Disentangling hexaploid genetics : towards DNA-informed breeding for postharvest performance in chrysanthemum
Geest, Geert van - \ 2017
Wageningen University. Promotor(en): R.G.F. Visser, co-promotor(en): U. van Meeteren; P.F.P. Arens. - Wageningen : Wageningen University - ISBN 9789463436427 - 142
chrysanthemum - plant breeding - postharvest quality - hexaploidy - polyploidy - quantitative trait loci - phenotypes - linkage mapping - metabolomics - polymorphism - dna - chrysanthemum - plantenveredeling - kwaliteit na de oogst - hexaploïdie - polyploïdie - loci voor kwantitatief kenmerk - fenotypen - koppelingskartering - metabolomica - polymorfisme - dna
DNA-informed selection can strongly improve the process of plant breeding. It requires the detection of DNA polymorphisms, calculation of genetic linkage, access to reliable phenotypes and methods to detect genetic loci associated with phenotypic traits of interest. Cultivated chrysanthemum is an outcrossing hexaploid with an unknown mode of inheritance. This complicates the development of resources and methods that enable the detection of trait loci. Postharvest performance is an essential trait in chrysanthemum, but is difficult to measure. This makes it an interesting but challenging trait to phenotype and detect associated genetic loci. In this thesis I describe the development of resources and methods to enable phenotyping for postharvest performance, genetic linkage map construction and detection of quantitative trait loci in hexaploid chrysanthemum.
Postharvest performance is a complicated trait because it is related to many different disorders that reduce quality. One of these disorders in chrysanthemum is disk floret degreening, which occurs after long storage. In chapter 2, we show that degreening can be prevented by feeding the flower heads with sucrose, suggesting carbohydrate starvation plays a role in the degreening process. To investigate the response to carbohydrate starvation of genotypes with different sensitivity to disk floret degreening, we investigated the metabolome of sugar-fed and carbohydrate-starved disk florets by 1H-NMR and HPAEC. We show that the metabolome is severely altered at carbohydrate starvation. In general, starvation results in an upregulation of amino acid and secondary metabolism. Underlying causes of genotypic differences explaining variation in disk floret degreening in the three investigated genotypes remained to be elucidated, but roles of regulation of respiration rate and camphor metabolism were posed as possible candidates.
In chapter 3, disk floret degreening was found to be the most important postharvest disorder after 3 weeks of storage among 44 white chrysanthemum cultivars. To investigate the inheritance of disk floret degreening, we crossed two genotypes with opposite phenotypic values of both disk floret degreening and carbohydrate content to obtain a population segregating for disk floret degreening. To phenotype the cultivar panel and the bi-parental population precisely and in a high throughput manner, we developed a method that quantified colour of detached capitula over time. This method was validated with visual observations of disk floret degreening during vase life tests. In a subset of the bi-parental population we measured carbohydrate content of the disk florets at harvest. The amount of total carbohydrates co-segregated with sensitivity to degreening, which shows that the difference in disk floret degreening sensitivity between the parents could be explained by their difference in carbohydrate content. However, the correlation was rather weak, indicating carbohydrate content is not the only factor playing a role.
In order to develop resources for DNA-informed breeding, one needs to be able to characterize DNA polymorphisms. In chapter 4, we describe the development of a genotyping array containing 183,000 single nucleotide polymorphisms (SNPs). These SNPs were acquired by sequencing the transcriptome of 13 chrysanthemum cultivars. By comparing the genomic dosage based on the SNP assay and the dosage as estimated by the read depth from the transcriptome sequencing data, we show that alleles are expressed conform the genomic dosage, which contradicts to what is often found in disomic polyploids. In line with this finding, we conclusively show that cultivated chrysanthemum exhibits genome-wide hexasomic inheritance, based on the segregation ratios of large numbers of different types of markers in two different populations.
Tools for genetic analysis in diploids are widely available, but these have limited use for polyploids. In chapter 5, we present a modular software package that enables genetic linkage map construction in tetraploids and hexaploids. Because of the modularity, functionality for other ploidy levels can be easily added. The software is written in the programming language R and we named it polymapR. It can generate genetic linkage maps from marker dosage scores in an F1 population, while taking the following steps: data inspection and filtering, linkage analysis, linkage group assignment and marker ordering. It is the first software package that can handle polysomic hexaploid and partial polysomic tetraploid data, and has advantages over other polyploid mapping software because of its scalability and cross-platform applicability.
