本文關(guān)鍵詞：基于2b-RAD技術(shù)的輔助基因組組裝和標(biāo)記分型研究　出處：《中國海洋大學(xué)》2015年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 2b-RAD技術(shù) 基因組組裝 標(biāo)記分型 全基因組選擇 櫛孔扇貝

【摘要】：非模式生物的遺傳資源相對匱乏,在這些物種中開展基因組范圍內(nèi)的遺傳學(xué)研究仍然非常困難。簡化基因組技術(shù)可以視為非模式生物的遺傳學(xué)研究的有利工具。該技術(shù)主要是通過降低基因組的復(fù)雜度來降低測序成本,被廣泛的應(yīng)用于遺傳圖譜構(gòu)建、數(shù)量性狀定位、群體遺傳學(xué)分析、系統(tǒng)進(jìn)化分析和輔助基因組組裝研究中。1、“橋接法”輔助基因組組裝策略本研究提出了一種“橋接法”基因組組裝策略：首先將2b-RAD分型技術(shù)引入傳統(tǒng)的Happy實(shí)驗(yàn)構(gòu)建高密度的2b-RAD圖譜,借助該圖譜可以對已有的Contigs序列進(jìn)行進(jìn)一步的升級。為了實(shí)現(xiàn)這一想法,我們不僅優(yōu)化了傳統(tǒng)的Happy實(shí)驗(yàn),同時還還提出了結(jié)合隨機(jī)抽樣技術(shù)的層次組裝算法。模擬數(shù)據(jù)顯示該組裝算法能夠?qū)M南芥全基因組的35,618個BsaXI標(biāo)簽組裝成40個Contigs,校正后的N50大小為4.1Mb(克隆長度為40kb,樣本量為100)；將人類1號染色體95,139個BsaXI標(biāo)簽組裝成16個Contigs,校正后的N50大小為14.4Mb(克隆長度為40kb,樣本量為100)。實(shí)際數(shù)據(jù)分析顯示層次組裝算法可以將擬南芥基因組內(nèi)34,753個BsaXI標(biāo)簽組裝成554個群,校正后的N50大小為224kb。在連接Contig方面,原始N50大小為54.1kb的Contig通過該軟件其N50可以提升到815kb,N50大小為183.4kb的Contig可以提升到1.03Mb,N50大小為552.7kb的Contig可以提升到3.7Mb,而且Contig之間連接的準(zhǔn)確率在98.1%-98.5之間。該低成本的輔助基因組組裝方案將在海洋生物復(fù)雜基因組組裝項(xiàng)目應(yīng)用中發(fā)揮重要作用。2、無參照基因組分型算法開發(fā)和應(yīng)用當(dāng)前簡化基因組技術(shù)的標(biāo)記分型軟件存在的缺點(diǎn)是：1)仿照有參照基因組的分型方法,無法排除基因組中重復(fù)序列對de novo分型造成的干擾；2)忽略了對顯性標(biāo)記的分型。本研究提出了一種混合泊松(正態(tài))分布模型對來自重復(fù)序列區(qū)域的序列進(jìn)行概率識別,并將該模型加入到已有的標(biāo)記分型軟件中形成新的分型算法iML。通過擬南芥和水稻基因組模擬數(shù)據(jù)分析表明iML方法比傳統(tǒng)的ML算法假陽性率低12%-23%。通過擬南芥2b-RAD數(shù)據(jù)和三刺魚的RAD-seq數(shù)據(jù)的驗(yàn)證表明iML方法比ML分型算法假陽性率低7%-17%(測序讀長為30bp)。此外本研究開發(fā)了RADtyping軟件,其不但整合了iML共顯性標(biāo)記分型算法,同時給出了處理顯性標(biāo)記的統(tǒng)計公式。通過擬南芥擬測交F1群體模擬數(shù)據(jù)顯示當(dāng)親本和子代的平均測序深度為20x時,兩類型標(biāo)記的分型準(zhǔn)確率可達(dá)98%。通過實(shí)際的兩套重復(fù)文庫分型結(jié)果發(fā)現(xiàn),共顯性標(biāo)記的分型一致性達(dá)96%。通過Sanger法驗(yàn)證顯示共顯性標(biāo)記的分型準(zhǔn)確率為96%,顯性標(biāo)記的準(zhǔn)確率為97%,這充分說明了RADtyping在標(biāo)記分型上具有較高的準(zhǔn)確率。3、2b-RAD技術(shù)在全基因組選擇中的應(yīng)用評估全基因組選擇技術(shù)實(shí)施的重要條件之一是要有大量的基因組范圍內(nèi)的遺傳標(biāo)記。2b-RAD技術(shù)雖然在分型成本上具有明顯的優(yōu)勢,其提供的標(biāo)記密度是否滿足水生生物全基因組選擇育種需求仍然是未知的。本研究根據(jù)蝦夷扇貝的基因組特征(包括基因組大小、雜合率、BsaXI酶切位點(diǎn)分布等)模擬了蝦夷扇貝的育種群體�？疾炝巳N不同標(biāo)記密度HD-SNPs(芯片密度),MD-SNPs(所有BsaXI酶切位點(diǎn)),LD-SNPs(帶有選擇性堿基的BsaXI位點(diǎn))對全基因組選擇育種值估計準(zhǔn)確率的影響。分析表明在不同的遺傳背景下MD-SNPs比HD-SNPs準(zhǔn)確率略低(3%)。在遺傳力在0.3～0.5左右時LD-SNPs在育種值準(zhǔn)確率估計上和MD-SNPs相當(dāng),但是標(biāo)記的分型成本僅為后者十分之一。隨后利用來源于3個家系的349個蝦夷扇貝育種群體,對殼高、殼長和殼寬三種性狀進(jìn)行全基因組選擇評估。家系間的育種值估計準(zhǔn)確率在0.15-0.3之間,家系內(nèi)的育種估計準(zhǔn)確率在0.23～0.36之間。上述分析表明2b-RAD技術(shù)是水生生物全基因組選擇項(xiàng)目中標(biāo)記分型的平臺首先。
[Abstract]:Non biological genetic resources are relatively scarce, it is still very difficult to carry out genetic studies within the genome in these species. Genetic research tool simplified genome technology can be regarded as non model organisms. This technology is mainly to reduce the cost by reducing the complexity of genome sequencing, is widely used in the construction of genetic map. QTL, genetic analysis, phylogenetic analysis and auxiliary genome assembly study.1, "bridging" auxiliary genome assembly strategy in this study presents a "bridging method" for group assembly strategy: first will build the high-density 2b-RAD map Happy experimental 2b-RAD typing technique into traditional, further with the help of Contigs to upgrade the existing sequence of the map. In order to realize this idea, we not only optimize the traditional Happy test, at the same time Also presented based on random sampling technology level assembly algorithm. Simulation data show that the algorithm can be assembled 35618 BsaXI tag genome assembly into 40 Contigs, corrected N50 size 4.1Mb (clone length 40KB, 100 samples); human chromosome 1 95139 BsaXI tag assembly 16 Contigs, N50 after correction of size 14.4Mb (clone length is 40KB, the sample size was 100). The actual data analysis showed that the level of assembly algorithm can be 34753 BsaXI tags within the Arabidopsis genome assembly into 554 groups, after