April 2010

The good news is …

We  now  have  a  short  list  of  genes  that  might  regulate  response  to  trypanosomiasis  as  well  as  a  large  set  of  gene5c  markers  for  African  ca9le  that  will  be  valuable  for  future  research.  

And the not so good news is …

Finding good and bad genes in Boran and N’Dama genome sequences

There  is  s5ll  a  lot  of  work  to  be  done  to  confirm  which  genes  are  involved  and  how  they  effect  the  disease  process.  

What makes Boran more susceptible to trypanosomiasis than N’Dama?

Boran (relatively susceptible) N’Dama (tolerant)

Boran cattle are much more susceptible to to trypanosomiasis than N’Dama. The gene mapping studies at ILRI in Nairobi showed that genes at ten places in the genome controlled the response to infection. A surprising observation was that both Boran and N’Dama had ‘Good’ and ‘Bad’ genes. The graphs under each animal show the effect of the ‘Bad’ genes below the midline and the effect of the ‘Good’ genes above the midline. The difference between the two breeds is that the ‘Good’ genes in N’Dama have a larger effect relative to the ‘Bad’ ones than Boran.

Cow Chromosomes

The black bars represent the 29 autosomes and two sex chromosomes of cattle. Coloured bars represent the position of the ten regions of the genome that contain genes that make a difference to how N’Dama and Boran respond to infection. Red, blue and pale blue bars show where there are genes controlling anemia, body weight and parasitaemia after infection. Each of the coloured bars cover 20-100% of a chromosome; these regions contain many genes making it difficult to identify the genes that make the difference.

PCV  

Body  weight  

Parasitaemia  

Hano5e  et  al  PNAS  2003  7443-­‐7448    

Next Generation sequencing to the rescue Next generation sequencing is transforming genetics. One ABI SOLiD instrument can generate 50Gb of sequence in a run, compared to only 3Gb in a human or cow genome. That means we can sequence each genome many times over to ensure highly accurate detection of differences between breeds.

We have sequenced DNA from 10 Boran from the ILRI ranch at Kapiti and 20 N’Dama from Guinea to discover the majority of the common differences between these breeds.

We can then look within the regions of the genome where we know that there are good trypanosomiasis genes in one breed or bad genes in the other and find differences within genes that could have an important effect on their phenotype.

Minor allele frequencies in N’Dama chromosome 16 where there is a ‘good’ gene. The dips in the graph are regions where there is very little variation in N’Dama, this could have been caused by selection for a beneficial variant of a gene either by the process of domestication or because one variant was better at resisting disease than another. The circle highlights the Tlr5 gene that is important in the innate immune response and could therefore have been selected to resist trypanosomiasis and / or other diseases. Less is known about the other genes that appear to be under selection but they may also be important

Consequence of Polymorphism N'Dama Boran Sahiwal 3 PRIME UTR 16,786 10,311 30,733 5 PRIME UTR 3,885 1,601 6,651 DOWNSTREAM 7,398 4,536 16,497 ESSENTIAL SPLICE SITE 2 267 1,029 INTRONIC 37,758 2,223 35,803 NON SYNONYMOUS CODING 29,350 10,454 45,344 SPLICE SITE 2,044 592 7,674 STOP GAINED 935 145 1,344 STOP LOST 25 11 41 SYNONYMOUS CODING 32,404 17,516 61,491 UPSTREAM 4,974 1,948 10,867 WITHIN MATURE miRNA 10 6 41 WITHIN NON CODING GENE 2,331 1,308 4,855

Table of the number of SNP with each type of effect on gene function. In addition to Boran and N’Dama we have also sequenced Sahiwal, a pure Bos indicus breed. Polymorphisms are differences between two DNA sequences. Only about 2% of DNA codes for genes, the rest either regulates when and where genes are switched on or they have no obvious purpose.

The differences between sequences that are most likely to effect an animal’s phenotype are within genes. And within genes the most important differences are ones that cause a difference in the protein sequence (NON SYNONYMOUS CODING) or effect how the components of the gene are assembled (ESSENTIAL SPLICE SITE) or disrupt a gene altogether (STOP GAINED), and these are highlighted in the table. We are looking at the regions of the genome where we know that the good and bad trypanosomiasis resistance genes are, to see which ones have these most important differences in them.

We are already testing the effect of a STOP GAINED polymorphism in the PON3 gene that could be responsible for large differences in expression of this gene. PON family genes have been shown to effect survival of mice after infection so this difference could be important.

DNA differences that effect phenotype

Trinity College Dublin