With the marker dosage scores of the bi-parental F1 population from the genotyping array and the developed methods to perform linkage analysis we constructed an integrated genetic linkage map for the hexaploid bi-parental population described in chapter 3 and 4. We describe this process in chapter 6. With this integrated linkage map, we reconstructed the inheritance of parental haplotypes for each individual, and expressed this as identity-by-descent (IBD) probabilities. The phenotypic data on disk floret degreening sensitivity that was acquired as described in chapter 3, was used in addition to three other traits to detect quantitative trait loci (QTL). These QTL were detected based on the IBD probabilities of 1 centiMorgan intervals of each parental homologue. This enabled us to study genetic architecture by estimating the effects of each separate allele within a QTL on the trait. We showed that for many QTL the trait was affected by more than two alleles.
In chapter 7, the findings in this thesis are discussed in the context of breeding for heterogeneous traits, the implications of the mode of inheritance for breeding and the advantages and disadvantages of polyploidy in crop breeding. In conclusion, this thesis provides in general a significant step for DNA-informed breeding in polysomic hexaploids, and for postharvest performance in chrysanthemum in particular.
The allo-octoploid strawberry: simply complex
Dijk, Thijs van - \ 2016
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Eric van de Weg. - Wageningen : Wageningen University - ISBN 9789462579637 - 185
fragaria ananassa - strawberries - polyploidy - microsatellites - linkage mapping - genome analysis - quantitative trait loci - genetic mapping - flowering - plant breeding - fragaria ananassa - aardbeien - polyploïdie - microsatellieten - koppelingskartering - genoomanalyse - loci voor kwantitatief kenmerk - genetische kartering - bloei - plantenveredeling
The garden strawberry (Fragaria x ananassa) is a fruit species that was developed through human intervention less than 300 years ago. Currently, it is the most important soft fruit in both production as well as value and renowned for its deliciousness. There are many challenges in growing such a delicate fruit, many of which have been overcome through improved cultivation techniques and breeding. The perishability of the product is, however, not the only challenge faced by strawberry breeders. In terms of genome composition, strawberry appears to have accumulated a wonderful array of obstacles to genetic studies. It is a vegetatively propagated allo-octoploid outbreeder, and only few crop species are worse of in this respect. Many of the molecular genetic ground work is therefore performed in its diploid ancestor, the woodland strawberry Fragaria vesca, which was sequenced in 2011. However, since nearly all strawberries that are eaten are octoploid, genetic research can’t linger at the wild diploids forever. In this thesis we developed new tools and analysis methods for genetic studies in the allo-octoploid strawberry and subsequently applied these methods in the detection of marker-trait associations.
The purpose of Chapter 2 was to develop a method to interpret the complex peak patterns generated by microsatellites in octoploid strawberry in such a way that we ended up with as much information as one would expect to retrieve from a microsatellite in a diploid system. This information could then be used to generate high quality linkage maps for the different sub-genomes and allow for easy alignment and comparison. We named the method MADCE, which stands for Microsatellite Allele Dose & Configuration Establishment. In the MADCE methodology, we first need to determine the dose of each allele present in an individual. For this we used the signal of fluorescent microsatellite peaks in relation to the total fluorescent signal generated by all peaks for that microsatellite. We then used the disomic inheritance of strawberry to establish the allelic configuration of each different homoeologue (subgenome). The repulsion of alleles from the same subgenome in offspring allowed us to form subgenomic pairs of alleles. We found that in single cross mapping populations, the deployment of our method was fairly easy due to the high number of offspring that can be used to establish repulsion between alleles. However, for pedigreed breeding germplasm this was another matter, as generally only few offspring were available. For this we added some additional tricks to the MADCE method, although some uncertainty about the configuration would remain for problematic lines and alleles.
In Chapter 3 we used the MADCE method from Chapter 2 to generate a genome wide linkage map for the Holiday x Korona (HxK) mapping population. This linkage map was to be used in subsequent experiments for QTL discovery as well as provide the strawberry community with a highly detailed map consisting not only of marker distances, but allele and haplotype configuration of the parents Holiday and Korona as well. The haplotype information revealed that inbreeding (homozygosity) levels in Holiday were similar to the levels expected from its pedigree, but that inbreeding levels of Korona were more than three times higher than expected, which could be resultant from selection pressure enacted by breeders. Selection pressure could also be causal to our discovery that the kinship between the two cultivars was twice as high as expected from their shared ancestry. Another discovery was a large inversion on one of the subgenomes of linkage group 2 (D). Up until the publication of our linkage map this inversion had not been reported in other linkage maps. Another innovation was our attempt at giving a biological or evolutionary meaning to the denomination of the linkage groups by arranging them according to similarity to the diploid ancestor F. vesca, based on F. vesca derived primer amplification efficiencies. The HxK map has been used in several (ongoing) research projects outside of our research group and has contributed to the development of the 90k Axiom SNP array for cultivated strawberry.
