# Molecular architecture of agronomical traits in sheep

Julie Demars, Laurence Drouilhet, Stéphane Fabre, Gwenola Tosser-Klopp

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Julie Demars, Laurence Drouilhet, Stéphane Fabre, Gwenola Tosser-Klopp. Molecular architecture of agronomical traits in sheep. École thématique. Cours européen pour étudiants et éleveurs "Genetics and genomics in livestock", Beni-Mellal - INRA-M, Morocco. 2020. hal-04803932

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# Molecular architecture of agronomical traits in sheep

J. Demars, L. Drouilhet, S. Fabre, G. Tosser-Klopp

GenPhySE unit - GENROC team
http://genphyse.toulouse.inra.fr/groups/genroc

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INRA
la science pour la vie, l'humain, la terre

INP ENSAT

envt

École nationale vétérinaire toulouse

<figure><img src="image_6.png" /></figure>

Université
de Toulouse

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Genetics and definition

* Science of **heredity**

* Genetics science studies **inheritance** of **traits** across generations and their variations
# Trait and Phenotype

* In genetics, the **phenotype** is the state of an observable and quantifiable trait

* Phenotype that can be measured **objectively**

* Values are **variable** within the population

* The phenotype is the result of the **action of the genes** that determine it, in an **environmental context**

$$P = \boxed{G} + E$$

* **Simple** or Monogenic trait vs. **Complex** or Polygenic trait
# Phenotypic variability

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>
## Simple or Monogenic trait

* A trait is called « Simple » if the various alternatives are **clearly distinguishable** from each other and are **few in number**

* Discontinuous distribution

* Expression of simple trait depends mainly on **major genes** that are slightly or none influenced by the environment

* Trait that can be summarized as a **binary** form
# Simple or Monogenic trait

<figure><img src="image_12.png" /><figcaption>## Hornless goat</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>## Horned goat</figcaption></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>
Complex or Polygenic trait

* A trait is called « Complex » if the various alternatives and are **numerous** in a population and each phenotype describes only a small number of individuals

• Discrete or continuous distribution

* A complex trait is usually dependent on **several genes** and is **highly influenced by the environment**

• Quantitative trait or QTL “Quantitative Trait Locus”
Complex or Polygenic trait

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>
The genome carries the genetic information

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>
# Genetic variability = polymorphisms

* **Macroscopic scale**

Chromosomes number

Duplications, rearrangments, deletions...

* **Intermediate scale**

Repeat sequences

Transposable elements

Short deletions, tandem repeats (*microsatellites*)

* **Microscopic scale**

Single Nucleotide Polymorphisms (**SNP**)

Insertion or deletion of 1 nucleotide (Indel)
Molecular markers

* Most common molecular markers : microsatellites, SNP

Microsatellites = (di-tri...)nucleotides in tandem repeats

SNP = Single Nucléotide Polymorphism

* Molecular marker ≠ Mutation

* A « good » molecular marker is neutral, polymorph, codominant, environment insensitive, non-epistatic, multi-allelic
# The linkage disequilibrium (LD)

## Quantification d'une association préférentielle de deux allèles

$$f_A = p$$

<figure><img src="image_20.png" /></figure>

$$f_G = q$$

$$p + q = 1$$

$$f_T = r$$

$$f_C = s$$

$$r + s = 1$$

<figure><img src="image_21.png" /></figure>

Situation en
équilibre

$$f_{AC} = ps$$

$$f_{AT} = pr$$

$$f_{GC} = qs$$

$$f_{GT} = qr$$
# The linkage disequilibrium (LD)

## Quantification d'une association préférentielle de deux allèles

$$f_A = p$$

<figure><img src="image_22.png" /></figure>

$$f_G = q$$

$$p + q = 1$$

$$f_T = r$$

<figure><img src="image_23.png" /></figure>

$$f_C = s$$

$$r + s = 1$$

<figure><img src="image_24.png" /></figure>

Situation en
déséquilibre

<figure><img src="image_25.png" /></figure>

$$f_{AT} = pr$$

$$f_{GC} = qs$$

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>
The size of LD will depend on ...

