Slide 1 GENOMIC RESEARCH PRESENTATION AT INTERNATIONAL YAK - - PowerPoint PPT Presentation

This is a 3 section presentation. Slides 1 thru 3 cover the Mechanics of The Generation Program. A detailed introduction was written by Larry Richards and communicated via IYAK Newsletter to the Membership. Explain the limitations of the Appendix B Program: Restricts the possible introduction of heterozygosity into the NAYR by requiring backcrossing to Foundation animals. Limitations on detection of Charolais hybrids by Dc testing when the dilution color drops out.

The GENOMIC RESEARCH PRESENTATION AT INTERNATIONAL YAK ASSOCIATION 2018 DENVER MEMBERSHIP MEETING

By Lawrence G. Richards Chair IYAK Genome Committee SPEAKER NOTES TEXT - In blue

Slide 1

Appendix G

1.0 All unknowns in North America (NA) may apply. 2.0 RegCom will oversee the Application Process. 3.0 Any Applicant that exceeds the 10 cattle allele threshold will automatically be denied registration. 4.0 One high quality photograph showing one side with the head facing the camera. 5.0 As the only white yaks in NA are from Charolais hybridization all white yaks will be rejected

• utright.

6.0 Applicants from known hybridization programs regardless of their color will be excluded based on the following:

• RegCom may mandate Parental Verification to all breeding age animals within the known

hybridization program.

• Any Applicant thus connected by PV to any known hybrid will thus be excluded.
• RegCom may require Dc or Dh testing.
• RegCom may interview involved personnel as part of their investigation.

7.0 For a IYAK member to knowingly misrepresent an Applicant (of any color) from a hybridization program will be considered a violation of the Code of Ethics resulting in immediate and irrevocable termination of IYAK membership. 8.0 For an IYAK member to knowingly sell as registrable with NAYR an Applicant (of any color) from a known hybridization program will also be considered a violation of the Code of Ethics resulting in the immediate and irrevocable termination of IYAK Membership. NOTES: Covers the 8 Rules of the Appendix G. Program and explain the cattle introgression testing by GeneSeek and the essence of the 10 allele threshold: A 15/16ths hybrid would have 12 (estimate) alleles and be rejected by the testing requirement for 10 or less alleles. A 1/32ths hybrid would have 6 alleles( estimate) and would slide thru. Hence Rules 5 thru 6. Testing 95 SNPs x 2 = 190 for a diploid organism.

Slide 2

Appendix-B Registry ry Progression - RECAP

a) B0 X B0 = B0 b) Foundation X B0 =B1 c) B2 X B0 = B1 d) B1 X B0 = B0 e) B1 X B1 =B1 f) Foundation X B1 = B2 g) B2 X B1 = B2 h) B2 X B2 = B2 i)Foundation X B2 = B3 (= Foundation) NOTES: At each level it is required to breed to an animal from the next higher level to advance in the progression. Exception B1 X B0 does not advance.

NOTE: As of 3/1/2016, no new animals were accepted into the Appendix-B Registry. Offspring of appendix- registered parents (B0, B1 and B2 animals) are still eligible for registration according to the Appendix-B Program Progression outlined above. Slide 3

Appendix -G Registry ry

• a. G0 - a yak without known sire and/or dam, which has no more than 10 cattle alleles (DNA tested by

GeneSeek), and has been approved by the Registration Committee (or Board of Directors in the situation the Registration Committee does not approve the yak for registration).

• b. G1 = G0xG0, G0xG1, G0xG2, G0xB0, G0xB1, G0xB2 or G0xFoundation
• AND has no more than 10 cattle alleles (DNA tested by GeneSeek) and has been approved by

the Registration Committee (or Board of Directors in the situation the Registration Committee does not approve the yak for registration).

• A G1 has one generation of pedigree, but does not have two complete generations of

pedigree.

