Towards marker assisted breeding in garden roses: from marker development to QTL detection

Over the last few decades the rose market in Eastern Europe showed a steady growth, which indicates that there is increasing demand for new cultivars that are adapted to the climate as well as to the customs and beauty criterion of that region. One of the possibilities to speed up breeding is to implement marker assisted selection (MAS). Implementation of MAS requires a specific infrastructure (molecular markers, knowledge on genetics of important traits, genetic maps) which is not yet available for tetraploid roses. In this thesis I developed some of the prerequisites for MAS in roses and discuss when and how MAS could have a positive effect on accelerating breeding and/or reducing the costs of the breeding process. The first step in understanding the structure of the genepool of garden roses was to evaluate the relatedness among available cultivars. For the first time genetic diversity among modern garden rose cultivars was evaluated (Chapter 2) using a set of 24 microsatellite markers covering most chromosomes. A total of 518 different alleles were obtained in a set of 138 rose cultivars. Genetic differentiation among types of garden roses (Fst=0.022) was four times that found among cut roses, and similar in magnitude to the differentiation among breeders, due to the fact that horticultural groups and breeders overlap largely in classification. In terms of genetic diversity cut roses can be considered as a subgroup of the garden roses. Winter hardy Canadian garden rose cultivars (Explorer roses) showed the least similarities to European roses, and introgression from wild species for winter hardiness was clearly visible. Roses of two breeding programmes (Harkness and Olesen) shared a similar genepool. Comparison of the differentiation among linkage groups indicated that linkage group 5 is potentially a region containing important QTLs for winter hardiness. Linkage group 6 contains the largest amount of genetic diversity, while linkage group 2 is the most differentiated among types of garden roses. Garden roses, as well as many other important crops (wheat, potato, strawberry, etc.) are polyploid. Genetic analyses of polyploids is complex as the same locus is present on multiple homologous chromosomes. SSR markers are suitable for mapping in segregating populations of polyploids as they are multi-allelic, making it possible to detect different alleles of the same locus on all homologous chromosomes. If a SSR marker gives fewer alleles than the ploidy level, quantification of allele dosages increases the information content. In Chapter 3 I showed the power of this approach. Alleles were scored quantitatively using the area under the peaks in ABI electropherograms, and allele dosages were inferred based on the ratios between the peak areas for two alleles in reference cases in which these two alleles occurred together. We resolved the full progeny genotypes, generated more data and mapped markers more accurately, including markers with “null” alleles. Even though SSR markers are one of the most appropriate marker systems for genetic studies in polyploids still few hurdles complicate (reduce) their implementation. The first major hurdle in developing microsatellite markers, the cloning step, has been overcome by next generation sequencing techniques. The second hurdle is the testing step to differentiate polymorphic from non-polymorphic loci. The third hurdle, somewhat hidden, is that only those polymorphic markers that detect a large effective number of alleles in the germplasm to be studied, are sufficiently informative to be deployed in multiple studies. Both selection steps are laborious and still done manually. In Chapter 4 I present a strategy in which we first screen sequence reads from multiple genotypes for repeats that show the most variation in length, and only these are subsequently developed into markers. We validated our strategy in tetraploid garden rose using Illumina paired-end transcriptome sequences of 11 roses. Out of 48 tested two markers did not amplify but all others were polymorphic. Ten loci amplified more than one locus, indicating duplicated genes or gene families. Completely avoiding this will be difficult, as the range of numbers of predicted alleles of highly polymorphic single- and multi-locus markers largely overlapped. Of the remainder, half were duplicates, indicating the difficulty of correctly filtering short sequence reads containing repeat sequences. The remaining 18 markers were all highly polymorphic, amplifying between 6 and 20 alleles in the 11 tetraploid garden roses. This strategy therefore represents a major step forward in the development of highly polymorphic microsatellite markers. Despite that garden roses are economically very important ornamentals, breeding is still mostly conventional, mainly due to tetraploidy and the lack of genetic maps and knowledge about the genetic base of important traits. Furthermore, crosses with unintended parents occur regularly and detection of these is not always straightforward, especially when genetically related varieties are used. Moreover, in polyploids detection of off-type offspring often relies on detecting differences in allele dosage rather than the presence of new alleles. In Chapter 5 I applied the WagRhSNP Axiom rose SNP array to generate 10,000s of SNPs for parentage analysis and to generate a dense genetic map in tetraploid rose. I described a method to separate progeny into putative populations which share parents, even if one of the parents is unknown, using PCO analysis and sets of markers for which allele dosages are incompatible. Subsequently, dense SNP maps were generated for a biparental and a self-pollinated mapping population with one parent in common. I confirmed a tetrasomic mode of inheritance for these crosses and created a starting point for implementation of marker-assisted breeding in garden roses by QTL analysis for important morphological traits (recurrent blooming and prickle shape). Winter hardiness is a complex trait and one of the most important limiting factors for garden rose growth and distribution in areas characterized by a continental climate. In Chapter 6 research was undertaken to determine the genetic regions underlying winter hardiness of garden roses, and to generate markers linked to them. For this purpose we exposed two segregating populations, RNDxRND and RNDxHP, to temperatures below -15C in a cold chamber and in the field in Serbia. The frost damage in the hardened plants was estimated directly at the phenotypic level (proportion of dieback) and at the non-visible physiological level indirectly (through the potential for meristem production in spring; regrowth). For winter hardiness we detected two tentative QTLs in the RNDxRND population and two tentative QTLs in the RNDxHP population, of which one was the same in both populations. The ability of plants to regrow in spring was associated to genomic regions on three linkage groups of the RNDxRND population, and on two different linkage groups in the RNDxHP population. A comparison of the ability for regrowth and level of damage caused by low temperature revealed that these two traits are inherited independently and that the final cold tolerance depends on the plant’s ability to withstand low temperature and to regrow fast in spring. In résumé, this thesis resulted in the development of basic tools (a fast strategy for polymorphic SSR marker development), basic methods/concepts for genetic analyses in polyploids (quantification of SSR allele dosage, distinguishing outliers from population in polyploid crops, dense SNP map generation and QTL study in tetraploids), and knowledge on genetics of important traits in rose (relatedness among modern garden roses (genetic diversity approach), mode of inheritance, occurrence of selfing, QTLs for morphological traits (recurrent blooming and prickle shape) and dissection of winter hardiness (level of damage caused by low temperature and regrowth)). Additionally, potential use of markers in every phase of rose breeding was discussed (Chapter 7). All these aspects contribute to a solid basis for marker assisted breeding in (garden) rose. </p