• Du nombre de génération : $D_{t+1} = D_0 (1-q)^t$

• De la **taille efficace** de la population : $E(r^2) = \frac{1}{(1+4N)}$

• Des **mécanismes méiotiques** comme les hot-spots de recombinaison
# The positional cloning strategy

The phenotype is the result of the **action of the genes**
that determine it, in an **environmental context**

$$P = G + E$$

Characterizing the G component and identifying
mutations underlying the phenotypic variability
The 3 components for positional cloning

* The studied trait (**phenotypes**)

* The genome variability (**molecular markers**)

* The protocol/design (**individuals**)

* The trait determinism

<figure><img src="image_28.png" /></figure>

Identifying a co-segregation of a DNA
polymorphism and a phenotype
# Number and type of molecular for positional cloning

* Markers **homogeneously spanned** along the genome

* Evolution of both **number** and **type** of molecular markers

* Progress related to **technological advances**

<table><thead><tr><th></th><th>1980</th><th>1990</th><th>2000</th><th>2010</th><th>2020</th></tr></thead><tbody><tr><td>Type</td><td>Microsatellites</td><td>SNPs</td><td>Polymorphisms</td><td></td><td></td></tr><tr><td>Number</td><td>200</td><td>1536, 60K, 500K...</td><td>All</td><td></td><td></td></tr><tr><td>Density</td><td>(≈ 1/15 Mb)</td><td></td><td>1 bp</td><td></td><td></td></tr><tr><td>Methodology</td><td>Size analysis</td><td>SNP array</td><td>Sequencing</td><td></td><td></td></tr><tr><td>Resolution</td><td>30 Mb</td><td>10 Mb à 1Mb</td><td>Mutation</td><td></td><td></td></tr></tbody></table>
# SNP array, SNP beadchip

* Common methodology, **quick, flexible and cheap**

* Simultaneous analysis of thousands of SNPs

* Reduced representation of the whole genome variability

* 2 benchmark technologies

- Affymetrix : **oligonucleotides array**

- Illumina : **allele-specific PCR**

AFFYMETRIX®

<figure><img src="image_29.png" /></figure>

Genomic DNA (250 ng)

<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

RE Digestion

Adapter
Ligation

Fragmentation
and End-Labeling

<figure><img src="image_33.png" /></figure>

Hybridization
& Wash

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

illumina®

<figure><img src="image_36.png" /></figure>

A

Infinium I
Allele-Specific Primer Extension

<figure><img src="image_37.png" /></figure>

B

Infinium II
Single Base Extension

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>
# The milestones for positional cloning

<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /></figure>

<figure><img src="image_43.png" /></figure>

<figure><img src="image_44.png" /></figure>
# Molecular architecture of fleece variation

Mol Biol Evol. 2017 Jul 1;34(7):1722-1729. doi: 10.1093/molbev/msx114.

## Genome-Wide Identification of the Mutation Underlying Fleece Variation and Discriminating Ancestral Hairy Species from Modern Woolly Sheep.

Demars J¹, Cano M², Drouilhet L¹, Plisson-Petit F¹, Bardou P¹,³, Fabre S¹, Servin B¹, Sarry J¹, Woloszyn F¹, Mulsant P¹, Foulquier D⁴, Carrière F⁴, Aletru M¹,⁵, Rodde N⁶, Cauet S⁶, Bouchez O¹,⁷, Pirson M¹,⁷, Tosser-Klopp G¹, Allain D¹.
# Fleece variation under the sheep domestication

* Variation in composition and structure of the fleece

* Hairy sheep vs. woolly sheep

<figure><img src="image_45.png" /></figure>

Huber, B. (2013), Introduction to Anthropology
# Fiber characteristics in hairy and woolly sheep

<figure><img src="image_46.png" /></figure>

wool

hair

heterotype

<figure><img src="image_47.png" /></figure>
# Hairy and woolly sheep in modern breeds

## • Hairy sheep

«sheeps whose coats consist of hair, more like that of cattle and goats, rather than wool», Sheep Production Handbook