In Chapter 4 we performed a QTL mapping study for disease resistance against the problematic pathogen Phytophthora cactorum, which causes crown rot in strawberry plants. In this study we used two different mapping populations: the Holiday x Korona (HxK) population from the previous chapter as well as E1998-142 x Elsanta (ExEls), developed more specifically for the purpose of finding resistance against P. cactorum. The HxK and ExEls populations were phenotyped over three years (2008, 2010 and 2011) under different seasons and conditions. The correlation between years for was quite low for both populations (ranging from 0.18 to 0.47), indicating a large environmental effect on disease pressure. Results from the QTL analysis showed that most QTLs were small in effect and only just above the statistical significance threshold. Only for ExEls we uncovered two QTLs with relatively high significance levels, but none were significant in all three years. Because of the high environmental influence, and the desire to have QTLs that are robust over environments, we used the average of all three years (AOTY) as an additional phenotype. When we used the AOTY trait, the QTL on LG7D became stronger than for any of the individual years. Whereas for the LG7C QTL the significance dropped to just below threshold levels. These results indicated that removing environmental noise through averaging over experiments is a good way to uncover the most reliable and therefore more valuable to a breeding program.
In Chapter 5 we investigated the genetics behind two different flowering habits that are grown commercially worldwide: seasonal flowering habit (SF) and perpetual flowering (PF) These varieties initiate flowering under long days, and can therefore produce fruit for a much longer period: throughout the summer and early fall. Evidence from literature and practical breeding suggested that PF is under dominant control. We decided to treat PF as a qualitative trait and divided two small mapping populations into PF and SF individuals. After screening several microsatellites, we found one locus that completely cosegregated with the PF trait at the bottom of LG4D. At the moment of mapping, a paper was published which mapped the same trait to the same location. We found that there were two very clear candidate genes within our QTL interval, FaCDF2 and FaFT2, which were homologous to genes that are major factors in the flowering pathway of Arabidopsis and many other plant species. We then sequenced the FaCDF2 gene from a number of distinct PF and SF cultivars. This resulted in the discovery of two quite distinct allelic variants, one of which was present in all PF cultivars. However this variant was also present in some of the SF cultivars, indicating that either FaCDF2 is not the causal gene, or that other loci can have a qualitative effect on the switch from SF to PF. We then performed microsatellite haplotyping on hundreds of cultivars and this revealed that all PF varieties of all origins carry the same haplotype in the PF QTL region, and that there weren’t any recombinations between the candidate genes FaCDF2 and FaFT2, which are 250kb apart on the physical genome. This makes it still undecided which of these two candidate genes are causal to the PF trait. Another interesting result from the haplotyping was that the complete PF haplotype was present with moderate frequency in SF varieties as well. Not only does this suggest a common origin, it also complicates the establishment of a theory for the mechanisms behind perpetual flowering in cultivated strawberry. So far we have not been able to establish whether the PF haplotype that is present in SF cultivars is functionally distinct from the PF haplotype in PF cultivars. All we know is that it does not confer perpetual flowering in these SF cultivars, and further experiments would be needed to find out the exact mechanism behind perpetual flowering.
In the general discussion (Chapter 6), the results of this thesis were placed in the broader context of science in general and plant breeding in particular.
Combining different pedigrees to fine-map QTL in the pig
Tortereau, F.J.D. - \ 2012
Wageningen University. Promotor(en): Martien Groenen; D. Milan, co-promotor(en): J. Riquet; Richard Crooijmans. - S.l. : s.n. - ISBN 9789461731418 - 173
varkens - stamboom - loci voor kwantitatief kenmerk - genetische kartering - overerving - nakomelingenonderzoek - rugspek - koppelingskartering - geslachtsverschillen - genetica - pigs - pedigree - quantitative trait loci - genetic mapping - inheritance - progeny testing - backfat - linkage mapping - sex differences - genetics
Pig domestication started around 10,000 years ago during the Neolithic age, independently in Europe and China, and most current pig breeds originate from these two areas. Among the 560 pig breeds that have been recorded over the world in 2007 by the FAO, only few of them have been intensively selected for production. Domestication and, more recently, pig breeders have relied on naturally occurring mutations to select individuals exhibiting favourable traits related to reproduction, growth, fatness, resistance to diseases and behaviour. In order to identify these mutations underlying the phenotypic variations of these traits, a number of QTL detection programs was set up, and thousands of QTLs have been detected in the 2000s. However, for only a few of them, fine-mapping has resulted in the identification of the causal polymorphism.
