Genetic Positioning of Centromeres Using Half-Tetrad

Analysis in a 4x-2x Cross Population of Potato

Park et al.Genetics 176: 85-94 (May 2007)

CENTROMERES

• Important functional elements of eukaryotic chromosomes– Ensure proper cell division – Ensure stable transmission of genetic material

• Determining their composition and structure can provide insight into their functional roles

• Identification of genetic position important for:– Distinguishing chromosome arms– Identifying proximal and distal markers or genes– Providing fixed positions in genetic maps

RECENT CENTROMERE RESEARCH

• Centromeres sequenced and studied extensively in: – Arabidopsis– Maize– Rice– Wheat

• Little sequencing has been reported for potato.

MAIN GOALS OF RESEARCH

• Use half-tetrad analysis (HTA) to localize centromeres on 12 potato chromosomes– Compare results of HTA with UHD map proposing

possible centromere positions based on marker density (van Os et al. 2006)

• Determine whether there is truly a single crossover per chromosome arm

Previous publications on these issues were theoretical proposals or

were limited in loci/chromosome number.

4x – 2x Population

• Male parent– Diploid– Produced numerically-

unreduced 2n pollen by first-division restitution (FDR)

• Female parent– Tetraploid

Tetraploid mapping population

FDR vs. SDRFDR

•Abnormal orientation of spindles before anaphase II

•Non-sister chromatids end up in the same nucleus

•Efficiently transmits heterozygosity of original genotype

SDR

•Premature cytokinesis before second meiotic division

•Sister chromatids end up in the same nucleus

FDR vs. SDR: Probability of Heterozygosity

FDR SDR

Centromere 100% 0%

Telomere 50% 100%

FDR vs. SDR: Probability of Heterozygosity

FDR SDR

Centromere 100% 0%

Telomere 50% 100%

van Os et al. 2006

• Proposed centromere position– based on strong clustering of AFLP markers

on UHD genetic map– AFLP markers tend to be clustered in

centromeric regions in several other species

Park et al. compared their HTA-based results with the proposed positions of van Os et al.

aaaa x ab

Nulliplex aaaa heterozygosity

NOT maintained

Simplex aaab heterozygosity MAINTAINED

Duplex aabb heterozygosity NOT maintained

aaaa x ab

Nulliplex aaaa heterozygosity

NOT maintained

Simplex aaab heterozygosity MAINTAINED

Duplex aabb heterozygosity NOT maintained

D = [ f(aaaa) + f(aabb)] x 100 cM

CROSSING OVER OCCURRED.

AFLP Marker Patterns

A1: Six nulliplex offspring genotypes

More crossing over, farther from centromere

A2: One nulliplex offspring genotype

Less crossing over, closer to centromere

Frequency of homozygosity

• Based on location on UHD map (van Os et al. 2006), grouped linkage groups

• Within linkage groups, arranged according to genetic position

• Calculated frequencies of alleles– 233 genotypes– Map position:

• [f(aa) + f(bb)] x 100• [(# homozygous alleles)/233] x 100

Combining results: HTA and UHD

Marker-to-centromere

distance

(% homozygosity)Gray box marks bin containing

centromere

Zero distance to centromere

Centromere Locations

Chromosome Bin1 13

2 1

4 35

5 46

6 17

7 68

8 22

9 31

10 64

12 49

Centromere Locations

Chromosome Bin1 13

2 1

4 35

5 46

6 17

7 68

8 22

9 31

10 64

12 49

Telocentric

predominantly terminal location of the centromere

Centromere Locations

Chromosome Bin1 13

2 1

4 35

5 46

6 17

7 68

8 22

9 31

10 64

12 49

What about chromosomes 3 and 11?

Chromosomes 3 and 11

• No markers with 100% heterozygosity

Approaching zero

Other funny stuff: Chromosomes 1 and 5

• 100% heterozygosity found in the wrong bin (chromosomes 1, 5)

• 99.6% heterozygosity found in the “centromere” bin (chromosome 1)

Cross-over frequency

• Chromosome 2 (telocentric)

– one arm analyzed

• Chromosome 4 (metacentric)

– one arm analyzed

• Chromosome 6 (metacentric)

– both arms analyzed

Only one crossover per chromosome arm?

Cross-over frequency

Boldface: all markers are heterozygous (centromeric)

Block a: no crossovers

Block b: one crossover

Block c: two crossovers

Number crossovers

No crossovers

One crossover

Two crossovers

Chr2 118 110 5

Chr4 167 58 8

chr6 135 93 5

per 233 genotypes

Cross-over frequency

• Confirms that 2n pollen originated through FDR– Since some markers were entirely heterozygous,

cannot have involved an SDR mechanism

• Fewer noncrossover events on telocentric chr2– Telocentric chromosome shows more crossover?– No. X-squared not significant.

• Second crossover per chromosome arm very rare – 5/233 for chr2, 8/233 for chr4, 5/233 for chr6– Indicates strong interference

Summary of Results

• Used HTA to localize centromeres of most potato chromosomes

• Confirmed centromere positions with those in UHD map (van Os et al. 2006)

• Marker density approach w/ UHD can be used for positioning centromeres

• HTA in potato is also powerful for positioning centromeres