Genome sequence of Hydrangea macrophylla and its application in analysis of the double flower phenotype.

One of the well-known floral abnormalities in flowering plants is the double-flower phenotype, which corresponds to flowers that develop extra petals, sometimes even containing entire flowers within flowers. Because of their highly priced ornamental value, spontaneous double-flower variants have been found and selected for in a wide range of ornamental species. Previously, double flower formation in roses was associated with a restriction of AGAMOUS expression domain toward the centre of the meristem, leading to extra petals. Here, we characterized the genomic region containing the mutation associated with the switch from simple to double flowers in the rose. An APETALA2-like gene (RcAP2L), a member of the Target Of EAT-type (TOE-type) subfamily, lies within this interval. In the double flower rose, two alleles of RcAP2L are present, one of which harbours a transposable element inserted into intron 8. This insertion leads to the creation of a miR172 resistant RcAP2L variant. Analyses of the presence of this variant in a set of simple and double flower roses demonstrate a correlation between the presence of this allele and the double flower phenotype. These data suggest a role of this miR172 resistant RcAP2L variant in regulating RcAGAMOUS expression and double flower formation in Rosa sp.

High-density linkage maps are vital to supporting the correct placement of scaffolds and gene sequences on chromosomes and fundamental to contemporary organismal research and scientific approaches to genetic improvement, especially in paleopolyploids with exceptionally complex genomes, eg, upland cotton ( Gossypium hirsutum L., “2n = 52”). Three independently developed intraspecific upland mapping populations were analyzed to generate 3 high-density genetic linkage single-nucleotide polymorphism (SNP) maps and a consensus map using the CottonSNP63K array. The populations consisted of a previously reported F2, a recombinant inbred line (RIL), and reciprocal RIL population, from “Phytogen 72” and “Stoneville 474” cultivars. The cluster file provided 7417 genotyped SNP markers, resulting in 26 linkage groups corresponding to the 26 chromosomes (c) of the allotetraploid upland cotton (AD)1 arisen from the merging of 2 genomes (“A” Old World and “D” New World). Patterns of chromosome-specific recombination were largely consistent across mapping populations. The high-density genetic consensus map included 7244 SNP markers that spanned 3538 cM and comprised 3824 SNP bins, of which 1783 and 2041 were in the At and Dt subgenomes with 1825 and 1713 cM map lengths, respectively. Subgenome average distances were nearly identical, indicating that subgenomic differences in bin number arose due to the high numbers of SNPs on the Dt subgenome. Examination of expected recombination frequency or crossovers (COs) on the chromosomes within each population of the 2 subgenomes revealed that COs were also not affected by the SNPs or SNP bin number in these subgenomes. Comparative alignment analyses identified historical ancestral At-subgenomic translocations of c02 and c03, as well as of c04 and c05. The consensus map SNP sequences aligned with high congruency to the NBI assembly of Gossypium hirsutum . However, the genomic comparisons revealed evidence of additional unconfirmed possible duplications, inversions and translocations, and unbalance SNP sequence homology or SNP sequence/loci genomic dominance, or homeolog loci bias of the upland tetraploid At and Dt subgenomes. The alignments indicated that 364 SNP-associated previously unintegrated scaffolds can be placed in pseudochromosomes of the NBI G hirsutum assembly. This is the first intraspecific SNP genetic linkage consensus map assembled in G hirsutum with a core of reproducible mendelian SNP markers assayed on different populations and it provides further knowledge of chromosome arrangement of genic and nongenic SNPs. Together, the consensus map and RIL populations provide a synergistically useful platform for localizing and identifying agronomically important loci for improvement of the cotton crop.

New Technologies, Tools and Approaches for Improving Crop Breeding

Identification of stable QTLs for seed oil content by combined linkage and association mapping in Brassica napus

