Pulmonary Hypertension Syndrome in Chicken: Peeking Under QTL Peaks

Pulmonary hypertension syndrome (PHS) is characterized a cascade of events resulting in cardiac anomalies including an enlarged heart, right ventricular hypertrophy, variable liver changes, and accumulation of fluid in the abdominal cavity (ascites). PHS is not a disease; it is a condition in which excess amount of ascitic fluids (a combination of lymph and blood plasma which has leaked from the liver) accumulate in the abdominal cavity which has prompted the common name</span><span lang=EN-US style='color:black'>"water belly"Several genetic and environmental factors such as altitude, temperature, lighting, ventilation, and nutritional factors seem to influence the development of the PHS. The work described in this dissertation is a first step towards the identification of genetic factors controlling PHS.</span>An experimental population was specifically generated with the aim to map QTL for a number of different economically important traits including PHS. The population is based on a so-called full-sib/half-sib design<span style='mso-spacerun:yes'> </span>based on three generations (G ₁ , G ₂ and G ₃ ) derived from a cross between two genetically different outcross broiler dam lines originated from the White Plymouth Rock breed. <strong>Chapter 2</strong> describes the initial mapping of a several QTL for traits related to PHS using a total genome scan. The total genome scan was performed on ten full sib families consisting of 20 G ₁ and 456 G ₂ birds which were typed with 420 microsatellite markers covering 24 autosomal chromosomes. Phenotypic observations were collected on 4202 G ₃ birds and a full sib across family regression interval mapping approach was used to identify the QTL. Statistical evidence for QTL was found on chicken chromosomes (GGA) 2, 4 and 6. Suggestive linkage was found on chromosomes 5, 8, 10, 27, and 28. Phenotypic measurements mostly were related to the condition of the heart (%RV, %TV, RATIO), appearance of the internal organs, ascites and survival. The most significant QTL that were identified are located on GGA2 for the traits, right and total ventricular weight as percentage of body weight (%RV and %TV). A related trait, the ratio of right ventricular weight as percentage to total ventricular weight (RATIO), reached the suggestive threshold on this chromosome.<o:p></o:p></span>The observed test statistics were not very high, which was to be expected in a G ₂ cross between two related breeds. The subsequent confirmation of the observed QTL in the next generations therefore, is an essential step before attempting to further narrow down the identified QTL intervals. The validation of the QTL on chromosomes 8, 10 and 28 and the confirmation of those on GGA8 and GGA10 is described in <strong>Chapter 3</strong> .</span>Validation was performed in generations G6 to G8 of the experimental cross. The population consisted of 19 full sib families (a combination of G ₆ /G ₇ and G ₇ /G ₈ ). In total 14 microsatellite markers were analyzed and twelve PHS-related traits measured on more than 1500 individuals. Significant results were found for the traits body weight at 2 and 5 weeks of age, right ventricular weight, RATIO (RV:TV) and total mortality. The most significant QTL were located on chromosome 8 for traits body weight at 5 wk of age ( ADL0278-MCW0351 ), right ventricular weight ( ADL0278-MCW0351 ) and RATIO (RV:TV) ( ROS0075-ADL0278 ) and on chromosome 10 for traits body weight at 2 wk of age ( MCW0035-ADL0102 ) and total mortality ( ADL0158-LEI0112 ). The QTL on GGA28 could not be confirmed. The validation and confirmation of the significant QTL on GGA2 and GGA4 are described in <strong>Chapter 4</strong> . This chapter also describes the further fine mapping of these two QTL using a combined linkage disequilibrium/linkage analysis approach (LDLA). Validation and fine mapping was performed on an advanced intercross line (AIL) created by random intercrossing in each generation from G ₃ onwards until G ₈ . In total 47 microsatellite markers were used, located within approximately 25 Mb and 16 Mb on GGA2 and GGA4, respectively. QTL originally detected in G ₂ /G ₃ generations were confirmed on the two chromosomes, for the traits RATIO on GGA2 and for BW5 _AS on GGA4. Additional QTLs were detected for the trait</span><span lang=EN-US style='font-family:"Monotype Corsiva";layout-grid-mode:line'>p</span><span lang=EN-US>CO ₂ on GGA2, and for BW3 _AS on GGA4. These two traits had not previously been measured in generation 3 of the original study. The combined LDLA approach enabled a 3-4 fold reduction of the size of the QTL interval.</span>The improvement of the physical map for GGA4 and the generation of a detailed comparative map between this chicken chromosome and human chromosomes 4 and X are described in Chapter 5</span></strong><span lang=EN-US style='letter-spacing:-.15pt'>. The mapping resolution of the physical map for GGA4 was improved by a combination of radiation hybrid (RH) mapping and BAC mapping. The ChickRH6 hybrid panel was used to construct an RH map of GGA4. Eleven microsatellites known to be located on GGA4 were included as anchors to the genetic linkage map for this chromosome. Based on the known conserved synteny between GGA4 and human chromosomes 4 and X, sequences were identified for the orthologous chicken genes from these human chromosomes by BLAST analysis. These sequences were subsequently used for the development of STS markers to be typed on the RH panel. Using a logarithm of the odds (LOD) threshold of 5.0, nine-linkage groups could be constructed which were aligned with the genetic linkage map of this chromosome. The resulting RH map consisted of the 11 microsatellite markers and 50 genes. To further increase the number of genes on the map and to provide additional anchor points for the physical BAC map of this chromosome BAC clones were identified for 22 microsatellites and 99 genes.</span><span lang=EN-US>The combined RH and BAC mapping approach resulted in the mapping of 61 genes on chicken GGA4 considerably increasing the resolution of the chicken-human comparative map for this chromosome. This enhanced comparative mapping resolution enabled the identification of multiple rearrangements between chicken chromosome 4 and human chromosomes 4q and Xp.</span>Finally, the results of this thesis are discussed within a broader context in <strong>Chapter 6</strong><span style='mso-bidi-font-weight:bold;mso-bidi-font-style:italic'>and in particular in relation to the recently published draft sequence of the chicken genome and the polymorphism map consisting of 2.8 million single nucleotide polymorphisms