## • Phenotypes (**hairy** vs. **woolly**) easily detectable on lambs

○ **Hairy** long coat ≈ 23.1 mm

<figure><img src="image_48.png" /><figcaption>St Croix</figcaption></figure>

<figure><img src="image_49.png" /><figcaption>Romanov</figcaption></figure>

<figure><img src="image_50.png" /><figcaption>Blackbelly</figcaption></figure>

<figure><img src="image_51.png" /><figcaption>Navajo-Churro</figcaption></figure>

○ **Woolly** short coat ≈ 8.3 mm

<figure><img src="image_52.png" /><figcaption>Texel</figcaption></figure>

<figure><img src="image_53.png" /><figcaption>Merino</figcaption></figure>

<figure><img src="image_54.png" /><figcaption>Berrichon du Cher</figcaption></figure>

<figure><img src="image_55.png" /><figcaption>Noire du Velay</figcaption></figure>

adapted from Allain et al. JAS 2013
# The fleece variation, a major agronomic trait

## • Important survival component

Less total mortality rate in long hairy bearing coats lambs

<figure><img src="image_56.png" /></figure>
# The fleece variation, a major agronomic trait

* **Important survival component**

* Less total mortality rate in long hairy bearing coats lambs

* More heat losses in woolly bearing coat lambs

* Long hairy coat lambs are more resistant to weather changes

* **Heritability of lamb birthcoat type**

* Binary trait, heritability = 0.85

* Continuous trait, heritability = 0.56

* **Sheep breeding permanent outdoor**

* Economical and agronomical trait = Importance to use in selection

* French Romane breed, high productive potential breed
# Description of the study design

## A familial strategy

The French Romane breed

Romanov
hairy

Berrichon du Cher
woolly

<figure><img src="image_57.png" /></figure>

<figure><img src="image_58.png" /></figure>

Romane

<figure><img src="image_59.png" /></figure>

Romane

<figure><img src="image_60.png" /></figure>

<figure><img src="image_61.png" /></figure>

Hairy lamb

<figure><img src="image_62.png" /></figure>

<figure><img src="image_63.png" /></figure>

Woolly lamb

<figure><img src="image_64.png" /></figure>

<figure><img src="image_65.png" /></figure>

## Animals

Half-sib QTL design in Romane
8 sires Romane * dams Romane

## Phenotype

Lenght of lamb's fleece (quantitative)
Type of lamb's fleece (binary)

<figure><img src="image_66.png" /></figure>
# Description of the study design

## A familial strategy

### The French Romane breed

<figure><img src="image_67.png" /></figure>

## Animals

Half-sib QTL design in Romane
8 sires Romane * dams Romane

## Phenotype

Lenght of lamb's fleece (quantitative)
Type of lamb's fleece (binary)

## Genotype

OvineSNP50 Genotyping BeadChip (n = 759)
Ovine Infinium® HD SNP BeadChip (n = 135)

## Methodology

Imputation
Genome-Wide Association Analysis (GWAS)
Linkage Analysis
# Two loci underlie fleece variation in Romane

## Genetic determinism

* 66% of hairy lambs vs. 33% of woolly lambs

* **Hairy dominant** allele from **Romanov (Q)**

* **Woolly recessive** allele from **Berrichon du Cher (q)**

<figure><img src="image_68.png" /></figure>

QTL<sub>Oar25</sub>

* 40% of phenotypic var.

* Interval ≈ 1,6 Mb

* 3/8 rams Qq

* Selective sweep

QTL<sub>Oar13</sub>

* 15% of phenotypic var.

