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PstI/MseI - Department of Plant Sciences

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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
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)
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
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
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
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
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
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
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)
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)
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
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 1st, 3.3
markers/pu in 2nd, 2.5 markers/pu in 3rd, 1.7 markers/pu
Marker density in short arm low in all subregions (0.2-1.1 markers/pu)
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
Possible explanations of differences
High level of recombination in proximal half of Allium
chromosomes suggests a high density of genes in this area
пѓЁGene density higher in distal regions for cereals
пѓЁRecombination 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
пѓЁSequence heterologies can significantly reduce
recombination rate
пѓЁLow degree of sequence identity between the two
parental homeologs
Possible explanations of differences
~58% of PstI/MseI markers proximally located on Allium
chromosomes
пѓЁPstI methylation-sensitive enzyme
пѓЁExpressed genes are typically hypomethylated
пѓЁPstI/MseI markers predominantly located in gene-rich
areas
Therefore, nonmethylated genic areas are found in more
proximal regions of chromosome
пѓЁContrasts with RFLP mapping results in cereals
Unlocking variability: inherent variation and
developmental traits of garlic plants originated
from sexual reproduction
Shemesh et al., 2008
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
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)
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 crosspollination
2 have same fragment pattern as mother, presumed to be selfings
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)
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 1st
and 2nd year of floral development
1st year seedlings can be used for study of blooming, avoiding field and
storage history effects
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
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
Effect of storage temperature on
ontogenesis of seedlings: Reproductive
traits
Transition to reproductive state greatly affected
Cold treatment required for flowering
Phenotypic variability under equal
growth conditions
In third year of development, populations segregated into 3 main
phenotypes: branching (20%), multifloral (60%), regular (20%)
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
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