• c. G2 = G1xG1, G1xG2, G1xB1,G1xB2 or G1xFoundation

AND has no more than 10 cattle alleles (DNA tested by GeneSeek) and has been approved by the Registration Committee (or Board of Directors in the situation the Registration Committee does not approve the yak for registration). A G2 has two generations of pedigree, but does not have three complete generations of pedigree

• d. G3 = Foundation = G2xG2, G2xB2 or G2xFoundation
• AND has no more than 10 cattle alleles (DNA tested by GeneSeek) and has been approved by

the Registration Committee (or Board of Directors in the situation the Registration Committee does not approve the yak for registration).

• A G3 has three generations of pedigree which qualifies it for registration in the Foundation

Registry. NOTES: Explain the progression. Must match or exceed the number of generations of recorded pedigree at both top and bottom of the pedigree to advance. All progeny must meet the introgression threshold testing at each level to advance. Even progeny from Foundation animals rejected if exceed the threshold. This requirement not held to the animals already in the Foundation or Appendix B Program. Market pressures will neutralize any Foundation animals producing

• ffspring exceeding the 10 allele

threshold. One NAYR with three entry points: Foundation. Appendix B. Appendix G. All lead to one Registry. The NAYR.

Slide 4

Payment

• 1. For a Foundation or Appendix-B Yak: Registration plus DNA analysis by

GeneSeek -$65 if the sire has had his DNA previously analyzed by GeneSeek.

• If the DNA of the sire has not been previously tested by

GeneSeek, there is a one- time $25 charge to process the sire so his DNA information will be in the GeneSeek data base for parentage testing.

• This requirement is to establish a Parental Verification link

between the UC Davis verified pedigree and the GeneSeek data base.

• If the DNA of the dam has not been previously been tested by

GeneSeek as a special concession, IYAK has agreed to pay the cost ($25) of the GeneSeek testing of the dam for the first year the GBPR is open.

• 2. For an Appendix-G Yak: Registration plus DNA analysis by GeneSeek-$65
• 3. For an Appendix-G Yak - G1,G2 or G3 (=Foundation): Registration plus

DNA analysis by GeneSeek -$65 if the sire has had his DNA previously analyzed by GeneSeek.

• If the DNA of the sire has not been previously tested by

GeneSeek, there is a one- time $25 charge to process the sire so his DNA information will be in the GeneSeek data base for parentage testing.

• This requirement is to establish a Parental Verification link between the

UC Davis verified pedigree and the GeneSeek data base. If Sire Is Not Known- Foundation, Appendix-B and Appendix-G:

• a. A second sire may be submitted at no cost if the sire's DNA has been

previously been processed by GeneSeek. If the sire's DNA has not been previously processed by GeneSeek, there will be a one-time charge of $25 for the sire's DNA to be processed by GeneSeek and entered into their data bank.

• b. Additional sires may be submitted. If their DNA has been previously

processed by GeneSeek, there will be a $10 charge per additional sire submitted for possible parental verification. If their DNA has not been previously processed by GeneSeek, there will be a one-time charge of $25 for each sire's DNA to be processed by GeneSeek and entered into their data bank. If Dam Is Not Known- Foundation, Appendix-B and Appendix-G:

• a. Testing for each additional dam is $50. This will include the GeneSeek DNA

analysis if the dam's DNA has not been previously analyzed by GeneSeek.

NOTE: Additional dams and sires after the fifth will be referred to the Registration Committee for review. Slide 5

DNA Replication

that can be opened to unravel the DNA helix for Replication and RNA Transcription. In contrast the deoxyribose phosphate polymer side chains (hand rails) are held together by high energy bonds that require enzymes to make or break. The enzyme DNA Polymerase (chain length 1100 aa) is the little machine that replicates each unwound chain to create 2 identical DNA molecules for cell division and egg and sperm production. The

• rdering of the new chains is determined by the base pairing of A (

adenine) to T (thymine) and G (guanine) to C(Cytosine) as seen in the sequence on the original unwound DNA strands. The reaction catalyzed by DNA polymerase is driven by the release

• f a pyrophosphate (double phosphate) from the nucleotide

triphosphate precursors ( A, T, G, C ) and accompanied by energy release which drives the reaction forward. All cell reactions are driven by the flow of energy. All this cellular energy starting with photosynthesis driven by the sun. ATP one of the 4 nucleotide precursors is one of the primary energy carriers in cellular

• rganisms.