At the First (1996) and Second (1997) International Strategy Meetings on Human DNA Sequencing, the attendees discussed and strongly endorsed the concept that human genomic DNA sequence produced by large-scale DNA sequencing centers funded by the Human Genome Project should release data as rapidly as possible. In the case of unfinished data, it was suggested that sequence assemblies of 1–2 kb in size should be released within 24 hr of generation. Finished data should be submitted to the public sequence databases on a similarly rapid time scale. Most of the funding agencies engaged in supporting the Human Genome Project have adopted policies that reflect the importance of rapid dissemination of genomic sequence data. Similarly the nucleotide sequence databases have made provision for the acceptance of the unfinished data as well as, of course, finished sequence data. At the 1998 meeting, the question was raised about similar data dissemination practices for genomic sequence data from other organisms. The attendees unanimously agreed that this was also critical and adopted the following statement as individual scientists. ‘‘As extensive determination of the genomic DNA sequence of several organisms proceeds, it is increasingly clear that sequence information has enormous and immediate scientific value, even prior to its final assembly and completion. Delaying the release of either unfinished or finished genomic DNA sequence data serves no useful purpose and actually has the effect of slowing the progress of research. Therefore, the attendees at the Third International Strategy Meeting on Human Genome Sequencing (Bermuda, Feb. 27–28, 1998) agreed unanimously to support, as individual scientists, the view that all publicly funded large-scale DNA sequencing projects, regardless of the organism, should deposit data immediately into the public domain, following the same guidelines that have previously been adopted by this group for human genomic sequence [see D.R. Bentley, Science 274: 533–534 (1996); http:// hugo.gdb.org/bermuda.htm]. The scientists attending this meeting will continue to adhere to these principles and urge all other scientists and policymaking groups involved in large-scale sequencing to adopt them as well.’’ This statement does not yet reflect, nor is it binding on, the policy of any funding agency. The attendees at the Third International Strategy Meeting on Human Genome Sequencing were Mark Adams, David Bentley, Bruce Birren, Martin Bobrow, Elbert Branscomb, Graham Cameron, Anthony V. Carrano, Ellson Chen, Francis Collins, David Cox, Mike Davies, Pieter de Jong, Richard Durbin, Glen Evans, Asao Fujiyama, Richard Gibbs, Eric Green, Phil Green, Mark Guyer, LaDeana Hillier, Ursula Hurtenbach, Elke Jordan, Jill Kent, Fumihiko Kikuchi, Steven Koonin, Jane Lamerdin, Eric Lander, Chris Martin, Richard McCombie, John McPherson, Michael Morgan, Richard Myers, James Ostell, Ari Patrinos, Jane Peterson, Bruce Roe, Jane Rogers, Andre Rosenthal, Yoshiyuki Sakaki, Barbara Skene, Edwin Southern, Jilly Steward, John Sulston, Mark Vaudin, Jeffrey Wadsworth, Robert Waterson, James Watson, Jean Weissenbach, Richard Wilson, and MarieLaure Yaspo.