* Interval ≈ 2 Mb

* 5/8 rams Qq

* Specific of Romane

Position on chromosomes
# The QTL<sub>Oar25</sub> maps to a 198 Kb interval

<figure><img src="image_69.png" /></figure>
# Fine-mapping of the QTL<sub>Oar25</sub>

## A familial strategy

The French Romane breed

* **Animals**

Half-sib QTL design in Romane

8 sires Romane * dams Romane

* **Phenotype**

Lenght of lamb's fleece (quantitative)

Type of lamb's fleece (binary)

* **Genotype**

OvineSNP50 Genotyping BeadChip (n = 759)

Ovine Infinium® HD SNP BeadChip (n = 135)

* **Methodology**

Identity By State Haplotype Identification

## A population strategy

Ancestral species and French breeds

* **Hairy ancestral species**

Ovis orientalis (n = 15)

Ovis musimon (n = 18)

Ovis canadensis (n= 3)

Ovis dalli (n = 2)

* **Hairy domestic breeds**

(6 breeds and 116 individuals)

Causse du Lot, Corse, Limousine, Manech
tête rousse, Rava, **Romanov**

* **Woolly modern breeds**

(18 breeds and 402 individuals)

Berrichon du Cher, Blanche du Massif Central,
Charmoise, Charollais, Île-de-France, Lacaune (lait),
Lacaune (viande), **Mérino d'Arles**, Mérino de
Rambouillet, Mourerous, Ouessant, Préalpes du Sud,
Rouge de l'Ouest, Roussin de la Hague, Suffolk,
Tarasconnaise, Texel, Vendéen
# Fine-mapping of the QTL<sub>Oar25</sub>

## A familial strategy

The French Romane breed

* **Animals**

Half-sib QTL design in Romane

8 sires Romane * dams Romane

* **Phenotype**

Lenght of lamb's fleece (quantitative)

Type of lamb's fleece (binary)

* **Genotype**

OvineSNP50 Genotyping BeadChip (n = 759)

Ovine Infinium® HD SNP BeadChip (n = 135)

* **Methodology**

Identity By State Haplotype Identification

## A population strategy

Ancestral species and French breeds

* **Animals**

**Hairy** ancestral species (n = 38)
(*Ovis musimon...*)

**Hairy** domestic breeds (n = 116)
(Romanov...)

**Woolly** modern breeds (n = 402)
(Berrichon du Cher, Merino d'Arles...)

* **Phenotype**

None (sheep breed standards)

* **Genotype**

Ovine Infinium® HD SNP BeadChip (n = 554)

* **Methodology**

Haplotypes in the QTL<sub>Oar25</sub> interval
# The QTL<sub>Oar25</sub> encompasses the IRF2BP2 gene

<figure><img src="image_70.png" /></figure>

<figure><img src="image_71.png" /></figure>
# The mutation is an insertion within IRF2BP2

## Whole Genome Sequencing

* 2 Romanov animals (**hairy** and $Q_{Oar25}/Q_{Oar25}$)

* 3 Berrichon du Cher animals (**woolly** and $q_{Oar25}/q_{Oar25}$)

* 2 QTL rams heterozygous ($Q_{Oar25}/q_{Oar25}$)

<figure><img src="image_72.png" /></figure>
# The insertion is an antisense EIF2S2 retrogene

* The ancestral hairy IRF2BP2<sup>wt</sup> allele

<figure><img src="image_73.png" /></figure>

* The modern woolly IRF2BP2<sup>asEIF2S2</sup> allele

<figure><img src="image_74.png" /></figure>
# The insertion segregates in the Romane breed

<figure><img src="image_75.png" /></figure>

1- QTL ram 1 Q<sub>Oar25</sub> / q<sub>Oar25</sub>

2- QTL ram 2 q<sub>Oar25</sub> / q<sub>Oar25</sub>

3- QTL ram 3 q<sub>Oar25</sub> / q<sub>Oar25</sub>

4- QTL ram 4 Q<sub>Oar25</sub> / q<sub>Oar25</sub>

5- QTL ram 5 q<sub>Oar25</sub> / q<sub>Oar25</sub>

6- QTL ram 6 Q<sub>Oar25</sub> / q<sub>Oar25</sub>

7- QTL ram 7 q<sub>Oar25</sub> / q<sub>Oar25</sub>

8- QTL ram 8 q<sub>Oar25</sub> / q<sub>Oar25</sub>

<figure><img src="image_76.png" /></figure>

Position on
chromosomes

## • Genotyping of the whole QTL protocol

o the signal disappeared when the insertion is used as a fixed effect

o the insertion explained 65% of the phenotypic variance
# The insertion discriminates hairy / woolly sheep