The 2 strands of the original DNA polymer are stereochemical

• pposites running in opposite directions, hence we see one DNA

polymerase enzyme replicating in one direction and the other DNA polymerase enzyme replicating in the other direction. (fun stuff that doesn’t relate to yak breeding tonight). Remember these bases and their pairing when we explain the new GeneSeek testing interpretations. NOTES: Yaks are diploid organism with 2 homologous chromosomes one paternal derived and one maternal derived. 30 chromosomes, 20,000 genes and 3 billion nucleotide pairs. Average 667 genes per chromosome (my assumption). Compare the double helix to a spiral staircase with 2 handrails held together by nucleotide base pairs (the steps). The nucleotide base pairs are AT, TA and GC and CG. The base pair bonds are weak hydrogen bonds

Slide 6

RNA Transcription

NOTES: RNA polymerase copies one strand of the DNA helix to create messenger RNA for the ultimate production of polypeptides (enzymes and enzyme subunits). Ribonucleotide Uracil replaces Thymine and binds with Adenine. DNA template determines the sequence of the messenger RNA molecule. The 2 separate strands would code for 2 different polypeptide chains. Which strand is copied is part of the control and regulation of the gene and is complicated.

Slide 7

Poly lypeptide Production

is selectively captured by an enzyme specific for it and binds it to a specific transfer RNA molecule. That specific transfer molecule has a 3 nucleotide sequence called an anti-codon that binds to a 3 nucleotide codon on the messenger RNA template. An enzyme peptidyl transferase then binds the selected amino acid to the growing polypeptide chain. Why do we care? Any disruption in the sequence of the base pairs in the DNA molecule can cause an altered phenotype in the animal. For example 2 years ago in my presentation we saw that the Charolais white dilution color was caused by a single base pair substitution in the Dc

• variant. Presumably homozygous in pure Charolais cattle and

resulting in white hybrid yaks. In Black and red angus cattle the ED is the black allele and e is the red allele. ED/ED is homozygous black and is black. e/e is the recessive homozygous red. ED/e is heterozygous black. E is dominant and is constantly producing the protein that produces black color even in the heterozygous state. The recessive e/e produces a nonfunctional protein and the animal is red. NOTES: Polypeptide production occurs on the ribosomes. A single stranded messenger RNA that carries the nucleotide sequence provided by the DNA template is read to produce a polypeptide. 3 sequential bases on the messenger RNA template form a codon. 20 amino acids are selectively bound to 20 specific transfer RNA molecules by 20 specific enzymes. That is each of 20 amino acids

Slide 8

Strategy for Marker Identification: Introgression has a really interesting solution

Slide 9

NOTES: Slide 9 shows how the scientists have arrived at a determination of cattle introgression within the genome of a specific yak. For review the scientists are looking at 95 SNP sites and given they are looking at both the paternal and maternal derived chromosomes then 190 alleles. Remember 10 alleles out of 190 is the threshold set for cattle introgression in our new Appendix G Registry. Slide 9 shows that at one-time yaks, American bison, gaur, banteng and cattle shared a common ancestor. The nucleotide sequence shown is a hypothetical presentation of an SNP site which shows Ancestral allele C common to yak, bison, gaur and banteng, whereas modern cattle have diverged to allele G at this site (homozygous G). In the common ancestor all of these including the cattle ancestor would be homozygous C. The slide shows the yak as heterozygous C/T and T is the Descendant yak allele for this site. For each of the 95 SNP sites the scientists have determined the Ancestral yak allele and the Descendent yak allele and the cattle allele. When we get to Slide 11 the SNP Table we will see at SNP site 9 this yak TFF Ono is heterozygous carrying one Ancestral Yak allele A and one Descendent Yak allele. Out of 188 alleles observed 77 are Descendent Yak alleles and 111 are Ancestral Yak alleles. 40.96 % of her alleles have evolved from the common ancestor. Extrapolating to the whole genome this reflects the history of evolution of this animal. Looking across the spectrum of the entire worldwide yak population would give an exact measure of the evolution of modern yaks from the common ancestor. Remember the genome is the blueprint for the phenotypic expression of the individual.

