LINKAGE AND GENETIC MAPPING IN EUKARYOTES

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LINKAGE AND GENETIC MAPPING IN EUKARYOTES

• Chapter 5

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INDEPENDENT ASSORTMENT

• Mendels law of independent assortment
• Alleles of different genes assort themselves
  independently during meiosis
• Genes reside upon chromosomes
• Chromosomes assort themselves independently
  during meiosis
• Chromosome theory of inheritance

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INDEPENDENT ASSORTMENT

• Each species contains thousands of genes
• Most species have at most a few dozen chromosomes
• Each chromosome must carry hundreds or thousands
  of genes
• Genes located close to each other on a chromosome
  do NOT assort independently during meiosis

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LINKAGE

• Genes located on the same chromosome display
  unique patterns of inheritance
• Data from such genetic crosses are used to
  construct genetic maps
• Describe the order of genes along the chromosome
• Newer strategies for gene mapping have largely
  replaced traditional means

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LINKAGE

• Eukaryotic chromosomes contain very long pieces
  of DNA
• Contains numerous genes
• Generally hundreds or thousands
• Two or more genes located on the same chromosome
  are linked

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LINKAGE

• The term linkage has two related meanings
• Two or more genes on the same chromosome are
  physically linked
• Two ore more genes on the same chromosome are
  genetically linked
• Tend to be transmitted as a unit

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LINKAGE

• The human karyotype has 46 chromosomes
• All genes on a single chromosome are physically
  linked to each other
• Chromosomes can be called linkage groups
• 22 autosomal linkage groups
• X linkage group
• Y linkage group
• Genes located far apart from each other on the
  same chromosome may assort independently
• Crossing over can occur between these genes

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LINKAGE

• One can study the transmission of two or more
  traits in a genetic cross
• e.g., Dihybrid cross, trihybrid cross, etc.
• The outcome of such a cross is dependent upon
  whether these genes are linked to each other
• Linkage affects the transmission of these alleles
  and traits

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LINKAGE

• Alleles for different genes may be linked along
  the same chromosome
• This linkage can be altered during meiosis
• Homologous chromosomes of diploid eukaryotic
  species can exchange pieces with each other
• Crossing over
• Occurs during prophase I of meiosis

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LINKAGE

• Sister chromatids associate with homologous
  sister chromatids
• Two pairs of sister chromatids form a bivalent
• A sister chromatid of one pair commonly crosses
  over with a sister chromatid from the other pair
• Can alter arrangements of linked genes
• Crossing over can lead to genetic recombination
• Alteration of the arrangement of linked alleles

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LINKAGE

• AB and ab gametes are parental
• Non-recombinant
• Ab and aB gametes are non-parental
• Recombinant

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BATESON PUNNETT, 1905

• Traits of sweet peas
• Flower color can be either purple or red
• Purple is dominant to red
• Pollen shape can be either oblong or round
• Oblong is dominant to round

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BATESON PUNNETT, 1905

• Purple, long (PPLL) x red, round (ppll)
• F1 offspring are purple, long (PpLl)
• F2 possess four different phenotypes
• 296 Purple long
• 19 Purple, round
• 27 Red, long
• 85 Red, round
• Observed ratio is 15.6 1.0 1.4 4.5
• Expected ratio of 9 3 3 1 is NOT seen!

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BATESON PUNNETT, 1905

• F2 generation
• Parental phenotypes were overrepresented
• Non-parental phenotypes were underrepresented
• These two genes do not assort independently
• Physically linked on the same chromosome
• The nature of this physical linkage was not
  apparent to Bateson and Punnett at this time

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THOMAS HUNT MORGAN

• Thomas Hunt Morgan
• Lord of the Flies
• Investigated several traits following an
  X-linked inheritance pattern
• Demonstrated that the genes governing these
  traits were located on the X chromosome
• Provided the first direct evidence that
  different genes are physically linked on the
  same chromosome

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THOMAS HUNT MORGAN

• Traits in Morgans cross
• The yellow body allele (y) is recessive to the
  gray body allele (y)
• The white eyed allele (w) is recessive to the red
  eyed allele (w)
• The miniature wing allele (m) is recessive to the
  normal winged allele (m)

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THOMAS HUNT MORGAN

• Traits in Morgans cross
• P generation yy ww mm x y w m Y
• F1 generation yy ww mm and y w m Y
• F2 generation
• 8 phenotypes present
• NOT equally represented

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THOMAS HUNT MORGAN

• Traits in Morgans cross
• P generation yy ww mm x y w m Y
• F1 generation yy ww mm and y w m Y
• F2 generation
• 758 y_w_m_
• 700 yywwmm
• 401 y_w_mm
• 317 yywwm_
• 16 y_wwmm
• 12 yyw_ m_
• 1 y_ wwm_
• 0 yy w_ mm

