whereas CS was susceptible to all those Bgt isolates. The powdery mildew resistance gene(s) in TA3465 was further mapped to the short arm of 4Sl and designated as Pm66.

Gene Duplication and Aneuploidy Trigger Rapid Evolution of Herbicide Resistance in Common Waterhemp.

An increase in gene copy number is often associated with changes in the number and structure of chromosomes, as has been widely observed in yeast and eukaryotic tumors, yet little is known about stress-induced chromosomal changes in plants. Previously, we reported that the EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene, the molecular target of glyphosate, was amplified at the native locus and on an extra chromosome in glyphosate-resistant Amaranthus tuberculatus Here, we report that the extra chromosome is a ring chromosome termed extra circular chromosome carrying amplified EPSPS (ECCAE). The ECCAE is heterochromatic, harbors four major EPSPS amplified foci, and is sexually transmitted to 35% of the progeny. Two highly glyphosate resistant (HGR) A. tuberculatus plants with a chromosome constitution of 2n = 32+1 ECCAE displayed soma cell heterogeneity. Some cells had secondary ECCAEs, which displayed size polymorphisms and produced novel chromosomal variants with multiple gene amplification foci. We hypothesize that the ECCAE in the soma cells of HGR A. tuberculatus plants underwent breakage-fusion-bridge cycles to generate the observed soma cell heterogeneity, including de novo EPSPS gene integration into chromosomes. Resistant soma cells with stable EPSPS amplification events as de novo insertions into chromosomes may survive glyphosate selection pressure during the sporophytic phase and are plausibly transmitted to germ cells leading to durable glyphosate resistance in A. tuberculatus This is the first report of early events in aneuploidy-triggered de novo chromosome integration by an as yet unknown mechanism, which may drive rapid adaptive evolution of herbicide resistance in common waterhemp.

Homoeologous recombination in the presence of Ph1 gene in wheat.

A crossover (CO) and its cytological signature, the chiasma, are major features of eukaryotic meiosis. The formation of at least one CO/chiasma between homologous chromosome pairs is essential for accurate chromosome segregation at the first meiotic division and genetic recombination. Polyploid organisms with multiple sets of homoeologous chromosomes have evolved additional mechanisms for the regulation of CO/chiasma. In hexaploid wheat (2n = 6× = 42), this is accomplished by pairing homoeologous (Ph) genes, with Ph1 having the strongest effect on suppressing homoeologous recombination and homoeologous COs. In this study, we observed homoeologous COs between chromosome 5Mg of Aegilops geniculata and 5D of wheat in plants where Ph1 was fully active, indicating that chromosome 5Mg harbors a homoeologous recombination promoter factor(s). Further cytogenetic analysis, with different 5Mg/5D recombinants, showed that the homoeologous recombination promoting factor(s) may be located in proximal regions of 5Mg. In addition, we observed a higher frequency of homoeologous COs in the pericentromeric region between chromosome combination of rec5Mg#2S·5Mg#2L and 5D compared to 5Mg#1/5D, which may be caused by a small terminal region of 5DL homology present in chromosome rec5Mg#2. The genetic stocks reported here will be useful for analyzing the mechanism of Ph1 action and the nature of homoeologous COs.

Physical Mapping of Amplified Copies of the 5-Enolpyruvylshikimate-3-Phosphate Synthase Gene in Glyphosate-Resistant Amaranthus tuberculatus.

Recent and rapid evolution of resistance to glyphosate, the most widely used herbicides, in several weed species, including common waterhemp (Amaranthus tuberculatus), poses a serious threat to sustained crop production. We report that glyphosate resistance in A tuberculatus was due to amplification of the 5-enolpyruvylshikimate-3-P synthase (EPSPS) gene, which encodes the molecular target of glyphosate. There was a positive correlation between EPSPS gene copies and its transcript expression. We analyzed the distribution of EPSPS copies in the genome of A tuberculatus using fluorescence in situ hybridization on mitotic metaphase chromosomes and interphase nuclei. Fluorescence in situ hybridization analysis mapped the EPSPS gene to pericentromeric regions of two homologous chromosomes in glyphosate sensitive A tuberculatus In glyphosate-resistant plants, a cluster of EPSPS genes on the pericentromeric region on one pair of homologous chromosomes was detected. Intriguingly, two highly glyphosate-resistant plants harbored an additional chromosome with several EPSPS copies besides the native chromosome pair with EPSPS copies. These results suggest that the initial event of EPSPS gene duplication may have occurred because of unequal recombination mediated by repetitive DNA. Subsequently, gene amplification may have resulted via several other mechanisms, such as chromosomal rearrangements, deletion/insertion, transposon-mediated dispersion, or possibly by interspecific hybridization. This report illustrates the physical mapping of amplified EPSPS copies in A tuberculatus.

