Bison: mitochondrial genomics

Introduction to bison conservation genomics

(to be continued)

Phylogeny: bison and yak are sister groups

(to be continued)

Interpreting bison CYTB variation

Bison mitochondrial genomes became well-represented at GenBank with the 1 Dec 10 release by the Derr group of 31 complete genomes from 6 herds including two woods bison (Bison bison athabascae) from the non-admixed Elk Island herd (along with various cow-bison hybrid and cow breed genomes). The cow-bison hybrids represent crossing of a bison male with a domestic cow (or rather a continuous line of female descent from such a cross) and so have strictly cow mitochondrial dna, not relevent to this section. The haplotype of all hybrids studied (from an unnamed private ranch in Montana, presumably Turner's Flying D) cluster with cow haplotype cHap32.

Bison accession numbers:
GU946976 GU946977 GU946978 GU946979 GU946980 GU946981 GU946982 GU946983 GU946984 GU946985 GU946986
GU946987 GU946988 GU946989 GU946990 GU946991 GU946992 GU946993 GU946994 GU946995 GU946996 GU946997
GU946998 GU946999 GU947000 GU947001 GU947002 GU947003 GU947004 GU947005 GU947006

The CYTB sequences retrieved from these genomic entries (they are not yet in the database used by blastp) show haplotype notation. The 15 previously existing bison sequences at GenBank (some just fragments are also provided. Older fragmentary sequences are demonstrably error-prone and will be used here only as support -- never as sole source -- of a polymorphism. Redundancy introduced via non-standard SwissProt (UniProt) entries also has to be manually removed -- the Swiss did no sequencing on their own, simply deriving protein sequences from existing GenBank entries. This leaves 5 older complete sequences for Bison bison and 4 fragments, 2 attributed to Bison bonasus and 1 fossil dna sequence from Bos primigenius to serve as outgroup (rather than an inbred domestic cow).

Here it is necessary to pick a terminology. This must accommodate NCBI taxonomy -- irregardless of its correctness -- because otherwise blastp searches cannot be restricted by taxon. Note although bison are definitely sistered with yak to the exclusion of all other extant species, that creates problems because yak has been put in the genus Bos. Many relic wild cattle have no english language common name but rather that of a local language. Terminology table must show synonyms to allow PubMed and google searches -- especially important in a fast-moving field to locate preprints and conference proceedings. The table below does not attempt to implicitly resolve any scientific issue; it simply states preferred terminology at this site along with synonyms in common use.

(editing to be continued)
Bison bison      plains bison
Bison athabascae woods bison
Bison bonasus    euro bison
Bison priscus    steppe bison
Bos primigenius  auroch (extinct except for Korean and Italian cattle with auroch mitochondrial genomes)
Bos grunniens    yak
Bos indicus 
zebu
kourey
Bos taurus common cow
gaur
wisent
Leptobos last common ancestor to cows and bison

Sequences are color clustered according to the phylogenetic tree above. bHap1 is not shown. Note the woods bison cannot be resolved from the plains bison even though the Elk Island woods bison are a relic herd that did not mix with 7,000 plains bison imported from the Flathead Reservation in Montana up to Canada's Wood Buffalo National Park in the 1920's.

>CYTB_bisBis.GU946988 bHap8 plains bison b973 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHAGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU946994 bHap11 plains bison b1031 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHAGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU946990 bHap10 plains bison b985 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHAGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU947000 bHap10 plains bison bFN5 Niobrara
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHAGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU946991 bHap10 plains bison b1005 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHAGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU947004 bHap17 plains bison bYNP1586 Yellowstone NP
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHAGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU946976 bHap2 plains bison b790 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHAGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU946977 bHap2 plains bison b853 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHAGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU946978 bHap2 plains bison b854 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHAGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU946981 bHap2 plains bison b880 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHAGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU946983 bHap2 plains bison b925 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHAGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU946984 bHap2 plains bison b929 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHAGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU946993 bHap2 plains bison b1029 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHAGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU946995 bHap2 plains bison b1050 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHAGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU946996 bHap2 plains bison b1051 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHAGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU947001 bHap2 plains bison bNBR1 National Bison Range
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHAGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU946986 bHap2 plains bison b959 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHAGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW
 
