1. The Integration of Recombination and Physical Maps in a Large-Genome Monocot Using Haploid Genome Analysis in a Trihybrid Allium Population Khrustaleva et al., 2005

2. Constructing integrated genetic and physical maps Four approaches : I.Construction of physical map by contig assembly of large insert DNA clones (BACs and YACs). II.Combination of in situ hybridization of BACs or YACs on plant chromosomes with recombination maps III.Use deletion or translocation lines to create physical landmarks on genomes and to relate these to recombination maps IV.Visualization of recombination points in interspecific hybrids via genomic in situ hybridization (GISH)

3. Research Goals I.Construct integrated physical and recombination maps of a large-genome monocot (Allium) II.Contribute to the study of chromosome organization in these species

4. Why this is important:  Enables a thorough analysis of recombination frequencies along Allium chromosomes  Allows for a comparison of genetic and physical distances  Makes it possible to study the physical distribution of two types of AFLP markers produced by restriction enzyme combinations sensitive (PstI/MseI) and nonsensitive (EcoRI/MseI) to methylation

5. Procedure  Utilized GISH and AFLP technology to construct integrated maps  Used Allium trihybrid population: Allium cepa X (Allium roylei x Allium fistulosum)  Compared AFLP profiles of individual genotypes with corresponding recombinant chromosomes  Constructed integrated physical and recombination maps of chromosome 5 and 8 for A. roylei and A. fistulosum simultaneously

6. GISH images:  Recombinant chromosome 5 centromeric region originated from A. roylei in 2 genotypes and from A. fistulosum in 5 genotypes  Recomb. Chromosome 8 centromeric region originated from A. roylei in 11 genotypes and from A. fistulosum in 1 genotype

7. Integrated physical and recombination maps for A.r. and A.f. chromosome 5  4 recombination sites on short arm and six on the long arm were used for physical mapping  In total, 10 physical recombination sites were integrated into the AFLP linkage group of chromosome 5

8. Integrated physical and recombination maps for A.r. and A.f. chromosome 8  4 recombination sites on proximal half of short arm and 10 on the long arm were used for physical mapping (8 located in interstitial part, 2 in distal part  In total, 14 physical recombination sites were integrated into the AFLP linkage map of chromosome 8

9. Relationship between genetic and physical distance: The physical distribution of recombination along chromosome 5  3 subregions with high recombination frequencies were found and no difference was found between the proximal and distal half of chromosome arms with respect to recombination frequency  1cM = 32.0 Mb in centromeric subregion; 1cM = 1.8 Mb in subregion with highest recombination frequency (70.6-72.6 pu)

10. Relationship between genetic and physical distance: The physical distribution of recombination along chromosome 8  Significant differences in recombination frequencies were found in proximal half of long arm and clear difference was found between the long and short chromosome arms with respect to recombination frequency  1cM = 31.3.0 Mb in centromeric subregion; 1cM = 1.4 Mb in subregion with highest recombination frequency (65.8-67.0 pu)

11. Physical distribution of AFLP markers for chromosome 5  Uneven distribution of the 64 AFLP markers from linkage group assigned to chromosome 5  Short arm: high density = 1.9 markers/pu in proximal subregion  Long arm: high density = 1.5 markers/pu in 2 subregions and 1.3 markers/pu in 1 subregion

12. Physical distribution of AFLP markers for chromosome 8  Uneven distribution of the 56 AFLP markers from 15 physical subregions on chromosome 8; 78.6% of markers on long arm  Proximal long arm: high density in 4 regions = 1.7 markers/pu in 1 st, 3.3 markers/pu in 2 nd, 2.5 markers/pu in 3 rd, 1.7 markers/pu  Marker density in short arm low in all subregions (0.2-1.1 markers/pu)

13. Conclusions: comparisons of Allium with other large-genome monocots Similarities:  Recombination hotspots restricted to few chromosomal regions  Physical density of markers corresponds closely to distribution of recombination  High degree of suppression of recombination in centromeric regions Differences:  Recombination predominantly occurs in proximal half of chromosome arm  ~58% of PstI/MseI markers occur in proximity of centromeric region