Seed quality traits (especially hardseededness and seed size) and resistance to disease (in particular powdery mildew caused by Erysiphe polygoni DC and charcoal rot caused by Macrophomina phaseolina (Tassi) Goid.) are key factors for the success and expansion of the mungbean (Vigna radiata (L.) Wilczek) industry in Australia. Understanding the inheritance of these characters will undoubtedly increase the efficiency of mungbean breeding programs and increase the potential to produce more desirable cultivars for both the local and overseas markets. Map based QTL analysis has recently become a powerful tool in the identification and manipulation of genes underlying important traits where little is known about them. With this in mind, two recombinant inbred (RI) populations segregating for seed characters and powdery mildew resistance were produced and regions of the mungbean genome conditioning these important traits were identified using QTL analysis. A genetic linkage map of mungbean consisting of 255 RFLP loci was developed using a RI population of 80 individuals. The population was derived from an inter-subspecific cross between the large, softseeded cultivar Berken (V. radiata subsp. radiata) and the completely hardseeded wild accession, ACC 41 (V. radiata subsp. Sublobata). The map, which comprised 13 linkage groups, spanned 738 cM with an average distance between markers of 3.0 cM and a maximum distance between linked markers of 15.4 cM. This map was compared to a previously published map of lablab (Lablab purpureus L.) using a common set of 65 RFLP probes. In contrast to some other comparative mapping studies among members of the Fabaceae, where a high level of chromosomal rearrangement has been observed, marker order between mungbean and lablab was found to be highly conserved. However, there was evidence that the two genomes had accumulated a large number of duplications/deletions after they diverged. To analyse seed characters, a framework set of 64 of the 255 RFLP clones and one morphological marker (leaf lobing) were selected to construct a linkage map using a population of 227 RI individuals from the same inter-subspecific cross. These markers were chosen to be located approximately 15 cM apart and/or at the distal regions of existing linkage groups. Using this population, a linkage map consisting of 79 loci was generated over 13 linkage groups, spanning a total distance of 685 cMwith an average distance between markers of 10.4 cM and a maximum distance of 20.7 cM. QTL analysis revealed four regions associated with hardseededness, with individual loci explaining between 4.9% and 23.2% of the variation. For seed size, 11 regions were identified, with individual loci explaining between 4.2% and 18.8% of the variation. Two of the loci involved in hardseededness co-located with those conditioning seed size, indicating that there may be a physiological effect of seed size on hardseededness in this population. A major locus that conferred resistance to powdery mildew in mungbean was also identified using a second population of 147 RI individuals derived from an intraspecific cross between Berken and the introduction line ATF 3640. Berken was susceptible and ATF 3640 highly resistant to the isolates of powdery mildew used in this study. To generate a linkage map for this population, 51 RFLP clones identified as being polymorphic from parental screening for the Berken x ACC 41 linkage map were selected to screen the Berken x ATF 3640 population. The 51 probes generated 52 mapped loci, which were used to construct a linkage map spanning 350 cM of the mungbean genome over 10 linkage groups. Using these markers, a major locus was identified that explained up to 88.3% of the total variation in the resistance response to the pathogen. To demonstrate the feasibility of developing user-friendly markers linked to important traits in mungbean, a marker closely linked to the powdery mildew resistance locus was then used to screen a BAC (bacterial artificial chromosome) library. Subclones of positive BAC clones were probed with oligonucleotides containing both AT and ATT repeats to identify those that contained putative microsatellites. Sequence analysis of positive subclones identified two unique sequences containing (AT)n repeats, one unique sequence containing an (ATT)n repeat and one unique sequence containing a compound repeat. A polymorphic SSR (simple sequence repeat) marker closely linked to the major locus conditioning powdery mildew resistance was generated from one of these. This marker could be used in breeding programs aimed at generating powdery mildew resistant cultivars. This approach could be used to develop more user-friendly markers for loci conditioning other important agronomic traits in mungbean.

BackgroundAdenoviruses are common pathogens infecting animals and humans. They are classified based on serology, or genome sequence information. These methods have limitations due to lengthy procedures or lack of infectivity data. Adenoviruses are easy to produce and amenable to genetic and biochemical modifications, which makes them a powerful tool for biological studies, and clinical gene-delivery and vaccine applications. Antibodies directed against adenoviral proteins are important diagnostic tools for virus identification in vivo and in vitro, and are used to elucidate infection mechanisms, often in combination with genomic sequencing and type specific information from hyper-variable regions of structural proteins.ResultsHere we describe a novel and readily useable method for cloning, expressing and purifying small fragments of hyper-variable regions 1-6 of the adenoviral hexon protein. We used these polypeptides as antigens for generating polyclonal rabbit antibodies against human adenovirus 3 (HAdV-B3), mouse adenovirus 1 (MAdV-1) and MAdV-2 hexon. In Western immunoblots with lysates from cells infected from thirteen human and three mouse viruses, these antibodies bound to homologous full-length hexon protein and revealed variable levels of cross-reactivity to heterologous hexons. Results from immuno-fluorescence and electron microscopy studies indicated that HAdV-B3 and MAdV-2 hexon antibodies recognized native forms of hexon.ConclusionsThe procedure described here can in principle be applied to any adenovirus for which genome sequence information is available. It provides a basis for generating novel type-specific tools in diagnostics and research, and extends beyond the commonly used anti-viral antibodies raised against purified viruses or subviral components.

Lisianthus (Eustoma grandiflorum) is an important floricultural crop cultivated worldwide. Despite its commercial importance, few DNA markers are available for molecular genetic research. In this study, we constructed a genetic linkage map and to detect quantitative trait loci (QTLs) for important agronomic traits of lisianthus. To develop simple sequence repeat (SSR) markers, we used 454-pyrosequencing technology to obtain genomic shotgun sequences and subsequently identified 8263 putative SSRs. A total of 3990 primer pairs were designed in silico and 1189 unique primer pairs were extracted through a BLAST search. Amplification was successful for more than 1000 primer pairs, and ultimately 278 SSR markers exhibited polymorphism between the two lisianthus accessions evaluated. Based on these markers, a genetic linkage map was constructed using a breeding population derived from crosses between the two accessions, for which flowering time differed (>140 days when grown under 20°C). We detected one QTL associated with flowering time (phenotypic variance, 27%; LOD value, 3.7). The SSR marker located at this QTL may account for variation in flowering time among accessions (i.e., three accessions whose nodes of the first flower were over 30 had late-flowering alleles of this QTL).