>CYTB_bisBis.GU946989 bHap9 plains bison b979 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHVGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU946997 bHap9 plains bison b1091 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHVGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU946982 bHap5 plains bison b897 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHVGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW
 
>CYTB_bisBis.GU946982 bHap5 plains bison b897 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHVGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU946987 bHap7 plains bison b961 Montana
MTSLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHVGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU946979 bHap3 plains bison b855 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHVGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU946992 bHap3 plains bison b1018 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHVGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU946992 bHap3 plains bison b1018 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHVGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU946998 bHap12 plains bison b1191 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHVGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU947003 bHap16 plains bison bTSBH1005 Texas State Bison Herd
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHVGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU946999 bHap13 plains bison b1428 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHVGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU947002 bHap13 plains bison bTSBH1001 Texas State Bison Herd
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHVGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisAth.GU947005 wHap15 woods bison wEI1 Elk Island
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHVGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU946980 bHap4 plains bison b877 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHVGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisBis.GU946985 bHap6 plains bison b935 Montana
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHVGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bisAth.GU947006 wHap14 woods bison wEI14 Elk Island
MTNLRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGMCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHVGRGLYYGSYTFLETWNIGVILLLTMMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIIMAIAMVHLLFLHETGSNNPTGISSDMDKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQMASIMYFLLILVLMPTAGTIENKLLKWDEFINITION

(editing to be continued)
YP_002791041  Bison bison
Q9T9C1        Bison bison
YP_003587278  Bison bonasus
ACE76876      Bos primigenius
YP_003541096  Bos primigenius
O20998        Bison bonasus
ADQ12704      Bison bonasus 61 ......T.....ETTAEF...................V......................  120
AAL85955      Bison bison
AAL85956      Bison bonasus
ADM87433      Bison bison 
AAW28803      Bison bison
AAW28804      Bison bison
AAW28802      Bison bonasus
AAN28295      Bison bison
CAA76013      Bison bonasus

Interpreting yak CYTB variation

Yaks are the closest living sister species to bison. Although 15,000 wild yaks still persist, they have been subject to very similar pressures to those experienced by bison: bottlenecks, population fragmentation, introgression from long domesticated yaks and hybridization with cattle. Adaptations specific to mitochondria may exist as yak live at altitudes exceeeding 3500 meters where average annual temperatures in rearing areas are –8°C, surviving winter temperatures of –40°C.

Because yaks provide the immediate outgroup for bison genetics (and vice versa), their parallel mitochondrial proteomics are investigated in depth here. This further enables reconstruction of their last common ancestor and correct placement of Pleistocene dna sequences.

left

Data availability for yaks was greatly improved by a Dec 2010 paper investigating yak phylogeographical structure and demographic history on the Qinghai-Tibetan Plateau. Complete mitochondrial genomes were determined for 48 domesticated and 21 wild yaks. The three lineages article supplemental established diverged at 420 kyr and 580 kyr in accordance with allopatric migration barriers created by two large plateau glaciations.

The wild yaks are found in all three branches of the tree (solid circles at left). Their entries at GenBank are apparently distinguished by a W (for wild) prefix, eg isolate W77 GQ464266. There is potential for confusion here because NCBI taxonomy uses Bos grunniens mutus subspecies notation for wild yak (a concept contradicted by the mixed distribution of wild and domestic yaks in the tree). Related concepts such as Bos mutus (Przewalski, 1883), Bos mutus grunniens, and Poephagus mutus also don't fit the facts.

Polymorphisms in wild yak cytochrome b sequences are the primary focus here as domestic yak may exhibit inbreeding issues and evolutionary artefacts. Consequently it is important to track which GenBank entries reference wild yaks.

Bos grunniens mutus has two GenBank entries relevent to CYTB: AAX53006 containing V195A, I348F otherwise lacking support and CAA76015, an older fragmentary sequence of no allelic interest. The Myanmar/Bhutan mithun sequence BAJ05329 attributed to Bos grunniens at GenBank has 12 differances but is 100% identical to 94 Bos indicus entries, ie the mitochondrial genome of this hybrid originated there.

The 21 new genome accessions of wild yak are GQ464266, GQ464265, GQ464264, GQ464263, GQ464262, GQ464261, GQ464260, GQ464259, GQ464258, GQ464257, GQ464256, GQ464255, GQ464254, GQ464253, GQ464252, GQ464251, GQ464250, GQ464249, GQ464248, GQ464247, GQ464246.