14. Possible explanations of differences  High level of recombination in proximal half of Allium chromosomes suggests a high density of genes in this area  Gene density higher in distal regions for cereals  Recombination events occur in gene-rich areas  Or, variation of recombination rates could be due to sequence heterologies in distal parts of A. roylei and A. fistulosum chromosomes  Sequence heterologies can significantly reduce recombination rate  Low degree of sequence identity between the two parental homeologs

15. Possible explanations of differences  ~58% of PstI/MseI markers proximally located on Allium chromosomes  PstI methylation-sensitive enzyme  Expressed genes are typically hypomethylated  PstI/MseI markers predominantly located in gene-rich areas  Therefore, nonmethylated genic areas are found in more proximal regions of chromosome  Contrasts with RFLP mapping results in cereals

16. Unlocking variability: inherent variation and developmental traits of garlic plants originated from sexual reproduction Shemesh et al., 2008

17. Traits of garlic (Allium sativum)  Agricultural garlic completely sterile, propagated only from cloves limits variation improvement of economically important traits restricted  High variability in garlic from Central Asia, main center of origin  Growth habit and flowering significantly affected by storage conditions environment during the current and previous growing seasons

18. Broadening the variability of garlic  Collected over 300 garlic landraces and wild populations across Central Asia (local genotypes possess economically useful traits lost during 10,000 years of domestication)  Over 30 accessions produced flowers and set seeds  Detailed characterization of seedlings’ development and assessment of inherent variation  Availability of seed allows the study of the complete life cycle, free of plant history (storage, growth temperatures of previous years affect vegetatively propagated plant development)

19. NBS profiling reveals polymorphism in seedling population  Extracted DNA from 28 seedlings and mother plant  10 fragments from mother were polymorphic in progeny  All but 2 had 1-8 foreign DNA fragments originating from cross- pollination  2 have same fragment pattern as mother, presumed to be selfings

20. Ontogeny of seedlings: Seed germination and juvenile state  Seed shape and color and seedling morphology typical to Allium  Germination lasted few weeks  Single or cluster bulbs formed at end of first growing season  Seedlings’ population varied in bulbing ability, bulbing time, and bulb traits (size, color, shape and clove number)

21. Ontogeny of seedlings: Reproductive state  Transition from vegetative to reproductive phase occurred after 6-22 foliage leaves and leaf primordia are produced  Garlic genepool highly variable  No morphological and developmental differences evident between 1 st and 2 nd year of floral development  1 st year seedlings can be used for study of blooming, avoiding field and storage history effects

22. Ontogeny of seedlings: Florogenesis and topset development  Florogenesis same as vegetatively propagated garlic  Flower primordia initiation followed by vegetative meristem initiation in inflorescence, forms thin leaves or small dormant bulbs (topsets)  Varied in number and size of topsets/leaves under equal environmental conditions

23. Effect of storage temperature on ontogenesis of seedlings: Vegetative traits  No effect on sprouting time, leaf growth, or time from planting to first occurrence of secondary growth  Bulbing, secondary sprouting of axillary buds strongly affected  Despite genetic variability, lower temperature produced bulbs after ~15 leaves, higher temperatures caused rotting prior to clove formation except one plant developed bulb after ~22 leaves

24. Effect of storage temperature on ontogenesis of seedlings: Reproductive traits  Transition to reproductive state greatly affected  Cold treatment required for flowering

25. Phenotypic variability under equal growth conditions In third year of development, populations segregated into 3 main phenotypes: branching (20%), multifloral (60%), regular (20%)

26. Conclusions  Confirmed the essential role of cross-pollination  Cross-pollinated plants highly heterozygous which is preferred genotypic state for seedling survival and plant vigor  Seed populations showed large variation in all morphological and physiological traits, similar variability as vegetatively propagated garlic  Variability by restored sexual reproduction may serve as rich source for genetic studies and breeding work, development of new and improved cultivars propagated from seed