Double flowers are one of the important objectives of ornamental plant breeding. Sagittaria sagittifolia is an aquatic herb in the Alismataceae family that is widely used as an ornamental plant in gardens. However, the reference genome has not been published, and the molecular regulatory mechanism of flower formation remains unclear. In this study, single molecule real-time (SMRT) sequencing technology combined with Illumina RNA-Seq was used to perform a more comprehensive analysis of S. sagittifolia for the first time. We obtained high-quality full-length transcripts, including 53,422 complete open reading frames, and identified 5980 transcription factors that belonged to 67 families, with many MADS-box genes involved in flower formation being obtained. The transcription factors regulated by plant hormone signals played an important role in the development of double flowers. We also identified an AP2 orthologous gene, SsAP2, with a deletion of the binding site for miR172, that overexpressed SsAP2 in S. sagittifolia and exhibited a delayed flowering time and an increased number of petals. This study is the first report of a full-length transcriptome of S. sagittifolia. These reference transcripts will be valuable resources for the analysis of gene structures and sequences, which provide a theoretical basis for the molecular regulatory mechanism governing the formation of double flowers.

Single or double flower type is one of the most important breeding targets in carnation (Dianthus caryophyllus L.). We mapped the D 85 locus, which controls flower type, to LG 85P_15–2 using a simple sequence repeat (SSR)-based genetic linkage map constructed using 91 F2 progeny derived from a cross between line 85–11 (double flower) and ‘Pretty Favvare’ (single flower). A positional comparison using SSR markers as anchor loci revealed that the map positions of the D 85 locus corresponded to the single locus controlling the single flower type derived from wild D. capitatus ssp. andrzejowskianus. We identified four co-segregating SSR markers on the D 85 locus. Verification of the SSR markers in commercial cultivars revealed that two of the four SSR markers (CES0212 and CES1982) were tightly linked to the D 85 locus, and amplified a 176-bp and 269-bp allele, respectively, which were common and unique to double flower cultivars. The map positions of the D 85 locus and the tightly linked SSR markers will be useful for determining the genetic basis of flower type and for marker-assisted breeding of carnations.

With advances in sequencing technology, a vast amount of genomic sequence information has become available. However, annotating biological functions particularly of non-protein-coding regions in genome sequences without experiments is still a challenging task. Recently deep learning-based methods were shown to have the ability to predict gene regulatory regions from genome sequences, promising to aid the interpretation of genomic sequence data. Here, we report an improvement of the prediction accuracy for gene regulatory regions by using the design of convolution layers that efficiently process genomic sequence information, and developed a software, DeepGMAP, to train and compare different deep learning-based models (https://github.com/koonimaru/DeepGMAP). First, we demonstrate that our convolution layers, termed forward- and reverse-sequence scan (FRSS) layers, integrate both forward and reverse strand information, and enhance the power to predict gene regulatory regions. Second, we assessed previous studies and identified problems associated with data structures that caused overfitting. Finally, we introduce visualization methods to examine what the program learned. Together, our FRSS layers improve the prediction accuracy for gene regulatory regions.

Most of the current QTL mapping methods are developed for the backcross and F2 populations. The inter-crossed F3 population is also popular and has been well devised in genetic study. As the F3 population can provide more recombinants than the backcross and F2 populations, it can facilitate the detection of closely linked QTL. In this paper, we propose a statistical model to estimate the effects and positions of QTL for the F3 population by considering the differences in the genome structure between the F3 and F2 populations. As the genotypes of QTL are usually unknown, the statistical model is a finite mixture model. By treating the mixture model as an incomplete data problem, the EM algorithm is implemented to obtain the maximum likelihood estimates of the parameters. Simulations were used to illustrate the performance of the proposed method and evaluate the relative efficiency in the F3 population (as comparing to that of the F2 population) in QTL mapping under several factors, such as sample size, genetic distance between QTL (and markers), the proportion of trait variance contributed by QTL (i.e. heritability). It is found that the QTL mapping by using the F3 population is more powerful and precise in separating closely linked QTL and in estimating the parameters as compare to the use of the F2 population, especially for small sample size, close genetic distance and low heritability. The paper can help outline a strategy of detecting tightly linked QTL for the advanced intercross populations to improve the resolution of genetic architecture of quantitative traits.

