親子測序ppt_11.15

1、Zhang Xuehai2015-11-9Background(背景介紹) 基因突變基因突變主要指DNA核苷酸順序，種類和數(shù)量的改變，是遺傳變異的基本來源。1、自發(fā)突變（偶然的復(fù)制、轉(zhuǎn)錄、修復(fù)時的錯配）；2、誘發(fā)突變（誘變劑）； 突變?yōu)檫z傳學(xué)研究突變?yōu)檫z傳學(xué)研究提供突變型，為育種工作提供提供突變型，為育種工作提供素材；素材； 突變是生物進(jìn)化的重要因素。突變是生物進(jìn)化的重要因素。 自發(fā)突變受一系列因素的影響、是一系列變化的結(jié)果，具有隨機性和稀有性（經(jīng)典認(rèn)識）。 隨著DNA測序技術(shù)的突破性進(jìn)展，使自發(fā)突變在基因組中的數(shù)量和分布有了精確的估計。確認(rèn)了自發(fā)突變在基因組中不是隨機分布的，突變率在突變熱點

2、是很高的，而突變熱點普遍存在于基因組中。（現(xiàn)在認(rèn)識），突變熱點是如何產(chǎn)生的？突變熱點是如何產(chǎn)生的？ 生物進(jìn)化的漫長過程中，造成生物多樣性的遺傳差異是如何產(chǎn)生、積累乃至保留下來的？Background(背景介紹)Indel誘變誘變假說假說（Tian et al, 2008 Nature）： 插入/缺失(Indel)兩側(cè)的堿基點突變出現(xiàn)的頻率較其他區(qū)域要高的多； Indel附近的突變率比一般自發(fā)突變率高10倍左右； 某一段DNA序列的突變率取決于該序列與Indel的距離，兩者有高度負(fù)相關(guān)；這種負(fù)相關(guān)關(guān)系在各個基因組中都是普通存在的。基于人、黑猩猩、恒河猴、小鼠、大鼠、果蠅、兩個水稻和三個釀酒酵母基

3、因組。從源頭上回答了遺傳變異是如何形成的。從源頭上回答了遺傳變異是如何形成的。經(jīng)典遺傳學(xué)：同源染色體對稱的等位基因系列；非對稱非對稱遺傳學(xué)遺傳學(xué)：同源染色體間基因的數(shù)量和位置是不同的，可能是某些疾病發(fā)生、雜種優(yōu)勢、自交退化等產(chǎn)生的遺傳基礎(chǔ)Background(背景介紹)1）自發(fā)突變的數(shù)量是由Indel的數(shù)量和密度所決定，因而基因組各區(qū)域的突變率很不相同。但I(xiàn)ndel本身是一種點突變，其發(fā)生有一定的隨機性，因而其誘發(fā)的突變也有一定的隨機性；自發(fā)突變的數(shù)量在Indel附近并不稀有，但遠(yuǎn)離Indel的區(qū)域是稀有的。2）找到了多數(shù)自發(fā)突變的發(fā)生根源，也就是說，生物多樣性的最初變異來源，主要是Indel

4、誘導(dǎo)產(chǎn)生的。3）Indel在編碼區(qū)間比堿基置換對基因功能的影響大得多，從而更易被自然選擇所淘汰，使保守區(qū)間的突變率降低。換句話說，自然選擇很大程度上是通過對Indel的選擇而實現(xiàn)的，自發(fā)突變率的高低很大程度上是自然選擇的結(jié)果。4）生物通過調(diào)節(jié)自身變異能力而適應(yīng)環(huán)境的能力，比我們原先想象的要大得多，即突變在進(jìn)化中的作用很大。 Evidence indicates that mutation rates vary almost 1,000-fold between species, from 1011 mutations per nucleotide site per generation in

5、some unicellular organisms to approximately 108 in primates (靈長類動物靈長類動物) (genome-wide averages). Mutation rates can vary between nucleotide sites and between members of the same species. Why mutations rates vary within genomes ？BackgroundConclusion Materials：Arabidopsis,riceandthehoneybee； Method：Pa