SNP Table

Slide 11

NOTES SLIDE 11: 23 of 95 SNPs shown. This testing compared the alleles at 95 SNP sites for alleles from Divergent cattle and Ancestral yaks and Descendent yaks. Ancestral yak alleles were from sequencing of banteng, bison and gaur before yaks diverged from them. Cattle diverged from yaks or vice-versa 4.5 million years ago. So the tested cattle alleles are only found in cattle. At each SNP they are looking at the alleles on both the maternal and the paternal chromosome. Thus at SNP 1 we see the animal is homozygous for the Ancestral Yak alleles where G represents the pair GC and thus GG represents GC/GC. At SNP 2 T represents TA and thus TT represents TA/TA and the animal is homozygous for the Descendent Yak Allele. Ted informs me that at each of these sites these are the surviving possible results. At both SNP 1 and SNP 2 there is no cattle allele found. In fact there are 0 cattle alleles found for this animal at any

• f the 95 sites.

Of the 200 legacy NAYR animals tested most animals tested between 0 and 2 cattle alleles with an average of 0.9% with one outlier at 4 alleles. 4 of 190 alleles is 2%. Comment on Neanderthal percentages in modern Caucasians (European derived humans). At SNP 9 this animal is heterozygous showing one Ancestral yak allele A and one Descendent yak allele G. In the 16 SNP Identification Code they use another code R to represent the heterozygous result AG. They speak in codes! The Ratio of Recent to Ancestral yak alleles is 0.69. This may bear some further investigation. I found this ratio slightly different in the results of the Chinese wild yaks to the Chinese domestic yaks.

Identification Animal Name: TFF Ono with IYAK Tag # (when provided). Sample ID: 16 SNP Identification Code GTGCTGTARGCCSTYA TFF Ono qualifies as an offspring of SIRE FW Escalade (1 Exclusions out of 93 genotypes checked) and the Dam is LDR Halleberry (0 Exclusions out of 94 genotypes checked) Cattle Introgression CattleAlleles =0 AncestralYakAlleles =111 RecentYakAlleles =77 CattleFraction =0.0 Coefficient of Inbreeding

• No. Heterozygous SNPs = 35

EstimationOfCOI =.0564 Other Features Mitochondrial DNA G - Yak Coat Color Y - Trim Fiber Gene A/T SAMPLE QUALITY TotalAssays= 98 TotalCalls= 97 CallRate =.990 CallRateThreshold =0.95 CallRateStatus=Pass

GeneSeek Yak Parentage and Cattle Introgression Test

NOTES: Our Condensed GeneSeek Yak Parentage and Cattle Introgression Report. Go back to the previous slide to see how the 16 SNP Identification Code is derived from the SNP Table. S and Y are 2 other conventions they use as seen in digits 13 and 15 the 2 other heterozygous sites. SNP Bar Code could be used for meat back tracking or as Genomic Based Brand Inspection for theft verification.