High throughput marker development and application in horticultural crops

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

Correlation and path analysis of grain yield and morphological traits in test–cross populations of maize

One of the goals of this paper was to determine correlation between grain yield, like the most important agronomic trait, and traits of the plant and ear that are influencing on the grain yield, in two test-cross populations, which are formed by crossing progenies of NSU(1) population after 17 cycles of phenotypic recurrent selection and two testers, 568/II NS and B73. At 568/II NS testcrosses, grain yield had the highest value of genotypic coefficient of correlations with kernel row number. In second studied population the highest value of coefficient of correlations also was found between grain yield and kernel row number, but that relation was negative. Path coefficient analysis provides more information among variables than do correlation coefficients. Because of that goal of this study also was founding the direct and indirect effects of morphological traits on grain yield. Desirable, high significant influence on grain yield, in path coefficient analysis, was found for ear height, in both studied populations. Plant height, in both testcross populations, kernel row number and oil content, at B73 testcrosses, has high significant undesirable effect on grain yield

Quantification of Allele Dosage in tetraploid Roses

Many important crops (wheat, potato, strawberry, rose, etc.) are polyploid. This complicates genetic analyses, as the same locus can be present on multiple homologous or homoeologous chromosomes. SSR markers are suitable for mapping in segregating populations of polyploids as they are multi-allelic, making it possible to detect different marker alleles of the same locus on multiple homologous r homoeologous chromosomes. If a SSR primer pair gives fewer alleles than the ploidy level, quantification of allele dosages increases information content. We show the power of this approach for the generation of a genetic map in a auto-tetraploid garden rose population. Alleles were scored quantitatively using the area under the peaks in ABI electropherograms, and allele dosages were inferred based on the ratios between the peak areas for two alleles in which these two alleles occurred together. We hereby start with some reference cases in which the ratio’s are determined between alleles that are in simplex condition. We thus resolved the full progeny genotypes, generated more data and mapped markers more accurately, including “null” alleles. The maps will be used for locating QTLs for winterhardiness in tetraploid roses