Whole genome sequence (WGS) information may soon be widely available to help clinicians personalize the care and treatment of patients. However, considerable barriers exist, which may hinder the effective utilization of WGS information in a routine clinical care setting. Clinical decision support (CDS) offers a potential solution to overcome such barriers and to facilitate the effective use of WGS information in the clinic. However, genomic information is complex and will require significant considerations when developing CDS capabilities. As such, this manuscript lays out a conceptual framework for a CDS architecture designed to deliver WGS-guided CDS within the clinical workflow. To handle the complexity and breadth of WGS information, the proposed CDS framework leverages service-oriented capabilities and orchestrates the interaction of several independently-managed components. These independently-managed components include the genome variant knowledge base, the genome database, the CDS knowledge base, a CDS controller and the electronic health record (EHR). A key design feature is that genome data can be stored separately from the EHR. This paper describes in detail: (1) each component of the architecture; (2) the interaction of the components; and (3) how the architecture attempts to overcome the challenges associated with WGS information. We believe that service-oriented CDS capabilities will be essential to using WGS information for personalized medicine.

Although many genetic factors have been successfully identified for human diseases in genome-wide association studies, genes discovered to date only account for a small proportion of overall genetic contributions to many complex traits. Association studies have difficulty in detecting the remaining true genetic variants that are either common variants with weak allelic effects, or rare variants that have strong allelic effects but are weakly associated at the population level. In this work, we applied a goodness-of-fit test for detecting sets of common and rare variants associated with quantitative or binary traits by using whole genome sequencing data. This test has been proved optimal for detecting weak and sparse signals in the literature, which fits the requirements for targeting the genetic components of missing heritability. Furthermore, this p value-combining method allows one to incorporate different data and/or research results for meta-analysis. The method was used to simultaneously analyse the whole genome sequencing and genome-wide association studies data of Genetic Analysis Workshop 18 for detecting true genetic variants. The results show that goodness-of-fit test is comparable or better than the influential sequence kernel association test in many cases.

High density genetic linkage maps that are extensively anchored to assembled genome sequences of the organism in question are extremely useful in gene discovery. To facilitate this process in perennial ryegrass (Lolium perenne L.), a high density single nucleotide polymorphism (SNP)- and presence/absence variant (PAV)-based genetic linkage map has been developed in an F2 mapping population that has been used as a reference population in numerous studies. To provide a reference sequence to which to align genotyping by sequencing (GBS) reads, a shotgun assembly of one of the grandparents of the population, a tenth-generation inbred line, was created using Illumina-based sequencing. The assembly was based on paired-end Illumina reads, scaffolded by mate pair and long jumping distance reads in the range of 3-40 kb, with >200-fold initial genome coverage. A total of 169 individuals from an F2 mapping population were used to construct PstI-based GBS libraries tagged with unique 4-9 nucleotide barcodes, resulting in 284 million reads, with approx. 1·6 million reads per individual. A bioinformatics pipeline was employed to identify both SNPs and PAVs. A core genetic map was generated using high confidence SNPs, to which lower confidence SNPs and PAVs were subsequently fitted in a straightforward binning approach. The assembly comprises 424 750 scaffolds, covering 1·11 Gbp of the 2·5 Gbp perennial ryegrass genome, with a scaffold N50 of 25 212 bp and a contig N50 of 3790 bp. It is available for download, and access to a genome browser has been provided. Comparison of the assembly with available transcript and gene model data sets for perennial ryegrass indicates that approx. 570 Mbp of the gene-rich portion of the genome has been captured. An ultra-high density genetic linkage map with 3092 SNPs and 7260 PAVs was developed, anchoring just over 200 Mb of the reference assembly. The combined genetic map and assembly, combined with another recently released genome assembly, represent a significant resource for the perennial ryegrass genetics community.