In chapter 1, the general introduction provides an overview of QTL detection in pig in relation to the molecular tools that are available for pig geneticists and to the different mapping strategies that can be used. Major limitations to QTL fine-mapping in pig (but also valid for other livestock species) concern the number of individuals, the number of informative genetic markers and the ability to detect non-additive QTLs.
To increase the statistical power by increasing the number of individuals, a combined linkage analysis is presented in chapter 2. To carry out this work, two pig F2 pedigrees comprising about a thousand individuals each and based on similar breeds (Large White and/or Landrace crossed with Meishan individuals) were combined. Both pedigrees had been developed in the late 1990s at INRA and WUR. Common QTLs segregating on SSC2, SSC4 and SSC6 were confirmed in the combined analysis, but QTLs that were specific to one pedigree disappeared or were detected at a lower significance threshold. Despite the limited benefits in term of the number of QTLs, the increase in the number of individuals, enabled us to separate two linked QTLs that were previously detected as a single one. False-positive QTLs were also detected as well as new QTLs characterised by a low frequency and/or a small effect. In addition, both pedigrees could be compared regarding the imprinting status at the IGF2-intron3-G3072A substitution, segregating on SSC2. The mutation was segregating within the European founders used in both pedigrees, Meishan individuals being all homozygous for the wild allele (G). This analysis, presented in chapter 3, shows that the structure of the pedigree (number of F1 individuals and size of half-sib families), the number of F1 heterozygous females at the IGF2 locus and the segregation of another QTL at a distance of 40 cM from the IGF2 locus influence the ability to detect imprinting at the IGF2 mutation. This spurious maternal effect can lead to incorrect conclusions regarding the imprinting status of the IGF2 mutation, with maternal effects being detected whereas they do not exist.
In order to fine-map the second QTL segregating on pig chromosome 2, a backcross design was set up. Sires that were finally progeny tested were all homozygous for a Meishan haplotype in the IGF2 region, so the phenotypic variation could not be due to the IGF2-intron3-G3072A mutation. Results from the progeny-testing presented in chapter 4 confirmed that a QTL underlying fatness traits was segregating on the short arm of SSC2. However, the size of the QTL interval could not be reduced because of epistatic interactions. These epistatic effects could be detected because full-sibs with Identical-by-descent haplotypes in the QTL regions were progeny-tested. This particular design could be analysed without the strong assumptions of the line-cross models (according to which QTL alleles are fixed within breeds), so interactions could be detected. The re-analysis of one of the two F2 pedigrees confirmed that a region on SSC13 interacts with the QTL segregating in SSC2, but other candidate regions still need to be considered.
The combined analysis of different pedigrees finally gives few benefits regarding the number of new QTLs that were detected. However, these combined analyses enabled to successfully consider non-additive effects such as imprinting and epistasis.
During the work described in this thesis, a major technological advance occurred for pig geneticists, with the commercialisation of the Illumina PorcineSNP60 Beadchip. With this tool, the number of genotypes that can be included in a QTL analysis tremendously increased. In order to properly use this new type of information, the order of the SNPs along the genome must be reliable. In chapter 5, the first high-density genetic map of the pig is presented. This genetic map was computed using information from in silico and RH mapping of the SNPs in combination with recombination rates between them and finally comprised 38,599 SNPs. Four pig pedigrees based on different breeds were analysed separately, and the analysis of the recombination rate along the pig genome highlighted that the more recombinant regions tend to cluster around the telomeres irrespective of the location of the centromere. Two of the four analysed pedigrees comprised enough male and female meiosis to construct sex-specific maps. Major sex-differences in recombination were observed with a higher recombination rate in the females only within GC-rich regions, with females exhibiting a much stronger correlation between recombination rate and specific sequence features. This new information will be of major importance when dealing with QTL fine-mapping and pig genome evolution.