Genetic diversity and differentiation in roses: A gardenrose perspective

tFor the first time genetic diversity among modern garden rose cultivars has been evaluated using a setof 24 microsatellite markers covering most chromosomes. A total of 518 different alleles were obtainedin the set of 138 rose cultivars and this led to the conclusion that in terms of genetic diversity cut rosescan be considered as a subgroup of the garden roses.Genetic differentiation among types of garden roses (Fst= 0.022) was four times that among cut roses,and similar in magnitude to the differentiation among breeders, due to the fact that horticultural groupsand breeders overlap largely in classification. Winter hardy Svejda’s cultivars (Canadian Explorer roses)showed the least similarities to European roses, and introgression from wild species for winter hardinesswas clearly visible. Roses of Harkness and Olesen shared a similar genepool. Comparison of the differen-tiation among linkage groups indicated that linkage group 5 is potentially a region containing importantQTLs for winter hardiness. Linkage group 6 contains the largest amount of genetic diversity, while linkagegroup 2 is the most differentiated among types of garden roses

Efficient development of highly polymorphic microsatellite markers based on polymorphic repeats in transcriptome sequences of multiple individuals

The first hurdle in developing microsatellite markers, cloning, has been overcome by next generation sequencing. The second hurdle is testing to differentiate polymorphic from non-polymorphic loci. The third hurdle, somewhat hidden, is that only polymorphic markers with a large effective number of alleles are sufficiently informative to be deployed in multiple studies. Both steps are laborious and still done manually. We have developed a strategy in which we first screen reads from multiple genotypes for repeats that show the most length variants, and only these are subsequently developed into markers. We validated our strategy in tetraploid garden rose using Illumina paired-end transcriptome sequences of 11 roses. Out of 48 tested two markers failed to amplify but all others were polymorphic. Ten loci amplified more than one locus, indicating duplicated genes or gene families. Completely avoiding duplicated loci will be difficult because the range of numbers of predicted alleles of highly polymorphic single- and multi-locus markers largely overlapped. Of the remainder, half were replicate markers (i.e., multiple primer pairs for one locus), indicating the difficulty of correctly filtering short reads containing repeat sequences. We subsequently refined the approach to eliminate multiple primer sets to the same loci. The remaining 18 markers were all highly polymorphic, amplifying on average 11.7 alleles per marker (range = 6 to 20) in 11 tetraploid roses, exceeding the 8.2 alleles per marker of the 24 most polymorphic markers genotyped previously. This strategy, therefore, represents a major step forward in the development of highly polymorphic microsatellite markers

High-density SNP-based genetic maps for the parents of an outcrossed and a selfed tetraploid garden rose cross, inferred from admixed progeny using the 68k rose SNP array

Dense genetic maps create a base for QTL analysis of important traits and future implementation of marker-assisted breeding. In tetraploid rose, the existing linkage maps includ

Using RNA-Seq to assemble a rose transcriptome with more than 13,000 full-length expressed genes and to develop the WagRhSNP 68k Axiom SNP array for rose (Rosa L.)

In order to develop a versatile and large SNP array for rose, we set out to mine ESTs from diverse sets of rose germplasm. For this RNA-Seq libraries containing about 700 million reads were generated from tetraploid cut and garden roses using Illumina paired-end sequencing, and from diploid Rosa multiflora using 454 sequencing. Separate de novo assemblies were performed in order to identify single nucleotide polymorphisms (SNPs) within and between rose varieties. SNPs among tetraploid roses were selected for constructing a genotyping array that can be employed for genetic mapping and marker-trait association discovery in breeding programs based on tetraploid germplasm, both from cut roses and from garden roses. In total 68,893 SNPs were included on the WagRhSNP Axiom array. Next, an orthology-guided assembly was performed for the construction of a non-redundant rose transcriptome database. A total of 21,740 transcripts had significant hits with orthologous genes in the strawberry (Fragaria vesca L.) genome. Of these 13,390 appeared to contain the full-length coding regions. This newly established transcriptome resource adds considerably to the currently available sequence resources for the Rosaceae family in general and the genus Rosa in particular

Development of the WagRhSNP AXIOM SNP Array Based on Sequences from Tetraploid Cut Roses and Garden Roses

Rose, as many other important ornamental, vegetable and field crops, is polyploid. This poses constraints in genetic analyses due to the occurrence of multiple alleles at marker and trait loci and the existence of multiple allele dosages. Developments in marker discovery (next generation sequencing), detection (SNP arrays) and analysis (software for dosage scoring) now make it feasible to develop high-density molecular marker maps for the homologous chromosomes in tetraploids separately, and thus perform QTL analysis in tetraploids. We developed a SNP array for rose to develop genetic maps in tetraploid garden roses and cut roses, which are to be used for inheritance studies and genetic mapping. Here we have indicated the general strategy followed for developing a SNP array and for scoring and using the SNP data generated, and elaborated on the activities undertaken to use the WagRhSNP Axiom array in rose. The array design is not proprietary but can be used by all researchers working in ros