MotivationPolyploid species carry more than two copies of each chromosome, a condition found in many of the world’s most important crops. Genetic mapping in polyploids is more complex than in diploid species, resulting in a lack of available software tools. These are needed if we are to realize all the opportunities offered by modern genotyping platforms for genetic research and breeding in polyploid crops.ResultspolymapR is an R package for genetic linkage analysis and integrated genetic map construction from bi-parental populations of outcrossing autopolyploids. It can currently analyse triploid, tetraploid and hexaploid marker datasets and is applicable to various crops including potato, leek, alfalfa, blueberry, chrysanthemum, sweet potato or kiwifruit. It can detect, estimate and correct for preferential chromosome pairing, and has been tested on high-density marker datasets from potato, rose and chrysanthemum, generating high-density integrated linkage maps in all of these crops.Availability and implementationpolymapR is freely available under the general public license from the Comprehensive R Archive Network (CRAN) at http://cran.r-project.org/package=polymapR.Supplementary information Supplementary data are available at Bioinformatics online.

BackgroundBulbous flowers such as lily and tulip (Liliaceae family) are monocot perennial herbs that are economically very important ornamental plants worldwide. However, there are hardly any genetic studies performed and genomic resources are lacking. To build genomic resources and develop tools to speed up the breeding in both crops, next generation sequencing was implemented. We sequenced and assembled transcriptomes of four lily and five tulip genotypes using 454 pyro-sequencing technology.ResultsSuccessfully, we developed the first set of 81,791 contigs with an average length of 514 bp for tulip, and enriched the very limited number of 3,329 available ESTs (Expressed Sequence Tags) for lily with 52,172 contigs with an average length of 555 bp. The contigs together with singletons covered on average 37% of lily and 39% of tulip estimated transcriptome. Mining lily and tulip sequence data for SSRs (Simple Sequence Repeats) showed that di-nucleotide repeats were twice more abundant in UTRs (UnTranslated Regions) compared to coding regions, while tri-nucleotide repeats were equally spread over coding and UTR regions. Two sets of single nucleotide polymorphism (SNP) markers suitable for high throughput genotyping were developed. In the first set, no SNPs flanking the target SNP (50 bp on either side) were allowed. In the second set, one SNP in the flanking regions was allowed, which resulted in a 2 to 3 fold increase in SNP marker numbers compared with the first set. Orthologous groups between the two flower bulbs: lily and tulip (12,017 groups) and among the three monocot species: lily, tulip, and rice (6,900 groups) were determined using OrthoMCL. Orthologous groups were screened for common SNP markers and EST-SSRs to study synteny between lily and tulip, which resulted in 113 common SNP markers and 292 common EST-SSR. Lily and tulip contigs generated were annotated and described according to Gene Ontology terminology.ConclusionsTwo transcriptome sets were built that are valuable resources for marker development, comparative genomic studies and candidate gene approaches. Next generation sequencing of leaf transcriptome is very effective; however, deeper sequencing and using more tissues and stages is advisable for extended comparative studies.

Construction of genetic linkage maps for lily was achieved using two populations, LA and AA that share one parent ÔConnecticut KingÕ. Three different molecular marker systems (AFLP TM , DArT and NBS profiling) were used in generating linkage maps for ÔConnecticut KingÕ. The LA and the AA populations consist of 20 and 21 linkage groups (LGs), respectively. Average density between markers was 3.9 cM for the LA and 5 cM for the AA population. Several horticultural traits were mapped for the first time in Lilium and showed to be single gene based. We propose to name these genes as LFCc for flower colour, lfs for flower spots, LSC for stem colour, lal for antherless phenotype and lfd for flower direction whereby upper and lower case names refer to dominant and recessive genes, respectively. Additionally, resistance to Lily mottle virus (LMoV) was mapped as a locus on LG AA10. For Fusarium resistance, the Kruskal-Wallis test identified six putative quantitative trait loci (QTL) in the AA population of which one QTL (explaining 25% of the variation in resistance) could be confirmed by interval mapping.

BackgroundWithin onion, Allium cepa L., the availability of disease resistance is limited. The identification of sources of resistance in related species, such as Allium roylei and Allium fistulosum, was a first step towards the improvement of onion cultivars by breeding. SNP markers linked to resistance and polymorphic between these related species and onion cultivars are a valuable tool to efficiently introgress disease resistance genes. In this paper we describe the identification and validation of SNP markers valuable for onion breeding.ResultsTranscriptome sequencing resulted in 192 million RNA seq reads from the interspecific F1 hybrid between A. roylei and A. fistulosum (RF) and nine onion cultivars. After assembly, reliable SNPs were discovered in about 36 % of the contigs. For genotyping of the interspecific three-way cross population, derived from a cross between an onion cultivar and the RF (CCxRF), 1100 SNPs that are polymorphic in RF and monomorphic in the onion cultivars (RF SNPs) were selected for the development of KASP assays. A molecular linkage map based on 667 RF-SNP markers was constructed for CCxRF. In addition, KASP assays were developed for 1600 onion-SNPs (SNPs polymorphic among onion cultivars). A second linkage map was constructed for an F2 of onion x A. roylei (F2(CxR)) that consisted of 182 onion-SNPs and 119 RF-SNPs, and 76 previously mapped markers. Markers co-segregating in both the F2(CxR) and the CCxRF population were used to assign the linkage groups of RF to onion chromosomes. To validate usefulness of these SNP markers, QTL mapping was applied in the CCxRF population that segregates for resistance to Botrytis squamosa and resulted in a QTL for resistance on chromosome 6 of A. roylei.ConclusionsOur research has more than doubled the publicly available marker sequences of expressed onion genes and two onion-related species. It resulted in a detailed genetic map for the interspecific CCxRF population. This is the first paper that reports the detection of a QTL for resistance to B. squamosa in A. roylei.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0879-0) contains supplementary material, which is available to authorized users.