6、rent-offspringsequencingstrategy； Conclusion：1、Mutationratesarehigherinheterozygotesandinproximitytocrossoverevents.2、Mutationsoccurdisproportionatelymoreofteninheterozygousthaninhomozygousdomains.3、Geneclustersunderpurifyingselection(commonlyhomozygous)andunderbalancingselection(mainlyheterozygous)

7、havelowandhighmutationrates,respectively.Theauthorssuggestthatextremely weak selection onthemutationratemaythereforenotbenecessarytoexplainwhymutationalhotandcoldspotsmightcorrespondtoregionsunderpositive/balancingandpurifyingselection,respectively.(也許并不需要用突變速度的極弱選擇來解釋為什么突變熱點和冷點可能會分別位于發(fā)生正向/平衡和純化選擇的區(qū)

8、域。)-強選擇會降低多樣性。MaterialandMethods(sequencing) Arabidopsis： 2 P0: Col(female), Ler(male) 26 P1: 17Col: Col1-2+Col3-5+Col3(1-12) 9 Ler: Ler2-4+ Ler1 + Ler1(1-5) 67 F2: 32 F4 : C52: 21+102=22 C64: 23+41=10The sample colored red is sequenced previously. Rice： 2 P0: 9311(paternal), PA64s(maternal) 6(5) P1

9、: 9311(-1,-2,-3), PA64s(-1,-2,-3) 1F1: LYP9_F1 12(11)F2: F2_33The sample colored red is poor sequencingand removed. Honey bees: 3 colonies 3 queens 43 drones(雄蜂) have been reported previouslyFigure1 Pedigree relationship of the sequenced Arabidopsis samples.Figure2 Schematic diagram of the detection

10、 of de novo mutations.Direct sequencing of genomes is the best way to detect de novo mutations, the error rate is high. We negate this by having several lines of quality control:1、Sequenced multiple independent DNA extractions from the same individual or inbred progeny of the individual, permitting

11、a mutation to be called only if replicates agree.2、we use a consensus approach, comparing each focal individual against all other relevant samples. For example, a presumptive mutation in an F2 progeny must be both called within a mutated sample and not called in both the sequenced parental genomes a

12、nd all other F2 progeny.These criteria ensure that the mutation must have arisen sometime after bolting in the F1, as all other F2 progeny share the same F1 parent.Sequencinghigh sequence quality: score30high coverage(6,000 for the sample cohorts and 40 for each sample) with at least five or more re

13、ads (must include both forward and reverse reads).False positive rates are negligible,while false negative rates are low.Toassesstheirreliability,severalstrategieswereapplied.First,Sangersequencing(100%):112sampledbasemutationsand43sampledindelspresentinF2plants.Confirmationrequiresthatthemutationbe

14、presentinthefocalindividualandabsentinbothparentalgenomes.Second,the32sequencedF4plants,derivedfromtwoF2plants(c52andc64with10mutationsobserved),confirmed100%oftheseF2mutationsatafrequencyof,73%(slightlyhigherthantheexpected62.5%).Third,werandomlysampled48F3plantsfromeachofthe21sampledF2plantsandsub

15、jectedtheseF3plantstoSangersequencing.Thisconfirmed99outof100sampledbasemutationsand24outof26indelspresentin F3 plants(92%).(P0-P1:7.410-9(=0.88/120Mb) vs7.010-9)3.6 fold2.8 fold9627332Number of mutations in Arabidopsisb,c Variation in the mutation rates as a function of heterozygosity proportion du

16、ring meiosis.Conculsion:Mutationsoccurdisproportionatelymoreofteninheterozygousthaninhomozygousdomains.Implicatingdiversityperseasacause.3.6 fold1.341.602.680.74Heterozygousblock,C64:48%,C52:61%.Variation of the mutation rates in heterozygosityNumber of mutations in rice4.09/1.2=3.4If heterozygosity