Single molecule mtDNA fiber FISH for analyzing numtogenesis.

Somatic human cells contain thousands of copies of mitochondrial DNA (mtDNA). In eukaryotes, natural transfer of mtDNA into the nucleus generates nuclear mitochondrial DNA (NUMT) copies. We name this phenomenon as "numtogenesis". Numtogenesis is a well-established evolutionary process reported in various sequenced eukaryotic genomes. We have established a molecular tool to rapidly detect and analyze NUMT insertions in whole genomes. To date, NUMT analyses depend on deep genome sequencing combined with comprehensive computational analyses of the whole genome. This is time consuming, cumbersome and cost prohibitive. Further, most laboratories cannot accomplish such analyses due to limited skills. We report the development of single-molecule mtFIBER FISH (fluorescence in situ hybridization) to study numtogenesis. The development of mtFIBER FISH should aid in establishing a role for numtogenesis in cancers and other human diseases. This novel technique should help distinguish and monitor cancer stages and progression, aid in elucidation of basic mechanisms underlying tumorigenesis and facilitate analyses of processes related to early detection of cancer, screening and/or cancer risk assessment.

Recurrent establishment of de novo centromeres in the pericentromeric region of maize chromosome 3.

Centromeres can arise de novo from non-centromeric regions, which are often called "neocentromeres." Neocentromere formation provides the best evidence for the concept that centromere function is not determined by the underlying DNA sequences, but controlled by poorly understood epigenetic mechanisms. Numerous neocentromeres have been reported in several plant and animal species. However, it has been elusive how and why a specific chromosomal region is chosen to be a new centromere during the neocentromere activation events. We report recurrent establishment of neocentromeres in a pericentromeric region of chromosome 3 in maize (Zea mays). This latent region is located in the short arm and is only 2 Mb away from the centromere (Cen3) of chromosome 3. At least three independent neocentromere activation events, which were likely induced by different mechanisms, occurred within this latent region. We mapped the binding domains of CENH3, the centromere-specific H3 histone variant, of the three neocentromeres and analyzed the genomic and epigenomic features associated with Cen3, the de novo centromeres and an inactivated centromere derived from an ancestral chromosome. Our results indicate that lack of genes and transcription and a relatively high level of DNA methylation in this pericentromeric region may provide a favorable chromatin environment for neocentromere activation.

A whole-genome, radiation hybrid mapping resource of hexaploid wheat.

Generating a contiguous, ordered reference sequence of a complex genome such as hexaploid wheat (2n = 6x = 42; approximately 17 GB) is a challenging task due to its large, highly repetitive, and allopolyploid genome. In wheat, ordering of whole-genome or hierarchical shotgun sequencing contigs is primarily based on recombination and comparative genomics-based approaches. However, comparative genomics approaches are limited to syntenic inference and recombination is suppressed within the pericentromeric regions of wheat chromosomes, thus, precise ordering of physical maps and sequenced contigs across the whole-genome using these approaches is nearly impossible. We developed a whole-genome radiation hybrid (WGRH) resource and tested it by genotyping a set of 115 randomly selected lines on a high-density single nucleotide polymorphism (SNP) array. At the whole-genome level, 26 299 SNP markers were mapped on the RH panel and provided an average mapping resolution of approximately 248 Kb/cR1500 with a total map length of 6866 cR1500 . The 7296 unique mapping bins provided a five- to eight-fold higher resolution than genetic maps used in similar studies. Most strikingly, the RH map had uniform bin resolution across the entire chromosome(s), including pericentromeric regions. Our research provides a valuable and low-cost resource for anchoring and ordering sequenced BAC and next generation sequencing (NGS) contigs. The WGRH developed for reference wheat line Chinese Spring (CS-WGRH), will be useful for anchoring and ordering sequenced BAC and NGS based contigs for assembling a high-quality, reference sequence of hexaploid wheat. Additionally, this study provides an excellent model for developing similar resources for other polyploid species.

