Download Ne - reproseed

Molecular genetic tools for management of
inbreeding, domestication and selection
Pierre Boudry
Objectives within ReproSeed
• Task 5.3 Quality of seed (S. Lapègue, Ifremer)
– 5.3.1 Genetic diversity of seed
-
Characterize the genetic diversity and allow traceability of C. gigas, M. edulis, and
P. maximus batches from hatcheries
Calculate the real number of genitors contributing to a seed production : effective
population size.
Comparative study of the genetic diversity of seed productions sampled (1) in the
wild and (2) in hatcheries.
This talk :
• General considerations about genetic diversty and parentage analyses
• ReproSeed results on P. maximus (R. Morvezen, LEMAR)
Molecular genetic tool box
Characteristics of a molecular marker:
- dominant / co-dominant
- mono-locus / multi-locus
- low throughput / high throughput : ease and cost to
genotype large number of markers on large number of
individuals.
Molecular genetic tool box
Evolution of techniques from the 80’s:
Ex. in C. gigas
- Allozymes : ≈ 10-15 loci
- RFLPs : mitochondrial and nuclear loci, low through-put
- AFLPs : hundreds of loci in “bulk”, dominant
- Microsatellites : ≈ 300 loci available, highly variable
- SNPs : ≈ > 3000 loci available, mostly biallellic
Molecular markers
•
Size polymorphism : microsatellites
(TCCA)7 : GAGGATGATCCATCCATCCATCCATCCATCCATCCATCGGATGG
(TCCA)11 : GAGGATGATCCATCCATCCATCCATCCATCCATCCATCCATCCATCCATCCATCGGATGG
Genotyping by electrophoresis of DNA molecules
•
Sequence polymorphism : SNPs
GAGGATGATTCGGATGG
GAGGATGAATCGGATGG
Genotyping by hybridization (e.g. microarrays), MALDI-TOF Mass spectrometry…
From 1 to > 900.000 loci.
Molecular genetic tool box
microsatellites
12 previouslyidentified loci
grouped in 4
multiplex sets
12 new loci
grouped in 3
multiplex sets
SNPs in C. gigas
Assessment of genetic variability
Parameters :
- Observed heterozygocity
- Allelic richness : number of alleles present in a given sample
taking into account its size
- Effective population size (Ne) or estimated breeding size (Nb)
Effective population size (Ne)
•
Ne is the number of breeding individuals corresponding to an observed
amount of genetic drift. It reflects the harmonic mean size over the population’s
entire history.
Wright (1938):
Ne =
4N – 2
Vk +2
Vk = variance in the number of gametes contributing to
the next generation
•
Ne is equivalent to the number of spawners that successfully and
equally contribute to the next generation
•
Ne is not necessary the number of breeding individuals
•
Ne is often much smaller than N
Genetic diversity in hatchery populations
•
Due to the high fecundity of molluscs, a few adults can generate very large
progenies, with small Ne
•
Estimates based on
- temporal variance in allelic frequencies (F) of neutral markers after t generations
in a sample of size S (Waples, 1989; Hedgecock et al., 1992):
E(F) = t/(2Ne) + 1/(2S0) + 1/(2St)
- observed number of offspring of male i and female j in a given sample
(Robertson, 1961) :
Parentage analyses
- Determine relatedness between pairs of individuals based on
shared alleles (identical by descent) at molecular markers :
• Offspring to its potential parent(s)
• Individuals of a population (i.e. relatedness)
7 bi-allelic loci
Offspring
Aa
BB
Cc
DD
Ee
ff
GG
Potential parents
Aa
aa
Aa
BB
BB
Bb
cc
Cc
cc
DD
Dd
DD
EE
EE
EE
Ff
Ff
Ff
GG
Gg
GG
Excluded parents
Aa
Aa
Aa
bb
BB
bb
cc
Cc
cc
dd
DD
DD
eE Ff GG
EE FF gg
EE Ff gg
Parentage analyses
Allocation success depends on number of loci and number of alleles
Simulations based on 20 parents
Bernatchez and Duchesne (2000)
Parentage analyses
Outcome : inferred relatedness between individuals
parent-offspring, full sibs, half sibs…
Methods and softwares:
• Maximum likelihood (CERVUS, PAPA,…)
– Looks for the most likely parental couple
– Always gives a solution !
• Exclusion (PROBMAX, VITASSIGN, FAP):
– Checks compatibility of offspring and parental
genotypes with Mendelian inheritance
– Highly sensitive to genotyping errors, but this can
be corrected with high power (>99%) of locus set
and allowing for mismatches
– No match ? genotyping error or “foreign” individual.
Parentage analyses
What for ?
• Assess reproductive success of parents
• Estimate Ne or Nb of populations
Assess / verify pedigree
Tracability
Control inbreeding of captive populations
Avoid inbreeding depression
Manage genetic diversity of exploited species
Impact on wild / native populations
- Gametes from each of the individuals used as female were fertilized by 3−4 males.
- 24 crosses were performed with 11 individuals used only as females, 13 used both
as females and as males, and 2 used only as males.
Parents
Offspring
Cervus 3.0 output
Ne = 33 (CI 95%, 31–69) by the SA method, and Ne = 24.3 (CI 95%, 22.4–26.3) by
the LD method (using COLONY).
•
Tinduff : 49 genitors
•
Roscanvel : wild
•
Le Fret : hatchery progeny
•
L’Auberlac’h : hatchery progeny
Le Fret : 82% assigned to their parents
L’Auberlac’h : only 12% assigned to the parental population BUT many full-sibs
Roscanvel : 1% assigned, 2 full-sibs
L’Auberlac’h sample was also born in the hatchery BUT from other parents
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6 microsatellite loci
Mean number of allele/locus : 12.7
9 males x 5 females
42 FS families
270 one-year old offspring analysed
4 traits recorded
Parentage analyses
What for ?
• Phenotypic data on related individuals allows to estimate
heritability of traits
Estimate heritability using pedigreed populations
wildly used in fish
New approaches in selective breeding
Walk-back selection : Doyle & Herbinger, 1994
The use of DNA fingerprinting for high-intensity
within-family selection in fish breeding
• Steps of proposed selection procedure:
o Phenotyping to select candidates
o Genotyping highest ranked candidates to identify their parents
o estimation of parental breeding value of parents (BLUP)
• Efficiency tested by simulation
Conclusions
• ‘Tools’ are now available - or can now be easily
developed - to assess diversity and perform
parentage assignment in aquacultured mollusks
species but their use is still relatively limited for
effective management of inbreeding,
domestication and selection of marine mollusks.
• The ‘next step’ will be the use of QTLs / genomewide approaches in selective breeding programs