<table><thead><tr><th>Breed</th><th>Genotyped animals (n)</th><th>Phenotype</th><th>Frequency (IRF2BP2<sup>wt</sup>)</th><th>Frequency (IRF2BP2<sup>asEIF2S2</sup>)</th></tr></thead><tbody><tr><td>Ovis orientalis</td><td>15</td><td>Hairy</td><td>1.00</td><td></td></tr><tr><td>Ovis musimon</td><td>18</td><td>Hairy</td><td>1.00</td><td></td></tr><tr><td>Ovis canadensis</td><td>3</td><td>Hairy</td><td>1.00</td><td></td></tr><tr><td>Ovis dalli</td><td>2</td><td>Hairy</td><td>1.00</td><td></td></tr><tr><td>Causses du Lot</td><td>20</td><td>Hairy</td><td>0.85</td><td>0.15</td></tr><tr><td>Corse</td><td>16</td><td>Hairy</td><td>1.00</td><td></td></tr><tr><td>Limousine</td><td>18</td><td>Hairy</td><td>0.94</td><td>0.06</td></tr><tr><td>Manech tête rousse</td><td>25</td><td>Hairy</td><td>1.00</td><td></td></tr><tr><td>Rava</td><td>19</td><td>Hairy</td><td>0.92</td><td>0.08</td></tr><tr><td>Romanov</td><td>18</td><td>Hairy</td><td>0.94</td><td>0.06</td></tr><tr><td></td><td>154</td><td></td><td>0.96</td><td>0.04</td></tr><tr><td>Berrichon du Cher</td><td>35</td><td>Woolly</td><td></td><td>1.00</td></tr><tr><td>Blanche du Massif Central</td><td>20</td><td>Woolly</td><td></td><td>1.00</td></tr><tr><td>Charmoise</td><td>22</td><td>Woolly</td><td></td><td>1.00</td></tr><tr><td>Charollais</td><td>22</td><td>Woolly</td><td></td><td>1.00</td></tr><tr><td>Île-de-France</td><td>23</td><td>Woolly</td><td></td><td>1.00</td></tr><tr><td>Lacaune (lait)</td><td>35</td><td>Woolly</td><td></td><td>1.00</td></tr><tr><td>Lacaune (viande)</td><td>34</td><td>Woolly</td><td></td><td>1.00</td></tr><tr><td>Mérino d'Arles</td><td>18</td><td>Woolly</td><td></td><td>1.00</td></tr><tr><td>Mérino de Rambouillet</td><td>27</td><td>Woolly</td><td></td><td>1.00</td></tr><tr><td>Mourerous</td><td>16</td><td>Woolly</td><td>0.06</td><td>0.94</td></tr><tr><td>Ouessant</td><td>18</td><td>Woolly</td><td></td><td>1.00</td></tr><tr><td>Préalpes du Sud</td><td>17</td><td>Woolly</td><td>0.03</td><td>0.97</td></tr><tr><td>Rouge de l'Ouest</td><td>16</td><td>Woolly</td><td></td><td>1.00</td></tr><tr><td>Roussin de la Hague</td><td>21</td><td>Woolly</td><td></td><td>1.00</td></tr><tr><td>Suffolk</td><td>18</td><td>Woolly</td><td></td><td>1.00</td></tr><tr><td>Tarasconnaise</td><td>15</td><td>Woolly</td><td></td><td>1.00</td></tr><tr><td>Texel</td><td>24</td><td>Woolly</td><td>0.04</td><td>0.96</td></tr><tr><td>Vendéen</td><td>21</td><td>Woolly</td><td></td><td>1.00</td></tr><tr><td></td><td>402</td><td></td><td>0.01</td><td>0.99</td></tr></tbody></table>
# The IRF2BP2/asEIF2S2 mRNA is transcribed

* The **IRF2BP2** transcript

<figure><img src="image_77.png" /></figure>

* The chimeric **IRF2BP2/asEIF2S2** transcript

<figure><img src="image_78.png" /></figure>

* Genotyping of RNA extracted from skin

<figure><img src="image_79.png" /></figure>
# A double-stranded EIF2S2 RNA is created