Slide 13

𝑑𝑝𝑗 = 1.0 − ℎ𝑓𝑢𝑓𝑠𝑝𝑨𝑧𝑕𝑝𝑢𝑓𝑈𝑝𝑢𝑏𝑚/(𝑂𝑡𝑗𝑢𝑓𝑡 ∗ 𝐹𝑦𝑞𝑓𝑑𝑢𝑓𝑒𝐼𝑓𝑢𝑡) 𝐹𝑦𝑞𝑓𝑑𝑢𝑓𝑒𝐼𝑓𝑢𝑡 = .3946

SNP No. SNP_ID Genotype 1 Bg_Bt_UMD3_chr1:11611152 G/G 2 Bg_Bt_UMD3_chr1:19603843 T/T 3 Bg_Bt_UMD3_chr1:106754991 G/G 4 Bg_Bt_UMD3_chr1:154872834 C/C 5 Bg_Bt_UMD3_chr2:35754741 T/T 6 Bg_Bt_UMD3_chr2:51851787 G/G 7 Bg_Bt_UMD3_chr2:73562811 T/T 8 Bg_Bt_UMD3_chr2:108669950 A/A 9 Bg_Bt_UMD3_chr2:135821825 A/G 10 Bg_Bt_UMD3_chr3:16782467 G/G 11 Bg_Bt_UMD3_chr3:31884274 C/C 12 Bg_Bt_UMD3_chr3:32162241 C/C 13 Bg_Bt_UMD3_chr3:55871907 C/G 14 Bg_Bt_UMD3_chr3:60501892 T/T 15 Bg_Bt_UMD3_chr3:72881652 C/T 16 Bg_Bt_UMD3_chr3:88807660 A/A 17 Bg_Bt_UMD3_chr4:6749340 C C/C 18 Bg_Bt_UMD3_chr4:31218839 G/G 19 Bg_Bt_UMD3_chr4:59985637 C/C 20 Bg_Bt_UMD3_chr4:68609356 A/T 21 Bg_Bt_UMD3_chr4:97170995 C/T 22 Bg_Bt_UMD3_chr5:72886737 A/A 23 Bg_Bt_UMD3_chr5:100853172 C/T 24 Bg_Bt_UMD3_chr5:119385958 C/C 25 Bg_Bt_UMD3_chr6:2829446 C/T 26 Bg_Bt_UMD3_chr6:35088167 G/T 27 Bg_Bt_UMD3_chr6:52296470 G/G 28 Bg_Bt_UMD3_chr6:66063680 C/T 29 Bg_Bt_UMD3_chr7:2908163 C C/C 30 Bg_Bt_UMD3_chr7:18126486 C/T 31 Bg_Bt_UMD3_chr7:49998354 G/G 32 Bg_Bt_UMD3_chr7:59924729 C/C 33 Bg_Bt_UMD3_chr7:61603492 A/A 34 Bg_Bt_UMD3_chr7:106143569 G/T 35 Bg_Bt_UMD3_chr8:35799123 C/T 36 Bg_Bt_UMD3_chr8:103151793 C/C 37 Bg_Bt_UMD3_chr8:105594647