correction of N50 size 224kb. in connection with Contig, the original N50 size 54.1kb Contig through the software N50 can be upgraded to 815kb N50, the size of 183.4kb Contig can be upgraded to 1.03Mb N50, the size of 552.7kb Contig can be upgraded to 3.7Mb, and the Contig connection between the accuracy in 98.1%- 98.5. Assist the genome assembly scheme of the low cost will play an important role in the marine biological complex.2 genome assembly project application, unmarked reference genome typing algorithm development and application of the simplified genome technology classification software shortcomings: 1) is modeled by a genotyping method of reference genome, to exclude the interference of repetition the genomic sequence of de novo type caused; 2) ignores the type of dominant markers. This study presents a mixed Poisson probability distribution model (normal) identification of repeat sequences from this area, and this model is added to the existing tag type software through the rice genome Arabidopsis and simulation data analysis showed that the iML method is better than the traditional ML algorithm of low false positive rate of 12%-23%. by RAD-seq data and 2b-RAD data of three Arabidopsis stickleback iML. classification algorithm to form new The results show that iML method is better than the ML type algorithm of low false positive rate 7%-17% (sequencing read length is 30bp). In addition to the research and development of the RADtyping software, which not only the integration of the iML co dominant markers typing algorithm, and gives a statistical formula to deal with dominant markers. The Arabidopsis pseudo testcross population F1 simulation data show that when the average depth of sequencing of the parent and filial generation was 20x, two sets of two types of repeat type library marking accuracy rate up to 98%. through the actual classification results, classification consistency of codominant markers was 96%. by Sanger method show that the accuracy rate of CO dominant labeled type was 96%, the accuracy rate of dominant the mark is 97%, which fully shows the important conditions of genomic selection technology for the implementation of the application of.3,2b-RAD technology RADtyping has high accuracy in marker typing in genomic selection in the evaluation is to have a large number of base Because the group within the scope of genetic markers although the.2b-RAD technology has obvious advantages in the classification of cost, the density of markers meet aquatic organisms genome selection breeding needs is still unknown. In this study, according to the characteristics of the genome of Yesso Scallop in Shell (including genome size, heterozygous ratio, BsaXI restriction site distribution simulation of the breeding population) Yesso Scallop in Shell. The effects of three kinds of different marker density HD-SNPs (chip density), MD-SNPs (all BsaXI restriction sites), LD-SNPs (BsaXI locus with selective base) on genomic selection influence accuracy of estimated breeding value. Analysis showed that the MD-SNPs in different genetic background than HD-SNPs the accuracy rate is slightly lower (3%). The genetic force in 0.3 ~ 0.5 LD-SNPs in breeding value estimation and the accuracy rate of MD-SNPs, but the cost is only 1/10 of the markers. Then use sources In 3 families, 349 breeding populations of Yesso Scallop in Shell, shell height, whole genome selection assessment of shell length and width of three characters. The accuracy of estimates between 0.15-0.3 among families in the breeding value of family breeding were accurately estimated in 0.23 ~ 0.36. The above analysis shows that 2b-RAD technology is the type of aquatic organisms genome selection marker platform project first.

【學(xué)位授予單位】：中國海洋大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：Q78

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