In terms of protein accessions (which will be shown at NCBI blastp output), these are ACU81659, ACU81646, ACU81633, ACU81620, ACU81607, ACU81594, ACU81581, ACU81568, ACU81555, ACU81542, ACU81529, ACU81516, ACU81503, ACU81490, ACU81477, ACU81464, ACU81451, ACU81438, ACU81425, ACU81412, ACU81399.

Of these, 16 fall in the main reference sequence group but 5 wild Tibetan plateau yaks exhibit polymorphisms that cannot be attributed to domestication. Two additional wild yaks from extreme NW China have additional double alleles but no associated PubMed publication. There is no overlap between wild yak polymorphism sites and the five of domestic yak. Alleles occurring in full length sequences are analyzed further below.

ACU81568 A017T       wild yak   isolate W50   GQ464259
ACU81399 I192T       wild yak   isolate W02   GQ464246
ACU81633 I192T       wild yak   isolate W75   GQ464264
ACU81555 D214N       wild yak   isolate W40   GQ464258
AAX53006 V195A I348F mutus      isolate Xinjiang01 unpublished Liu,Q Wu,M Li,Y 
AAX53007 V195A I348F mutus      isolate Xinjiang02 unpublished Liu,Q Wu,M Li,Y
ACU81529 V329M       wild yak   isolate W1313 GQ464256

ABI15999 V039I A067T domestic yak              fragment   PUBMED:17257194 Poephagus
ABI16000 V039I A067T domestic yak              fragment   PUBMED:17257194 Poephagus
ACU82153 A084T       domestic yak isolate HY5
ACU82101 V098L       domestic yak isolate HY1
AAU89116 I118T       domestic yak             =SP:Q5Y4Q0  PUBMED:16942892
ACU81711 I118T       domestic yak isolate HZ3 
ACU81737 I118T       domestic yak isolate MQ1
AAS93096 I118T       domestic yak              fragment   PUBMED:17257194
AAS93099 I118T       domestic yak              fragment   PUBMED:17257194

Although the mitochondria encodes the usual 20 amino acids, only a subset of physio-chemically similar residues (the reduced alphabet) ever appear at a given position in a given protein. This subset describes the acceptable substitutions that do not significantly disrupt protein functionality. Discovery of this reduced alphabet can be achieved with greater sensitivity when the number of available species and their individual sequences multiplicities are high. For mitochondrial proteins, that sensitivity is 1 in 10,000 (0.01% occurrence frequency) for a given amino acid.

Interpretive certainty is never attained without experimentation but improves (up to a point) with more sequence data. Here it is important to check whether certain less common substitutions have persisted over evolutionary time in a phylogenetically coherent manner (ie a sub-clade) or are novel adaptations perhaps in conjunction with a co-evolving residue at another site (or another protein, perhaps even nuclear-encoded). After these considerations, the remaining rare changes are either deleterious or sequencing error. Polymorphism significance can be pursued at the xray structural level for only 3 of the 13 mitochondrial proteins (CYTB, COX2, COX1) and even this is complicated in the case of CYTB by its oligomeric association with 3 nuclear encoded proteins.

Aligning CTYB from the 70 complete yak mitochondrial genomes available on 1 Dec 10 shows variation at just 9 sites along the protein (ie 9 nsSNPs). These are quickly found when the web alignment tool retains input sequence order, displays residues identical to the top sequence as dots, gaps fragmentary data correctly, and allows a wide display permitting effective cross-species comparisons.

Yak and bison -- despite being sister species -- share variation only at one site, position 98. Here yak is exclusively valine with the exception of a single deleterious occurrence (see below) of leucine, whereas bison have a mix of valine and alanine (which otherwise is very rare at this position in mammals), ie the ancestral residue was valine. Thus no lineage sorting occurred at any amino acid position in CYTB at the time these species diverged. Lineage sorting however may be important in the overall evolution of the Bovini: 53 ancient polymorphisms (at the dna level) are said to have persisted since Bos and Bison diverged from Bubalus 5–8 million years ago.