• 38

Bg_Bt_UMD3_chr9:8416809 A/G 39 Bg_Bt_UMD3_chr9:36039743 A/A 40 Bg_Bt_UMD3_chr9:96035965 A/A 41 Bg_Bt_UMD3_chr9:97200189 A/A 42 Bg_Bt_UMD3_chr10:13448721 A/G 43 Bg_Bt_UMD3_chr10:72325948 T/T 44 Bg_Bt_UMD3_chr10:101587185 A/A 45 Bg_Bt_UMD3_chr11:10319205 G/G 46 Bg_Bt_UMD3_chr11:34588924 C/T 47 Bg_Bt_UMD3_chr11:42099862 C/T 48 Bg_Bt_UMD3_chr11:75398487 G/G 49 Bg_Bt_UMD3_chr11:83618697 A/A 50 Bg_Bt_UMD3_chr12:16628394 C/C 51 Bg_Bt_UMD3_chr12:38918421 G/T 52 Bg_Bt_UMD3_chr12:65478052 G/T 53 Bg_Bt_UMD3_chr13:14128040 C/C 54 Bg_Bt_UMD3_chr13:16431857 C/C 55 Bg_Bt_UMD3_chr13:36437915 C/T 56 Bg_Bt_UMD3_chr14:3368905 C/C 57 Bg_Bt_UMD3_chr14:20015146 C/T 58 Bg_Bt_UMD3_chr14:35695388 C/C 59 Bg_Bt_UMD3_chr14:51388662 T/T 60 Bg_Bt_UMD3_chr15:1612812 C/C 61 Bg_Bt_UMD3_chr15:24038406 A/C 62 Bg_Bt_UMD3_chr15:31899012 C/C 63 Bg_Bt_UMD3_chr15:39249514 A/A 64 Bg_Bt_UMD3_chr16:17627607 G/T 65 Bg_Bt_UMD3_chr16:27346407 T/T 66 Bg_Bt_UMD3_chr16:37235898 G/T 67 Bg_Bt_UMD3_chr17:6749112 A/T 68 Bg_Bt_UMD3_chr17:31022749 A/A 69 Bg_Bt_UMD3_chr17:48657788 C/C 70 Bg_Bt_UMD3_chr17:50526845 C/C 71 Bg_Bt_UMD3_chr17:73228099 G/T 72 Bg_Bt_UMD3_chr18:22272606 A/G 73 Bg_Bt_UMD3_chr18:31899459 A/G 74 Bg_Bt_UMD3_chr19:53556396 C/C 75 Bg_Bt_UMD3_chr19:63181970 A/G 76 Bg_Bt_UMD3_chr20:6979616 T/T 77 Bg_Bt_UMD3_chr20:22394132 T/T 78 Bg_Bt_UMD3_chr20:52343058 C/G 79 Bg_Bt_UMD3_chr21:8541469 T/T 80 Bg_Bt_UMD3_chr21:35371951 C/C 81 Bg_Bt_UMD3_chr22:3842135 G/T 82 Bg_Bt_UMD3_chr22:21618015 C/C 83 Bg_Bt_UMD3_chr22:40850741 A/A 84 Bg_Bt_UMD3_chr22:48005412 C/T 85 Bg_Bt_UMD3_chr24:62505702 G/G 86 Bg_Bt_UMD3_chr25:158052 C/C 87 Bg_Bt_UMD3_chr25:42661806 T/T 88 Bg_Bt_UMD3_chr26:10857224 C/T 89 Bg_Bt_UMD3_chr26:12336398 T/T 90 Bg_Bt_UMD3_chr27:13550559 A/G 91 Bg_Bt_UMD3_chr27:16057108 G/T 92 Bg_Bt_UMD3_chr28:5406231 T/T 93 Bg_Bt_UMD3_chr28:18571823 T/T 94 Bg_Bt_UMD3_chr29:16271334 G/G 95 Bg_Bt_UMD3_chr29:21414616 A/G

𝑑𝑝𝑗 = 1.0 − 35/(94 ∗ .3946) Count of SNPs 95 -1 failure, 35 measured hets 𝑑𝑝𝑗 = 0.0564 Slide 14

NOTES SLIDE 14: Shows all 95 SNP results with the heterozygous sites blocked in yellow. 35 measured heterozygous sites from the 95. One not read at site 37. Result 94 measured sites used in calculation. COI calculation formula. Distribution of SNPs per chromosome ranges from 2 to 7. All SNPs from chromosome 1 thru 3 used for the SNP ID. Homozygosity and Heterozygosity 2 sides of same coin. Positive COI result more homozygous. Negative result more heterozygous. Slide shows possibility of comparing 2 or more animals side by side for homozygosity at 95 SNP sites. Could delete the SNP ID information and run Genotypes side by side. Discussion of some limitations of this raw data in regards to homozygosity. The limitation is 95 SNPs from 3 billion in the yak genome.

• Dr. Ted Kalbfleisch plans to run this 95 SNP test against the 4 Chinese yaks with whole genome sequences (WGS) and compare the result from 95 SNPs against

calculations from the WGS. It would cost a fortune to compare the WGS of 2 animals to predict the heterozygosity of a paired mating. What we have with 95 SNPs may be of some value in comparing breeding choices. I believe all these 95 SNPs are from between the genes and are not correlated to any observable phenotypic traits. Summary we have great new test. Take advantage of this new Appendix Registry and bring your unregistered yaks into the program. GeneSeek was impressed by the sophistication of the approach developed by Drs. Michael Heaton and Ted Kalbfleisch. Ted Kalbfleisch Associate Professor

• Dept. of Biochemistry and Molecular Genetics

School of Medicine, University of Louisville, Kentucky

• Dr. Michael P. Heaton

U.S. Meat and Animal Research Center (USMARC) Clay Center, Nebraska