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THOMAS HUNT MORGAN

• High proportions of parental allele combinations
• Nonparental combinations also did exist
• 758 y_w_m_
• 700 yywwmm
• 401 y_w_mm
• 317 yywwm_
• 16 y_wwmm
• 12 yyw_ m_
• 1 y_ wwm_
• 0 yy w_ mm

? Parental
? Parental
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THOMAS HUNT MORGAN

• Morgans conclusions
• All three genes are located on the X chromosome
• Tend to be transmitted together as a unit
• Nonparental combinations of alleles resulted
  from crossing over
• Physical exchange of segments between X
  chromosomes
• Crossover frequency is dependent upon the
  distance between the genes
• Fewer crossovers between genes very close
  together

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THOMAS HUNT MORGAN

• Morgans conclusions
• Crossover frequencies between linked genes
  reflect the distance between these genes
• 758 y_w_m_
• 700 yywwmm
• 401 y_w_mm
• 317 yywwm_
• 16 y_wwmm
• 12 yyw_ m_
• 1 y_ wwm_
• 0 yy w_ mm

? Parental (no crossovers)
? Parental (no crossovers)
? Crossover between w and m
? Crossover between w and m
? Crossover between y and w
? Crossover between y and w
? Double crossover (y w, w m)
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THOMAS HUNT MORGAN

• Morgans conclusions
• Crossover frequencies reflect the distance
  between these genes
• No crossovers (most likely)
• 758 y_w_m_
• 700 yywwmm
• Crossover between w and m (fairly likely)
• 401 y_w_mm
• 317 yywwm_
• Crossover between y and w (unlikely)
• 16 y_wwmm
• 12 yyw_ m_
• Double crossover (very unlikely)
• 1 y_ wwm_
• 0 yy w_ mm

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CHI SQUARE ANALYSIS

• Chi square analysis can evaluate the fit between
  a hypothesis and observed experimental data
• Can be used to determine if data from a dihybrid
  vross is consistent with linkage or independent
  assortment
• Standard hypothesis Genes are not linked
• Determine whether data fit this hypothesis
• Low chi square value
• Cannot reject hypothesis
• Infer that genes assort independently
• High chi square value
• Reject hypothesis
• Conclude that the genes are linked

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CHI SQUARE ANALYSIS

• Insert chi square problem here

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CREIGHTON McCLINTOCK

• Obtained direct evidence that genetic
  recombination is due to crossing over
• Performed crosses in maize (Zea mays)
• Crosses involved parental chromosomes with
  unusual structural features
• Homologous chromosomes could be microscopically
  distinguished
• Recombinant offspring could be correlated with
  microscopically observable exchanges in segments
  of homologous chromosomes

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CREIGHTON McCLINTOCK

• Creighton and McClintock used a normal and an
  abnormal version of maize chromosome 9
• Abnormal version possessed two microscopically
  detectable features
• Darkly staining knob at one end
• Extra piece of a chromosome at the other end
• Translocation of a portion of chromosome 8

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CREIGHTON McCLINTOCK

• Creighton and McClintock studied genes at both
  ends of chromosome 9
• A gene governing colored (C) or colorless (s)
  kernels was located near the knobbed end of the
  chromosome
• A gene governing starchy (Wx) or waxy (wx)
  endosperm texture was located near the other end
  of the chromosome

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CREIGHTON McCLINTOCK

• Creighton and McClintocks cross
• Parent A
• Knobbed/translocated chromosome 9 (C wx)
• Cytologically normal chromosome 9 (c Wx)
• Parent B
• Two cytologically normal copies of chromosome 9
• Genotype cc Wxwx

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CREIGHTON McCLINTOCK

• Creighton and McClintocks cross
• Parent A can produce four different gametes
• Two are non-recombinant (parental)
• Two are recombinant
• Recombinant offspring always corresponded to
  microscopically observable exchanges between
  homologous chromosomes

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CREIGHTON McCLINTOCK

• Pairing chromosomes, heteromorphic in two
  regions, have been shown to exchange genes
  assigned to these regions

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MITOTIC CROSSING OVER

• Several rounds of mitosis generally follow
  fertilization in multicellular organisms
• Mitosis normally does not involve the pairing of
  homologous chromosomes
• Crossing over does not normally occur during
  mitosis
• Mitotic crossing over does sometimes occur
• Mitotic recombination

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MITOTIC CROSSING OVER

• Mitotic recombination
• Rare
• Produces recombinant chromosomes
• May possess a new combination of alleles
• Some happen early in development
• Produces a patch of tissue in the adult
• e.g., Unusual patches on Drosophila bodies are
  due to mitotic recombination

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MITOTIC CROSSING OVER

• yy snsn x y sn Y
• Yellow vs. grey body
• Singed vs. normal bristles
• yy snsn zygote formed
• Develops into embryo, then adult
• Wild-type coloration and bristles