Homoeologous recombination-based transfer and molecular cytogenetic mapping of powdery mildew-resistant gene Pm57 from Aegilops searsii into wheat.

KEY MESSAGE: Pm57, a novel resistant gene against powdery mildew, was transferred into common wheat from Ae. searsi and further mapped to 2S s #1L at an interval of FL0.75 to FL0.87. Powdery mildew, caused by the fungus Blumeria graminis f. sp. tritici, is one of the most severe foliar diseases of wheat causing reduction in grain yield and quality. Host plant resistance is the most effective and environmentally safe approach to control this disease. Tests of a set of Chinese Spring-Ae. searsii (SsSs, 2n = 2x = 14) Feldman & Kislev ex K. Hammer disomic addition lines with a mixed isolate of the powdery mildew fungus identified a novel resistance gene(s), designed as Pm57, which was located on chromosome 2Ss#1. Here, we report the development of ten wheat-Ae. searsii recombinants. The wheat chromosomes involved in five of these recombinants were identified by FISH and SSR marker analysis and three of them were resistant to powdery mildew. Pm57 was further mapped to the long arm of chromosome 2Ss#1 at a fraction length interval of FL 0.75 to FL 0.87. The recombinant stocks T2BS.2BL-2Ss#1L 89-346 (TA5108) with distal 2Ss#1L segments of 28% and 89(5)69 (TA5109) with 33% may be useful in wheat improvement. The PCR marker X2L4g9p4/HaeIII was validated to specifically identify the Ae. searsii 2Ss#1L segment harboring Pm57 in T2BS.2BL-2Ss#1L against 16 wheat varieties and advanced breeding lines, and the development of more user-friendly KASP markers is underway.

Chromatin-associated transcripts of tandemly repetitive DNA sequences revealed by RNA-FISH.

Tandemly repetitive DNA sequences, also named satellite repeats, are major DNA components of heterochromatin and are often organized as long arrays in the pericentromeric, centromeric, and subtelomeric regions of eukaryotic chromosomes. An increasing amount of evidence indicates that transcripts derived from some satellite repeats play important roles in various biological functions. We used a RNA-fluorescence in situ hybridization (RNA-FISH) technique to investigate the transcription of the four well-characterized satellite repeats of maize (Zea mays), including the 180-bp knob repeat, the telomeric (TTTAGGG)n repeat, the 156-bp centromeric repeat CentC, and a 350-bp subtelomeric repeat. Although few transcripts derived from these four repeats were found in the expressed sequence tag and RNA-seq databases, RNA-FISH consistently detected the transcripts from three of the four repeats on interphase nuclei, suggesting that the transcripts from the three repeats are largely integrated into chromatin. The transcripts from the knob and telomeric repeats were mapped to the related DNA loci. In contrast, the transcripts from the CentC repeats were mainly localized to the nucleolus, although nucleoplasmic CentC transcripts were also detectable. The nucleolus and nuclear RNAs appeared to be important for the nuclear localization for at least one centromeric protein, Mis12. We demonstrate that RNA-FISH is a powerful tool to assess the level of transcription as well as to physically map the nuclear locations of the transcripts derived from satellite repeats.

Exploring the tertiary gene pool of bread wheat: sequence assembly and analysis of chromosome 5M(g) of Aegilops geniculata.

Next-generation sequencing (NGS) provides a powerful tool for the discovery of important genes and alleles in crop plants and their wild relatives. Despite great advances in NGS technologies, whole-genome shotgun sequencing is cost-prohibitive for species with complex genomes. An attractive option is to reduce genome complexity to a single chromosome prior to sequencing. This work describes a strategy for studying the genomes of distant wild relatives of wheat by isolating single chromosomes from addition or substitution lines, followed by chromosome sorting using flow cytometry and sequencing of chromosomal DNA by NGS technology. We flow-sorted chromosome 5M(g) from a wheat/Aegilops geniculata disomic substitution line [DS5M(g) (5D)] and sequenced it using an Illumina HiSeq 2000 system at approximately 50 × coverage. Paired-end sequences were assembled and used for structural and functional annotation. A total of 4236 genes were annotated on 5M(g) , in close agreement with the predicted number of genes on wheat chromosome 5D (4286). Single-gene FISH indicated no major chromosomal rearrangements between chromosomes 5M(g) and 5D. Comparing chromosome 5M(g) with model grass genomes identified synteny blocks in Brachypodium distachyon, rice (Oryza sativa), sorghum (Sorghum bicolor) and barley (Hordeum vulgare). Chromosome 5M(g) -specific SNPs and cytogenetic probe-based resources were developed and validated. Deletion bin-mapped and ordered 5M(g) SNP markers will be useful to track 5M-specific introgressions and translocations. This study provides a detailed sequence-based analysis of the composition of a chromosome from a distant wild relative of bread wheat, and opens up opportunities to develop genomic resources for wild germplasm to facilitate crop improvement.