Finally, in the general discussion presented in chapter 6, arguments toward further fine-mapping of QTLs in pig are given despite the increasing interest in genomic selection. Despite major improvements that have been made in the development of high-density SNP chips, efforts are still needed to overcome the biases linked to the design of the chips. In parallel to the development of high-density genotyping tools, few improvements were made regarding phenotyping. In this final chapter, various programs dedicated to the description of highly precise phenotypes and to the development of homogenous phenotyping practices are presented. Such programs, in combination with the development of appropriate genotyping tools, will facilitate the detection of causal variants. These efforts that are still necessary are not only required for pig but also to most livestock species for which QTL fine-mapping is still needed.
A molecular cytogenetic study of intergenomic recombination and introgression of chromosomal segments in lilies (Lilium)
Nadeem Khan, M. - \ 2009
Wageningen University. Promotor(en): Richard Visser; Jaap van Tuyl. - [S.l. : S.n. - ISBN 9789085853800 - 121
lilium - cytogenetica - recombinatie - genomen - introgressie - hybridisatie - hybriden - genetische kartering - polyploïdie - koppelingskartering - lilium - cytogenetics - recombination - genomes - introgression - hybridization - hybrids - genetic mapping - polyploidy - linkage mapping
Lilies (Lilium L.) are one of the most important ornamental bulbous crops for cut flower industry
grown extensively in The Netherlands for last few decades. The genus Lilium consists of seven
different sections with about 80 species. The species within genus Lilium (2n = 2x = 24)
comprise a range of desirable and complementary characters. Besides being an important
horticultural crop, lily (Lilium) also serves as an interesting model plant for molecular
cytogenetic research and introgression breeding for several reasons like, i). Lily is a model crop
for interspecific hybridization and it includes plants of different taxonomic species each of which
possess valuable horticultural traits that need to be combined in the new cultivars. ii) Through
careful selection n and 2n gametes can be obtained in interspecific hybrids. iii) The genomes of
different species are so well differentiated genetically that the parental chromosomes can be
clearly identified in the F1 hybrids as well as in the progenies through DNA in situ hybridization
techniques. iv) The chromosomes are large enough and the number and position of
homoeologous recombination sites can be clearly detected. v) The large and easily identified
chromosomes in different lily species could be a potential source for the cytological mapping of
the Lilium genomes. Taking advantage of these favourable attributes of lily, a molecular
cytogenetic investigation was conducted to evaluate the amount of recombination and
introgression of characters between Longiflorum - Asiatic (LA) and Oriental - Asiatic (OA)
hybrids through the use of n and 2n gametes.
For this purpose different F1 Longiflorum × Asiatic (LA) and Oriental × Asiatic (OA)
hybrids were backcrossed with different Asiatic cultivars. Ovule and embryo rescue techniques
were employed to get backcross (BC) progenies. Most of the F1 LA appeared to be sterile but
some hybrids were able to produce only 2n gametes in considerable frequencies. However, in
rare occasions it was also found that normal meiosis took place in few of the LA hybrids which
resulted into the formation of normal n gametes. Ploidy level and intergenomic recombination
was studied in LA interspecific hybrids in order to assess the possibility of functional n gametes
and their potential use in introgression at diploid level in lily. A total of 104 BC1 LA
interspecific lily hybrids were obtained, 27 diploids (2n = 2x = 24), 73 triploids (2n = 2x = 36)
and 4 aneuploids (2x – 1, 2x + 2 or 2x + 3). Similarly, triploid BC1 (LAA) plants were
backcrossed to diploid Asiatic parents. As a result 14 diploid BC2 progenies were produced. The
intergenomic recombination and amount of introgression of respective genome (L and A) was
assessed in these diploid genotypes through GISH (Genomic in situ Hybridization). Extensive
intergenomic recombination was found among the chromosomes in LA hybrids. A large of
amount of L- genome was transmitted from F1 LA hybrids to their subsequent BC1 progenies.
However, very few segments of L- genome were introgressed from the BC1 diploid and triploid
(LAA) plants to the BC2 progenies (Chapter 2). GISH identifies a considerable amount of
recombination events amongst different interspecific lily hybrids (LA and OA) obtained from
functional 2n gametes. Based on recombination sites on different chromosomes identified by
GISH, cytological maps of three genomes of Lilium were constructed. For this purpose, BC
progenies of two diploid interspecific hybrids of lily, viz., Longiflorum × Asiatic (LA) and
Oriental × Asiatic (OA) were used. The BC progenies of LA hybrids consisted of both triploid
(2n = 3x = 36) and diploid (2n = 2x = 24) with some aneuploid genotypes and those of OA
hybrids mostly consisted of triploid (2n = 3x = 36) and some aneuploid genotypes. In LA
hybrids 248 recombination sites were cytologically localized on 12 different chromosomes of
each genomes (i.e., L and A). Similarly, 116 recombinant sites were marked on 12 chromosomes
each from the BC progenies of OA hybrids (O and A genomes). The distances of the
recombination sites from the centromeres are measured (in micrometres). Based on these
recombination sites four cytological maps were constructed. Since an Asiatic parent was
involved in both hybrids, viz., LA and OA, two maps were constructed for A genome which
were indicated as Asiatic (L) and Asiatic (O) and one each for Longiflorum (A) and Oriental (A)
genomes (Chapter 3).