<figure><img src="image_80.png" /></figure>

## RNAseA protection assay

○ ribonuclease that cleaves only ssRNA (EIF2S2 dsRNA is protected)

○ *IRF2BP2*<sup>wt</sup>/IRF2BP2<sup>wt</sup> versus IRF2BP2<sup>asEIF2S2</sup>/IRF2BP2<sup>asEIF2S2</sup>

<figure><img src="image_81.png" /><figcaption>① IRF2BP2 ssRNA</figcaption></figure>

<figure><img src="image_82.png" /></figure>

<figure><img src="image_83.png" /><figcaption>② EIF2S2 ssRNA</figcaption></figure>

<figure><img src="image_84.png" /></figure>

<figure><img src="image_85.png" /><figcaption>③ EIF2S2 dsRNA</figcaption></figure>

<figure><img src="image_86.png" /></figure>
# The dsRNA alters EIF2S2 and IRF2BP2 stability

<figure><img src="image_87.png" /></figure>
# Molecular architecture of prolificacy in Grivette and Olkuska breeds

PLoS Genet. 2013 Apr;9(4):e1003482. doi: 10.1371/journal.pgen.1003482. Epub 2013 Apr 25.

Genome-wide association studies identify two novel BMP15 mutations responsible for an atypical hyperprolificacy phenotype in sheep.

Demars J¹, Fabre S, Sarry J, Rossetti R, Gilbert H, Persani L, Tosser-Klopp G, Mulsant P, Nowak Z, Drobik W, Martyniuk E, Bodin L.
Sheep as a valuable model to study prolificacy

<table><thead><tr><th>Gene (Chromosome)</th><th>Mutation</th><th>Name (Fecundity)</th><th>Phenotype Heterozygous</th><th>Homozygous</th><th>Founder Breed</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="6">BMP15 (OARX)</td><td>V299D</td><td>Inverdale, <i>FecX</i><sup>I</sup></td><td>Hyper prolific</td><td>Sterile</td><td>Romney</td><td>Galloway et al.</td></tr><tr><td>Q291ter</td><td>Hanna, <i>FecX</i><sup>H</sup></td><td>Hyper prolific</td><td>Sterile</td><td>Romney</td><td>Galloway et al.</td></tr><tr><td>S367I</td><td>Belclare, <i>FecX</i><sup>B</sup></td><td>Hyper prolific</td><td>Sterile</td><td>Belclare</td><td>Hanrahan et al.</td></tr><tr><td>Q239ter</td><td>Galway, <i>FecX</i><sup>G</sup></td><td>Hyper prolific</td><td>Sterile</td><td>Belclare and Cambridge</td><td>Hanrahan et al.</td></tr><tr><td>C321Y</td><td>Lacaune, <i>FecX</i><sup>L</sup></td><td>Hyper prolific</td><td>Sterile</td><td>Lacaune</td><td>Bodin et al.</td></tr><tr><td>ΔP154S159</td><td>Rasa Aragonesa, <i>FecX</i><sup>R</sup></td><td>Hyper prolific</td><td>Sterile</td><td>Rasa Aragonesa</td><td>Martinez-Royo et al.; Monteagudo et al.</td></tr><tr><td>BMPR1B (OAR6)</td><td>Q249R</td><td>Booroola, <i>FecB</i><sup>B</sup></td><td>Hyper prolific</td><td>Hyper prolific</td><td>Booroola Merino, Garole and Javanese</td><td>Mulsant et al.; Souza et al.; Wilson et al.</td></tr><tr><td rowspan="3">GDF9 (OAR5)</td><td>S395F</td><td>High Fertility, <i>FecG</i><sup>H</sup></td><td>Hyper prolific</td><td>Sterile</td><td>Belclare and Cambridge</td><td>Hanrahan et al.</td></tr><tr><td>S427R</td><td>Thoka, <i>FecG</i><sup>T</sup></td><td>Hyper prolific</td><td>Sterile</td><td>Icelandic</td><td>Nicol et al.</td></tr><tr><td>F345C</td><td>Embrapa, <i>FecG</i><sup>E</sup></td><td>Hyper prolific</td><td>Hyper prolific</td><td>Santa Inês</td><td>Silva et al.</td></tr><tr><td>δ4GALNT2 (OAR11)</td><td>g.36938224T>A<br/>g.37034573A>G</td><td>Lacaune, <i>FecL</i></td><td>Hyper prolific</td><td>Hyper prolific</td><td>Lacaune</td><td>Drouilhet et al.</td></tr></tbody></table>
# Description of the study design