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MITOTIC CROSSING OVER

• yy snsn zygote develops into embryo
• Mitotic recombination occurs in one embryonic
  cell
• Recombinant chromosomes possess new
  combinations of alleles

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MITOTIC CROSSING OVER

• Mitotic recombination occurs in one embryonic yy
  snsn cell
• Cell divides to produce two adjacent cells
• Different genotypes
• yy snsn yy snsn
• Mitotic division expands each cell into a patch
  of cells
• yy snsn ? grey, singed
• yy snsn ? yellow, normal
• Twin spot

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GENETIC MAPPING

• Genetic mapping
• Gene mapping
• Chromosome mapping
• Determines linear order and distance between
  genes linked along the same chromosome
• Each gene has its own locus at a particular
  site within a chromosome

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GENETIC MAPPING

• Genetic maps are useful in many ways
• Allows an understanding of the overall complexity
  and genetic organization of a particular species
• Comparison of genetic maps between different
  species improves our understanding of their
  evolutionary relationships

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GENETIC MAPPING

• Genetic maps are useful in many ways
• Information can be used to diagnose and
  potentially to treat mapped human diseases
• Helps genetic counselors predict the likelihood
  of couples transmitting inherited diseases
• Can provide plant and animal breeders with
  important information for selective breeding
  programs

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GENETIC MAPPING

• The linear order of genes can be deduced from the
  frequency of non-parental offspring
• Genetic linkage maps can be produced
• Useful for analyzing organisms easily crossed to
  produce numerous offspring in short periods of
  time
• e.g., Drosophila, several plant species, etc.
• Traditional mapping approaches are difficult for
  many organisms
• e.g., Humans, etc.
• Alternative methods of gene mapping have been
  developed
• See chapter 20

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GENETIC MAPPING

• The frequency of recombination is correlated to
  the distance between genes
• Crossovers are unlikely between two genes very
  close together on a chromosome
• Tightly linked genes
• Few recombinant offspring will be produced
• Crossovers are more likely between two genes
  located far apart on a chromosome
• Alleles of such genes are more likely to
  recombine
• Many recombinant offspring will be produced

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GENETIC MAPPING

• One can distinguish recombinant from parental
  offspring when conducting a testcross
• An individual heterozygous for two or more genes
  is crossed to an individual homozygous
  recessive for these genes

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GENETIC MAPPING

• se se se se testcross
• Parental offspring
• 542 se se
• 537 se se
• Recombinant offspring
• 76 se se
• 75 se se

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GENETIC MAPPING

• Distances between genes are measured in map units
• 1 map unit equals 1 recombination frequency
• Also called centiMorgans
• In honor of Thomas Hunt Morgan

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GENETIC MAPPING

• The map distance between two genes can be
  calculated
• (Recombinant offspring / total offspring) x 100
• (76 75 / 542 537 76 75) 100
• (151 / 1230) 100
• 12.3 map units
• 12.3 centiMorgans

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STURTEVANTS GENETIC MAPS

• Alfred Sturtevant
• Constructed the first genetic map in 1911
• Undergraduate in T. H. Morgans lab
• Studied the inheritance of six recessive X-linked
  alleles (of five different genes)
• y (yellow body color)
• w (white eye color)
• w-e (eosin eye color)
• v (vermilion eye color)
• m (miniature wings)
• r (rudimentary wings)

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STURTEVANTS GENETIC MAPS

• Sturtevants hypothesis
• The distance between genes can be estimated from
  the proportion of recombinant offspring
• Sturtevants experiment
• Various crosses between females heterozygous for
  two genes and doubly recessive males
• Pairwise testcrosses

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STURTEVANTS GENETIC MAPS

• Sturtevants data
• Percent recombinant offspring calculated for each
  pair of alleles

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STURTEVANTS GENETIC MAPS

• Interpreting the data
• Map distances likely to be more accurate between
  genes that are closely linked
• y w are 1.0 map unit apart
• v and r are 3.0 map units apart
• r and m are 23.9 map units apart
• w and v are 29.7 map units apart

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STURTEVANTS GENETIC MAPS

• Interpreting the data
• Other features of the data allowed the
  determination of gene order
• v is between w and r
• w and r show a 33.7 recombinant frequency
• w and v show a 29.7 recombinant frequency
• v and r show a 3.0 recombinant frequency
• etc.

