Lecture 6 - Establishing genomics platform for crop species Flashcards

Question

How can you deal with inter homolog polymorphisms when mapping a genome?

Answer 1

Have extra codes in polyploidy nucleotide bases which correspond to a mixture of two bases- ambigutity codes Y corresponds to a mixture of C and T bases S corresponds to a mixture of G and C bases Because this is a polymorphism between homologus loci the polymorphism is present in both cultivar types Interhomolog polymorphisms can be ignored if have sequenced deeply enough If different between cultivar SNP it is a hemi-SNP

Answer 2

Can use illumi mRNA sequencing Reads from two cultivars (e.g. two oilseed rape cultivars) and maps onto a reference sequence Transcript assembly acts as reference e.g. brassica transcriptome) for the identification of SNP mRNA derived sequences often used - cheaper Call potential varient alleles Interhomolog polymorphisms called against the reference sequence can be filtered out as they would occur in both cultivars Main source of error of using next gen sequencing to look at SNP markers: if have very thin coverage of sequence reads won't be sure if there is enough depth of sequencing to have captured both types of bases in that particular region

Answer 3

1. Need to make a high density linkage map with more than 50-100 linkage markers. 2. Start with two variations that are as genetically divergent from each other as can be found. Cross these and produce F1 generation. 3. self the hybrid back and develop recombinant inbred lines. (using many rounds of single seed descent/capture process - get spores and grow up to produce mapping population which then are invarient and can be maintained indefinately). 4. Once a population has been generated individuals need to be sequenced e.g. through mRNA sequencing. 5. Call alleles for each of the individual plants by mapping the sequence reads to the reference sequence and calling dependent on whether have mixed bases or single bases and looking for what varies across a population. 6. Construct a genetic linkage map using appropraite software. 7. For each polymorphic marker give each line in a population a score of a or b (corresponding to the allele or marker which occurs in that particular line - where a referes to a female parent in the original crossover and b refers to the male parent) 8. This is then used by computational processing to find the order in the genome to which these markers fit best 9. Generates a high density SNP linkage map. 10. If done based of mRNA sequences shows that the polymorphisms are within genes.

Answer 4

TNDH linkage map with around 21000 SNP markers for 527 recombination lines Large number of markers generally limited by the number of recombination bins present within the sequenced population. Although around 21000 markers, they will simplify to a number of different patterns and scoring strings which will represent the recombination bins across the population.

Answer 5

Have vertical strings that correspond to an allele and when stops, represents a recombination point within the population. Markers are in the order which the occur in the genome Can look for sequence similarity in other genomes using these markers and see regions of high colinearity e.g. between arabidopsis and brassica enome

Answer 6

found around 9000 brassica genes coevolved directly Used genetic linkage map by SNP Most SNPs have sequence similarity with arabidopsis Colour coding gives block colinearity between genomes consistent with diagonals on genome plot

Answer 7

No genome sequence for Brassica napus but there is for an ancestral species. For genes in which a polymmorphism occurs, can find through the sequence similarity in the gene sequence of scaffold of the ancestral sequence As know the order of the markers (from previos comparison of brassica napus with arabidopsis) to identify and associate te scaffolds of other species to the constructed linkage maps 125 000 transcripts anchored to the pseudomolecules (comprising B. rapa and B. oleracea genome sequence scaffolds) - represents the hypothetical gene order in oil seed rape

Answer 8

1. Take genome sequence scaffold of target species and anchor mapped markers to scaffold 2. Leads to the identification of a lot of chimeric scaffold assembled 3. Quality can be improved by splitting and rearranging scaffolds to match them to the correct parts of the genome 4. These assemble in pseudomolecules which represent genome organisation. 5. Take all genes from the species/transcript assemblies and find sequence similarites by BLAST to anchor the transcripts to the pseudomolecules 6. Hypothetical order of genes 7. Can then align genes of species of interest to sequence similarity Need higher density linkage map the smaller the sequence scaffolds

Answer 9

Use a more distant species to organise genes of the species of interest Exploit colinear genomes at least on a short range in order to infer gene orders e.g. comaprative genomics of wheat with Brachypodium 4677 genes were anchored directly by SNP mapping to high density genetic linkage map (by genetic linking of transcriptomr SNP gene markers) Enables an analysis of the colinearity of the genomes of hexaploid wheat and B. distachyon Identify the best sequence map of wheat transcripts to genome sequence scaffold of related species (B. distachyon) Relatively simple rearrangment of sequences as the B. distachyon sequence is highly contiguos and cereal genomes show fewer rearrangements than those of brassicas e.g. Once have an organisation of brachypodium genome into pseudomolecules can use sequence similarity to position the rest of the genome and identify a hypothetical organisation in the wheat genome of identified wheat genes. 112479 transcripts anchored to pseudomolecules (comprising B.distachyon genome sequence scaffolds) to represent the hypotetical gene order in wheat

Answer 10

Use genome sequence scaffolds of related species, the closer related the species the better the related genome is as proxy