* **Populations**

* French **Grivette** breed and Polish **Olkuska** breed

* **Major gene for ovulation suspected**

* Litter Size (LS) measured in Grivette : **High LS≥2.7** vs. **Normal LS≤1.8**

* Ovulation Rate (OR) measured in Olskuska : **High OR≥3.0** vs. **Normal OR≤2.0**

* **Case (hyper prolific) vs. Control (normal) design**

* Genotyping using the **54K SNP array**

* Filtering for Call Rate (>0.99), Call Freq (>0.98), HWE (≤10⁻⁶) and MAF (<0.01)

* Comparison of allelic frequencies
Description of the study design

<table><thead><tr><th></th><th colspan="2">Initial Design</th><th colspan="2">Final Design</th></tr><tr><th></th><th>Grivette</th><th>Olkuska</th><th>Grivette</th><th>Olkuska</th></tr></thead><tbody><tr><td><strong>Individuals (n)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Total</td><td>40</td><td>64</td><td>39</td><td>63</td></tr><tr><td>Control</td><td>11</td><td>35</td><td>11</td><td>34</td></tr><tr><td>Case</td><td>29</td><td>29</td><td>28</td><td>29</td></tr><tr><td>Call Rate &lt; 0,98</td><td>1</td><td>1</td><td></td><td></td></tr><tr><td><strong>Phenotype</strong></td><td><em>Litter Size</em></td><td><em>Ovulation Rate</em></td><td><em>Litter Size</em></td><td><em>Ovulation Rate</em></td></tr><tr><td>Total</td><td>2,75 (0,88)</td><td>2,75 (1,40)</td><td>2,75 (0,89)</td><td>2,77 (1,40)</td></tr><tr><td>Control</td><td>1,51 (0,27)</td><td>1,60 (0,30)</td><td>1,51 (0,27)</td><td>1,61 (0,30)</td></tr><tr><td>Case</td><td>3,22 (0,46)</td><td>4,14 (0,81)</td><td>3,24 (0,46)</td><td>4,14 (0,81)</td></tr><tr><td><strong>Markers</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Total</td><td>54241</td><td>54241</td><td>47290</td><td>46451</td></tr><tr><td>Call Freq &lt; 0,98</td><td>4142</td><td>3595</td><td></td><td></td></tr><tr><td>Position Pb</td><td>84</td><td>84</td><td></td><td></td></tr><tr><td>HWE &lt; 1E-06</td><td>0</td><td>159</td><td></td><td></td></tr><tr><td>MAF &lt; 0,01</td><td>2725</td><td>3952</td><td></td><td></td></tr></tbody></table>
# Genome Wide Association Study (GWAS) for prolificacy

<figure><img src="image_88.png" /></figure>

<figure><img src="image_89.png" /></figure>

<figure><img src="image_90.png" /></figure>

<figure><img src="image_91.png" /></figure>
# Haplotypic clustering and association

<figure><img src="image_92.png" /><figcaption>◄ BMP15</figcaption></figure>

1.3Mb

$Fr_{case}$=0.95 vs. $Fr_{control}$=0.54
$P_{corrected}$=9.90E-03

1.3Mb

$Fr_{case}$=0.76 vs. $Fr_{control}$=0.27
$P_{corrected}$=9.90E-04
# BMP15 coding sequence polymorphisms