17、 causes an increase in the mutation rate. We test this by comparing progeny derived from heterozygous and homozygous parents in our two plant species.Population-wide intragenomic diversity is commonly reported to be higher in genomic domains of high crossing-over,Figure The relationship between nucl

18、eotide diversity (Col versus Ler) and recombination rate. (67 F2 and 32 F4 plants)high recombination rates to also be domains with high mutation rates, possibly because recombination is mutagenicFigure Relationship between the mutation and recombination rate. Conclusion : recombination is mutagenic

19、or that both mutation and recombination preferentially occur in high diversity domains.Relationship between the nucleotide diversity/mutation and recombination rateHoney bee: mutation happens more commonly in the vicinity of crossovers. Table Mutations in coding and non-coding regions in Arabidopsis

20、 and rice.Whichcannotbeaccountedforintermsofdifferencesintrinucleotidecontent.two reasons may be ：1、underestimation of the mutation rate in coding sequences, possibly due to purifying selection；2、a lower mutation rate in the transcribed sequence, possibly owing to transcription-coupled repair. The l

21、evel of diversity within the parentsConclusion: The level of diversity within the parents surrounding mutation sites is higher than the genome average (0.39% between two parents).(換句話說：多樣性與離突變位點的距離負(fù)相關(guān))Figure Relationship between mutation and distance to polymorphic sites.Figure Relationship between

22、nucleotide diversity and the distance to the de novo mutations.Spectra of nucleotide substitutions in Arabidopsis and riceConclusion：1、More transitions(轉(zhuǎn)換) than transversions(顛換).2、Mutations are disproportionately common at GC-rich nucleotide triplets (see Supplementary table). Figure Spectra of nuc

23、leotide substitutions in Arabidopsis and rice.Distribution of the mutations on the chromosomes The grey vertical bars :the position of all collected mutations. Blue shadow :the mutation number /each window. The red lines: The average pairwise diversity among the published 80 Arabidopsis ecotypes.Fig

24、ure Distribution of the mutations on the chromosomes. Howheterozygositymightpromotemutation?- poor-pairing modelFigure(left) A schematic diagram of the genomic structures and the possible pairings of two homologous chromosomes during the meiosis at two mutated LRR-TM genes (top) and one mutated NBS-

25、LRR gene (bottom).AT3G23110:TCAT3G23120:Del:Cnon-synonymous mutationframeshiftDiscussion Proposeapositive-feedback loop(正向反饋循環(huán)正向反饋循環(huán)),wherebyhighlevelsofmolecularvariationinanindividualfacilitatetheproductionofmorevariation.(mutation&recombination VS diversity, chicken-and-egg problem) Thisisaph

26、enomenon.Howheterozygositymightpromotemutation?Whatismechanism?(poor-pairing model,especiallyforindels,)Heterozygote instability (HI)：HI influences both the rate and distribution of mutations Ifrecombinationmagnifiesthemutationrate,ratesshouldbereducedonchromosomesthatcannotrecombine?suchastheXandYo

27、fhumanmalesandallthechromosomesofmalefruitflies.Iftheauthorshypothesisiscorrect.Totestwhetherheterozygosityperseisadirectdeterminantofthemutationrate.Honeybee:malescontainonlyasetofchromosomesinheritedfromtheirmothersmutationratesshouldbelowerinmalesthanintheirheterozygoussisters.DiscussionDiscussio

28、n It is worth considering how the approximately 3.6-fold difference in mutation rate between inbred and outbred strains found in the current study compares with variation among individuals in normal populations. The mutation rates in two inbred lines of fruit fly differ by around 2.3-fold, and these rates are slightly higher than those of outbred flies. (why?) Furthermore, humans and chimpanzees, which are highly homozygous, have extremely high mutation rates. Of course, there are many biological differences between these species, so caution must be taken not to over interpret these observ