With a view to generate genetic variation via homoeologous recombination in BC
progenies of LA and OA hybrids the most logical approach was the use of 2n gametes. 63 BC1
LA (LA × AA or AA × LA) and 53 OA (AA × OA) progeny plants were obtained after unilateral
sexual polyploidization. 16 genotypes from F2 LA populations were obtained after bilateral
sexual polyploidization through sib-mating of F1 LA hybrids. GISH was employed for the
identification of the parental genomes, mode of origin of these progenies and measurement of the
introgression in different interspecific lily hybrids. Most of the BC1 progeny plants (LA and OA)
had originated through 2n gametes by First Division Restitution (FDR) mechanism. However,
there were 12 genotypes in LA hybrids and four genotypes in OA hybrids that originated through
2n gametes formation as the result of Indeterminate Meiotic Restitution (IMR). A higher amount
of recombination was found in LA hybrids as compared to OA hybrids. Intergenomic
recombination was also determined in the sib-mated F2 LA population. In this case both parents
had contributed gametes with the somatic number of chromosomes (i.e., 2n-2n) thus confirming
the event of bilateral sexual polyploidization in interspecific LA hybrids. Based on these results,
the relevance of interspecific lily hybrids obtained from uni- and bilateral sexual
polyploidization leading to allotriploid and allotetraploid formation in interspecific lily hybrids is
discussed in the context of introgression and mapping (Chapter 4). Molecular markers are an important tool for the construction of genetic linkage maps, as the first step in the genetic
dissection of the required traits leading to crop improvement followed by the marker assisted
breeding in different plants. Lilium has one of the largest genome in plant kingdom and genetic
mapping in lilies is constrained by its large genome. DArT (Diversity Array Technology), a
molecular marker technique can detect and type DNA variation at several hundred genomic loci
in parallel without relying on genome sequence information. The DArT technique was developed
for Longiflorum × Asiatic (LA) lily hybrids to enable an efficient and effective genetic mapping
with the production of a large numbers of markers in microarrays-based assay. The restriction
enzyme PstI + TaqI combination generated the largest frequency of polymorphic genomic
representations for a genotyping array. Genomic representations from 88 F1 LA plants were used
to assemble a DArT genotyping microarray. A total of 687 DArT markers were developed and
382 polymorphic markers were mapped on 14 main linkage groups which is two more then the
haploid chromosome number (i.e. n = 12). The resulting linkage map with 382 DArT markers
spanned 1329 cM (3.5 cM/marker on average). The results highlighted the potential of DArT as
a genetic technique for genome profiling in the context of molecular breeding and genomics,
especially in crops with large genome sizes where other techniques proved to be less useful
The results of the present investigation are of practical implication. These results show
the advantages of the n gametes and their subsequent progenies which opened a new approach of
lily breeding ‘the analytic breeding’ in the allopolyploids. It also shows the possibility of using
certain triploid hybrids for further breeding. A comparison has been made between different
types of interspecific crosses, the amount of intergenomic recombination and introgressions of
chromosomal segments obtained after unilateral sexual polyploidization. Furthermore, bilateral
sexually polyploidization via sib-mated F1 hybrids producing 2n gametes has been proven. The
use of allotetraploids obtained from bilateral sexual polyploidization could be a novel approach
in the breeding of LA-hybrids. These allotetraploid with recombinant chromosomal segment
may be a potential source to generate genetic variation in subsequent progenies. The molecular
cytogenetic GISH and FISH techniques proved to be a powerful tool useful for the construction
of cytogenetic maps in interspecific crosses in crops with large genomes sizes like lily. These
techniques are also used for the identification and integration of genetic maps with chromosome
maps. FISH also helps to monitor the introgressed chromosome segment or marker of interest in
the subsequent progenies. Application of the DArT technique proved to be an effective method
to construct genetic linkage maps especially crops (like Lilium) with large genome sizes where
other techniques might be less useful.