Genome wide characterization of simple sequence repeats in watermelon genome and their application in comparative mapping and genetic diversity analysis.

BACKGROUND: Microsatellite markers are one of the most informative and versatile DNA-based markers used in plant genetic research, but their development has traditionally been difficult and costly. The whole genome sequencing with next-generation sequencing (NGS) technologies provides large amounts of sequence data to develop numerous microsatellite markers at whole genome scale. SSR markers have great advantage in cross-species comparisons and allow investigation of karyotype and genome evolution through highly efficient computation approaches such as in silico PCR. Here we described genome wide development and characterization of SSR markers in the watermelon (Citrullus lanatus) genome, which were then use in comparative analysis with two other important crop species in the Cucurbitaceae family: cucumber (Cucumis sativus L.) and melon (Cucumis melo L.). We further applied these markers in evaluating the genetic diversity and population structure in watermelon germplasm collections. RESULTS: A total of 39,523 microsatellite loci were identified from the watermelon draft genome with an overall density of 111 SSRs/Mbp, and 32,869 SSR primers were designed with suitable flanking sequences. The dinucleotide SSRs were the most common type representing 34.09 % of the total SSR loci and the AT-rich motifs were the most abundant in all nucleotide repeat types. In silico PCR analysis identified 832 and 925 SSR markers with each having a single amplicon in the cucumber and melon draft genome, respectively. Comparative analysis with these cross-species SSR markers revealed complicated mosaic patterns of syntenic blocks among the genomes of three species. In addition, genetic diversity analysis of 134 watermelon accessions with 32 highly informative SSR loci placed these lines into two groups with all accessions of C.lanatus var. citorides and three accessions of C. colocynthis clustered in one group and all accessions of C. lanatus var. lanatus and the remaining accessions of C. colocynthis clustered in another group. Furthermore, structure analysis was consistent with the dendrogram indicating the 134 watermelon accessions were classified into two populations. CONCLUSION: The large number of genome wide SSR markers developed herein from the watermelon genome provides a valuable resource for genetic map construction, QTL exploration, map-based gene cloning and marker-assisted selection in watermelon which has a very narrow genetic base and extremely low polymorphism among cultivated lines. Furthermore, the cross-species transferable SSR markers identified herein should also have practical uses in many applications in species of Cucurbitaceae family whose whole genome sequences are not yet available.

Molecular Cytogenetic Mapping of Satellite DNA Sequences in Aegilops geniculata and Wheat.

Fluorescence in situ hybridization (FISH) provides an efficient system for cytogenetic analysis of wild relatives of wheat for individual chromosome identification, elucidation of homoeologous relationships, and for monitoring alien gene transfers into wheat. This study is aimed at developing cytogenetic markers for chromosome identification of wheat and Aegilops geniculata (2n = 4x = 28, UgUgMgMg) using satellite DNAs obtained from flow-sorted chromosome 5Mg. FISH was performed to localize the satellite DNAs on chromosomes of wheat and selected Aegilops species. The FISH signals for satellite DNAs on chromosome 5Mg were generally associated with constitutive heterochromatin regions corresponding to C-band-positive chromatin including telomeric, pericentromeric, centromeric, and interstitial regions of all the 14 chromosome pairs of Ae. geniculata. Most satellite DNAs also generated FISH signals on wheat chromosomes and provided diagnostic chromosome arm-specific cytogenetic markers that significantly improved chromosome identification in wheat. The newly identified satellite DNA CL36 produced localized Mg genome chromosome-specific FISH signals in Ae. geniculata and in the M genome of the putative diploid donor species Ae. comosa subsp. subventricosa but not in Ae. comosa subsp. comosa, suggesting that the Mg genome of Ae. geniculata was probably derived from subsp. subventricosa.