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STURTEVANTS GENETIC MAPS

• Interpreting the data
• As the percentage of recombinant offspring
  approaches 50, this value becomes less accurate
  as a measure of map distance

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GENETIC MAPPING

• Sturtevant used multiple dihybrid testcrosses to
  make his genetic maps
• Trihybrid crosses can be used to determine the
  order and distance between linked genes

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GENETIC MAPPING

• Trihybrid crosses
• Cross true-breeding strains differing in three
  alleles
• Perform a testcross with an F1 female
• Crossovers can produce recombinant gametes in F1
  females
• Collect data from the F2 generation
• Eight possible phenotypes
• 2 parental, 6 recombinant
• Construct linkage map

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GENETIC MAPPING

• Trihybrid testcrosses yield eight possible
  phenotypes in the F2 generation
• 2 parental
• 6 recombinant
• 2 from a single crossover
• 2 from another single crossover
• 2 from a double crossover

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GENETIC MAPPING

• One can compute the likelihood of double
  crossovers in trihybrid testcrosses
• Product rule is used
• Observed numbers of double crossovers are often
  lower than expected numbers
• Result of positive interference

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GENETIC MAPPING

• Interference
• The occurrence of a crossover decreases the
  probability that a second crossover will happen
  nearby
• The first crossover interferes with the ability
  to form a second crossover in the immediate
  vicinity
• Interference 1 coefficient of coincidence (C)
• C observed double crossovers / expected doubles

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MAPPING IN HAPLOID EUKS

• Certain species of lower eukaryotes have also
  been used in mapping studies
• e.g., Unicellular algae and fungi
• May be unicellular or multicellular
• Spend part of their life cycle in a haploid state

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MAPPING IN HAPLOID EUKS

• Fugal cells are typically haploid
• Reproduce asexually by mitosis
• Can reproduce sexually by the fusion of two
  haploid cells to form a zygote
• Zygote can proceed through meiosis to produce
  four haploid spores
• This group of four spores is called a tetrad
• (Not to be confused with a tetrad of four sister
  chromatids)
• In some species, meiosis is followed by a single
  round of mitosis to produce eight spores

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MAPPING IN HAPLOID EUKS

• Fugal cells are typically haploid
• Reproduce asexually by mitosis
• Can reproduce sexually by the fusion of two
  haploid cells to form a zygote
• Zygote can proceed through meiosis to produce
  four haploid spores
• This group of four spores is called a tetrad
• (Not to be confused with a tetrad of four
  sister chromatids)

This is an alga, not a fungus, but the life cycle
is similar.
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MAPPING IN HAPLOID EUKS

• Ascomycetes
• Sac fungi
• Products of a single meiotic division are
  contained within a sac known as an ascus
• Each ascus contains four (or eight) spores
• These spores are called ascospores
• Asci can be dissected and each haploid spore
  studied

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TETRADS

• The arrangement of spores within an ascus varies
  between species
• Unordered tetrads
• Spores of a tetrad randomly mix together
• e.g., Saccharomyces cerevisiae, Chlamydomonas
  rheinhardii, etc.
• Ordered tetrads
• Tight ascus prevents spores from mixing
• e.g., Neurospora crassa, etc.

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TETRADS

• The arrangement of spores within an ascus varies
  between species
• Unordered tetrads
• Spores of a tetrad randomly mix together
• e.g., Saccharomyces cerevisiae, Chlamydomonas
  rheinhardii, etc.
• Ordered tetrads
• Tight ascus prevents spores from mixing
• e.g., Neurospora crassa, etc.

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ORDERED TETRADS

• Ordered tetrads
• Position and order of spores within the ascus
  reflects their relationship to each other as they
  were produced
• Allows mapping of genes relative to the
  centromere
• Centromere is cytologically visible
• Correlates a genes location with cytological
  characteristics of a chromosome

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ORDERED TETRADS

• Ordered tetrads
• Segregation patterns can be used to map genes
  relative to the centromere

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UNORDERED TETRADS

• Unordered tetrads
• Contain products of a single meiosis event
• Spores are randomly arranged within ascus
• Can be used to map genes in dihybrid crosses

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UNORDERED TETRADS

• Ura arg x ura-2 arg-3
• Three possible combinations of four haploid cells
• Four spores with the parental allele combination
• Parental ditype (PD)
• Four spores with the nonparental allele
  combination
• Nonparental ditype (NPD)
• Two parental and two nonparental allele
  combinations
• Tetratype (T)

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UNORDERED TETRADS

• Independent assortment
• 50 PD, 50 NPD are expected
• Linkage
• No crossover yields PD
• Single crossover yields T
• Double crossovers can yield PD, T, or NPD

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UNORDERED TETRADS

• Double crossovers can yield PD, T, or NPD
• Four chromatid double crossover yields NPD
• Three chromatid double crossover yields T
• Two chromatid double crossover yields PD

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UNORDERED TETRADS

• Data from a tetrad analysis can be used to
  calculate the map distance between two linked
  genes
• Map distance is calculated as the percentage of
  offspring that carry recombinant chromosomes
• Map distance (NPD ½ T / total asci) 100
• (single X tetrads 2 double X / total
  tetrads) 100