Gerbera hybrida is an economically important cut flower. In the production and transportation of gerbera with unavoidable periods of high relative humidity, grey mould occurs and results in losses in quality and quantity of flowers. Considering the limitations of chemical use in greenhouses and the impossibility to use these chemicals in auction or after sale, breeding for resistant gerbera cultivars is considered as the best practical approach. In this study, we developed two segregating F1 populations (called S and F). Four parental linkage maps were constructed using common and parental specific SNP markers developed from expressed sequence tag sequencing. Parental genetic maps, containing 30, 29, 27 and 28 linkage groups and a consensus map covering 24 of the 25 expected chromosomes, could be constructed. After evaluation of Botrytis disease severity using three different tests, whole inflorescence, bottom (of disc florets) and ray floret, quantitative trait locus (QTL) mapping was performed using the four individual parental maps. A total of 20 QTLs (including one identical QTL for whole inflorescence and bottom tests) were identified in the parental maps of the two populations. The number of QTLs found and the explained variance of most QTLs detected reflect the complex mechanism of Botrytis disease response.Electronic supplementary materialThe online version of this article (doi:10.1007/s11032-016-0617-1) contains supplementary material, which is available to authorized users.

BackgroundSNP (Single Nucleotide Polymorphism) markers are rapidly becoming the markers of choice for applications in breeding because of next generation sequencing technology developments. For SNP development by NGS technologies, correct assembly of the huge amounts of sequence data generated is essential. Little is known about assembler's performance, especially when dealing with highly heterogeneous species that show a high genome complexity and what the possible consequences are of differences in assemblies on SNP retrieval. This study tested two assemblers (CAP3 and CLC) on 454 data from four lily genotypes and compared results with respect to SNP retrieval.ResultsCAP3 assembly resulted in higher numbers of contigs, lower numbers of reads per contig, and shorter average read lengths compared to CLC. Blast comparisons showed that CAP3 contigs were highly redundant. Contrastingly, CLC in rare cases combined paralogs in one contig. Redundant and chimeric contigs may lead to erroneous SNPs. Filtering for redundancy can be done by blasting selected SNP markers to the contigs and discarding all the SNP markers that show more than one blast hit. Results on chimeric contigs showed that only four out of 2,421 SNP markers were selected from chimeric contigs.ConclusionIn practice, CLC performs better in assembling highly heterogeneous genome sequences compared to CAP3, and consequently SNP retrieval is more efficient. Additionally a simple flow scheme is suggested for SNP marker retrieval that can be valid for all non-model species.

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In this paper we present an overview of current developments in sequencing that offer the possibility to generate large numbers of markers in ornamental crops. The prospects of this new sequence technology for the application of markers in breeding of outcrossing and/or polyploid crops are discussed using examples in rose and lily. MOLECULAR MARKERS IN BREEDINGMarker development in ornamentals has been lagging behind compared to marker development in large agricultural and horticultural crops. This is partly due to the fact that there are many different ornamental crops. As markers need to be developed for each of these crops separately, the costs related to development and the time needed to develop them were obstacles. In addition, morphological characteristics are important during ornamental breeding of new cultivars, and these can be assessed without the use of markers. This is slowly changing, as it becomes more important to develop cultivars that have growth characteristics suitable for specific environments, and breeding also has to focus on traits that are difficult to assess and/or are controlled by multiple loci (quantitative traits), including stem production, time to flowering, flower size, and disease resistances. For instance, Fusarium resistance in lily is controlled by six putative QTLs (Shahin et al., 2010). Markers for each of these QTLs would make it possible to select progeny plants that have inherited the combination of these six loci. As this can already be done in a seedling stage, this would speed up the breeding process, especially in bulbous ornamentals, which have a long juvenile phase (3 years in lily, 5 years in tulip). In the case of introgression of disease resistances from wild relatives, markers can also be used to assist selection against wild germplasm unlinked to the desired QTLs. PROGRESS IN SEQUENCINGIn the past five years, the emergence of massively parallel sequencing technologies has dramatically reduced time and costs for sequencing. These developments will continue and sequencing will become cheaper while fragment lengths will increase. Importantly, parallel sequencing can be done for many targets, or even on the complete genomic DNA, without prior knowledge of the DNA. Hence it now becomes feasible to generate large amounts of DNA sequence information for species for which little prior sequence information exists, and to mine these sequences for polymorphisms that form the basis of the development of molecular markers. These molecular markers can be applied for marker-assisted breeding (MAB) and identification of cultivars and hybridization events. It offers great opportunities for ornamental crops, for which few molecular markers are currently available.