The summary table of yak CYTB amino acid polymorphisms below arises from alignment of 5000 full-length mammalian cytochrome b orthologs. Red indicates deleterious mutation, green a possibly acceptable change but of restricted distribution and fitness, and blue a near-neutral substitution. It can be seen that the smallish yak population sampled (21 wild, 48 domestic added in Aug 10 to 3-4 previously available) already contains 5 deleterious alleles in CYTB which represents only 10% of the mitochondrial proteome.

The changes can also be displayed in context by coloring the appropriate residues in a reference sequence relative to a composite sequence consolidating all the polymorphisms from distinct animals (no one animal has more than two of the 9; V195A + I348F occurs in two animals). This latter sequence is quite useful in comparing polymorphism sites across species as explained in the annotation tricks section.

>CYTB_bosGruR Bos grunniens cytochrome b ref seq taken as gi|147744503 
MTNIRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGVCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHVGRGLYYGSYTFLETWNIGVILLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIITAIAMVHLLFLHETGSNNPTGISSDADKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLVADLLTLTWIGGQPVEHPYIIIGQLASIMYFLLILVLMPTAGTIENKLLKW

>CYTB_bosGruP Bos grunniens composite polymorphisms: A017T A084T V098L I188T I192T V195A D214N V329M I348F
MTNIRKSHPLMKIVNNTFIDLPAPSNISSWWNFGSLLGVCLILQILTGLFLAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHTNGASMFFICLYMHLGRGLYYGSYTFLETWNIGVTLLLTVMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFIITATAMAHLLFLHETGSNNPTGISSNADKIPFHPYYTIKDILGALLLILALMLLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAI
LRSIPNKLGGVLALAFSILILALIPLLHTSKQRSMIFRPLSQCLFWTLMADLLTLTWIGGQPVEHPYFIIGQLASIMYFLLILVLMPTAGTIENKLLKW

   A017T       A084T     V098L     I118T     I192T     V195A     D214N     V329M     I348F  
  927  A    4,994  A   4522  V   4309  I     94  I   4528  V   4429  D   4610  V   4232  I
 4018  S        3  T    430  I    667  S   4353  L    427  I    512  N    188  T    651  V
   46  T        1  P     34  M     14  I    505  M     25  T     43  E    133  A     63  T
    3  L        1  V     11  A      1  T     31  T      4  G      8  S     44  I     45  M
    3  M                  1  L                3  F      4  M      2  Y     22  M      4  N
    1  F                  1  N                2  V      1  A      1  H      2  G      2  F
    1  P                                      1  A                          1  E      1  A                       
                                              1  S        

A017T: At position 98, the mammalian reduced alphabet consists primarily of serine with yak alanine also well represented at 18%. Threonine occurs in 46 sequences so cannot be sequence error or serious mutation. Bulk seems to be the main criterion at this site rather than polarity -- threonine though polar is bulkier residue than serine or alanine. To determine whether it has arisen multiple times or just in one clade, the phylogenetic distribution of the 46 occurrences needs consideration.

It can be seen from the graphic at left that A017T has arisen multiple times with no common denominator (such as high elevation lifestyle) but -- with the exception of monotremes -- never in a deep stem ancestor. That is, A017T occurs here and there but only in recently speciated clades. This suggests that while not lethal, over time it gets replaced by more adaptive serine or alanine.

A017T
  927  A
 4018  S
   46  T
    3  L
    3  M
    1  F
    1  P

V098L: At position 98, the reduced alphabet consists of valine 90% of the time regardless of mammalian clade with the similar (branched chain aliphatic) isoleucine having substantial dispersed representation at nearly 9%. The 430 species in which it occurs are scattered incoherently within mammal clades, meaning that it has arisen independently many times. V098I may be slightly suboptimal as there is an evident bias (at some level) against equal occurence. It likely co-exists with valine in most non-bottlenecked populations of mammals, observed if enough individuals of a given species are sequenced.

However leucine, the seemingly similar third aliphatic residue, occurs one once despite being but a single base change transition away from the dominant residue. Were leucine a near-neutral substitution, its incidence would be vastly higher. Thus the change V098L reported for yak represents either a deleterious mutation or an unprecedented adaptation (eg to high altitude) or sequencing error in GenBank entry ACU82101. The same can be said for the more overtly radical change V098N in lemur AAS00156.