<table><thead><tr><th rowspan="2">Variant</th><th rowspan="2">Base change</th><th rowspan="2">Coding Base (bp)</th><th rowspan="2">Coding residue (bp)</th><th rowspan="2">Amino Acid Change</th><th colspan="6">Genotype</th></tr><tr><th>Controls</th><th>C/T (0)</th><th>T/T (11)</th><th>C/T (0)</th><th>T/T (28)</th></tr></thead><tbody><tr><td colspan="10"><strong>Grivette</strong></td></tr><tr><td>ΔL10</td><td>ΔCTT</td><td>28-30</td><td>10</td><td>Deletion Leu</td><td>-/- (6)</td><td>-/+ (5)</td><td>+/+ (0)</td><td>-/- (27)</td><td>-/+ (1)</td><td>+/+ (0)</td></tr><tr><td>P249P</td><td>C>T</td><td>747</td><td>249</td><td>Unchanged Pro</td><td>C/C (0)</td><td>C/T (0)</td><td>T/T (11)</td><td>C/C (0)</td><td>C/T (0)</td><td>T/T (28)</td></tr><tr><td><strong>T317I=FecX<sup>Gr</sup></strong></td><td><strong>C>T</strong></td><td><strong>950</strong></td><td><strong>317</strong></td><td><strong>Thr>Ile</strong></td><td><strong>C/C (2)</strong></td><td><strong>C/T (6)</strong></td><td><strong>T/T (3)</strong></td><td><strong>C/C (0)</strong></td><td><strong>C/T (2)</strong></td><td><strong>T/T (26)</strong></td></tr><tr><td colspan="10"><strong>Olkuska</strong></td></tr><tr><td>ΔL10</td><td>ΔCTT</td><td>28-30</td><td>10</td><td>Deletion Leu</td><td>-/- (0)</td><td>-/+ (7)</td><td>+/+ (26)</td><td>-/- (0)</td><td>-/+ (0)</td><td>+/+ (29)</td></tr><tr><td>V135G</td><td>T>G</td><td>404</td><td>135</td><td>Val>Gly</td><td>T/T (33)</td><td>T/G (1)</td><td>G/G (0)</td><td>T/T (29)</td><td>T/G (0)</td><td>G/G (0)</td></tr><tr><td><strong>N337H=FecX<sup>O</sup></strong></td><td><strong>A>C</strong></td><td><strong>1009</strong></td><td><strong>337</strong></td><td><strong>Asn>His</strong></td><td><strong>A/A (16)</strong></td><td><strong>A/C (18)</strong></td><td><strong>C/C (0)</strong></td><td><strong>A/A (1)</strong></td><td><strong>A/C (9)</strong></td><td><strong>C/C (19)</strong></td></tr></tbody></table>
Novel **BMP15** Fecundity mutations

<figure><img src="image_93.png" /></figure>
Novel BMP15 Fecundity mutations

<figure><img src="image_94.png" /></figure>
Effect of mutations on the prolificacy

<figure><img src="image_95.png" /><figcaption>BMP15<sup>T317I</sup> (FecX<sup>Gr</sup>) Genotype</figcaption></figure>

<figure><img src="image_96.png" /><figcaption>BMP15<sup>N337H</sup> (FecX<sup>O</sup>) Genotype</figcaption></figure>

<figure><img src="image_97.png" /><figcaption>BMP15<sup>T317I</sup> (FecX<sup>Gr</sup>) Genotype</figcaption></figure>

<figure><img src="image_98.png" /><figcaption>BMP15<sup>N337H</sup> (FecX<sup>O</sup>) Genotype</figcaption></figure>
Effect of mutations on the BMP15 protein

<figure><img src="image_99.png" /></figure>

<figure><img src="image_100.png" /></figure>

<figure><img src="image_101.png" /></figure>

BMP15 Amino Acids Position

BMP15^{T317I} = FecX^{Gr}

BMP15^{N337H} = FecX^{0}
Effect of mutations on the BMP15 signalling

<figure><img src="image_102.png" /></figure>
Model of the ovulation quota control

<figure><img src="image_103.png" /></figure>
<figure><img src="image_104.png" /></figure>

Merci de votre attention

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INRAO
la science pour la vie, l'humain, la terre

INP ENSAT

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école nationale
vétérinaire
toulouse

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Université
de Toulouse

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