V098L
4522	V most common amino acid at position 98 of CYTB
 430	I
  34	M
  11	A bison
   1	L yak
   1	N lemur

(analysis to be continued)
I118T
 1843  L
  404  V
   87  A
   61  M
    6  T (all yak)
    1  S
    1  F

(analysis to be continued)
I192T  
   94  I
 4353  L
  505  M
   31  T
    3  F
    2  V
    1  A
    1  S

(analysis to be continued)
V195A  
 4528  V
  427  I
   25  T
   11  X
    4  G
    4  M
    1  A

(analysis to be continued)
D214N  
 4429  D
  512  N
   43  E
    8  S
    4  X
    2  Y
    1  H

(analysis to be continued)
V329M  
 4610  V
  188  T
  133  A
   44  I
   22  M
    2  G
    1  E

(analysis to be continued)
I348F  
 4232  I
  651  V
   63  T
   45  M
    4  N
    2  F
    1  A

Kilo-sequence alignment tricks

New sequencing technologies have greatly affected the amount of mammalian mitochondrial genomic data available at GenBank. Five years ago, it was acceptable to publish population-level D loop sequences accompanied by a few fragmentary coding reads; today, a publication might offer 60-70 entire mitochondrial genomes. This favors evolutionary study of mitochondrial proteins over comparative genomics of nuclear genome products because the latter is still restricted to around 50 species (Dec 2010) almost all incompletely sequenced.

Many long-standing issues such as introgression, historic bottlenecks, population mixing, accrual of deleterious coding variants, hard polytomies, and lineage sorting during speciation can now be approached and resolved, especially with the increasing sequencing of end-Pleistocene frozen dna. This may allow more enlightened management of endangered species such as bison where populations reached rock bottom -- recovering numbers is not enough if genomic integrity is still at risk.

However, the flood of data raises significant issues in extraction of significant information: it is not instructive to align the tens of thousands of sequences available for each of 13 mitochondrial proteins -- that give a an intractable array of 3789 amino acids by 12500 sequences, enough to fill 20 x 100 = 2000 screens on the largest possible computer monitor. That data must be distilled down somehow to take-away information.

This section explains a practical desktop protocol for extracting the 'reduced phylogenetic alphabet' at each residue of the mitochondrial proteome. The method depends heavily on current capabilities of Blastp at NCBI and so may not be completely stable to changes made there over time.

First note that tBlastn cannot be used against the nr or wgs nucleotide databases at NCBI (or with Blat at UCSC) since the significantly different genetic code of mammalian mitochondria is no longer supported as a parameter option. Other oddities involve missing terminal nucleotides that are added before translation. However mitochondrial dna is usually translated sensibly at GenBank protein entries.

The vertebrate mitochondrial code:

TTT F Phe      TCT S Ser      TAT Y Tyr      TGT C Cys  
TTC F Phe      TCC S Ser      TAC Y Tyr      TGC C Cys  
TTA L Leu      TCA S Ser      TAA * Ter      TGA W Trp  
TTG L Leu      TCG S Ser      TAG * Ter      TGG W Trp  

CTT L Leu      CCT P Pro      CAT H His      CGT R Arg  
CTC L Leu      CCC P Pro      CAC H His      CGC R Arg  
CTA L Leu      CCA P Pro      CAA Q Gln      CGA R Arg  
CTG L Leu      CCG P Pro      CAG Q Gln      CGG R Arg  

ATT I Ile      ACT T Thr      AAT N Asn      AGT S Ser  
ATC I Ile i    ACC T Thr      AAC N Asn      AGC S Ser  
ATA M Met i    ACA T Thr      AAA K Lys      AGA * Ter  Bos can use ATA as initiation codon
ATG M Met i    ACG T Thr      AAG K Lys      AGG * Ter  

GTT V Val      GCT A Ala      GAT D Asp      GGT G Gly  
GTC V Val      GCC A Ala      GAC D Asp      GGC G Gly  
GTA V Val      GCA A Ala      GAA E Glu      GGA G Gly  
GTG V Val i    GCG A Ala      GAG E Glu      GGG G Gly  

    AAs  = FFLLSSSSYY**CCWWLLLLPPPPHHQQRRRRIIMMTTTTNNKKSS**VVVVAAAADDEEGGGG
  Start  = --------------------------------MMMM---------------M------------
  Base1  = TTTTTTTTTTTTTTTTCCCCCCCCCCCCCCCCAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGG
  Base2  = TTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGG
  Base3  = TCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAG