本文選題：桃 + SNP標(biāo)記��； 參考：《中國(guó)農(nóng)業(yè)科學(xué)》2017年23期

【摘要】：【目的】挖掘與桃抗蚜性狀緊密連鎖的SNP位點(diǎn)及候選基因,為桃抗性分子標(biāo)記輔助選擇育種奠定基礎(chǔ),并為進(jìn)一步揭示桃抗蚜性狀的遺傳基礎(chǔ)和分子機(jī)制提供依據(jù)�！痉椒ā恳詠�(lái)源于‘粉壽星’的抗蚜桃‘01-77-3’為母本,栽培品種‘中油桃13號(hào)’為父本,雜交獲得F1代分離群體進(jìn)行基因定位。以‘96-5-1’(抗蚜)為母本,‘10-7’(感蚜)為父本雜交獲得F1分離群體作為驗(yàn)證定位位點(diǎn)準(zhǔn)確性的材料。參考桃基因組序列(Prunus persicaGenome v2.0.a1)開(kāi)發(fā)基于Sanger測(cè)序的SNP標(biāo)記,在親本(‘01-77-3’‘中油桃13號(hào)’)及各4個(gè)子代中PCR擴(kuò)增后進(jìn)行Sanger測(cè)序,獲得候選SNP后,擴(kuò)大群體驗(yàn)證,實(shí)現(xiàn)對(duì)抗性基因的初步定位。對(duì)親本(‘01-77-3’‘中油桃13號(hào)’‘96-5-1’‘10-7’)進(jìn)行覆蓋度約為70×的全基因組深度測(cè)序,并基于重測(cè)序數(shù)據(jù),在初定位區(qū)間內(nèi)開(kāi)發(fā)基于Sanger測(cè)序與HRM分析的SNP標(biāo)記,篩選多態(tài)性標(biāo)記,對(duì)‘01-77-3’ב中油桃13號(hào)’雜交后代141株實(shí)生苗進(jìn)行基因分型,以獲得與桃抗蚜型基因緊密連鎖的分子標(biāo)記。通過(guò)雙親(‘96-5-1’‘10-7’)表型與基因型一致,在精細(xì)定位區(qū)間內(nèi)開(kāi)發(fā)與桃抗蚜性狀緊密連鎖的In Del位點(diǎn),以驗(yàn)證In Del標(biāo)記與抗蚜表型是否連鎖。最后,參考桃基因組分析定位區(qū)間的候選基因�！窘Y(jié)果】通過(guò)人工接種觀察蚜蟲(chóng)對(duì)桃F1后代單株新梢危害表明,抗蚜與感蚜比例接近1㑳1(P值為0.556;χ~2為0.348),符合孟德?tīng)栠z傳規(guī)律。基于Sanger測(cè)序結(jié)果,初步將抗蚜基因定位在桃基因組Pp01_38011783與Pp01_47231340之間,物理距離約為9.22 Mb。親本全基因組深度測(cè)序分別產(chǎn)生Clean data 17.109 Gb,平均覆蓋度為75.19×,在Pp01初定位區(qū)間內(nèi)開(kāi)發(fā)基于HRM與Sanger測(cè)序的SNP標(biāo)記共29對(duì),篩選后得到11對(duì)緊密連鎖的標(biāo)記�；蚍中徒Y(jié)果表明,與桃抗蚜基因緊密連鎖的標(biāo)記位于桃基因組Pp01的45.66 Mb和46.12 Mb處,Pp01_45.66處等位基因?yàn)門(mén)和C,Pp01_46.12處等位基因?yàn)镚和C,遺傳距離分別為1.4 c M、2.1 c M;與抗蚜基因完全連鎖的標(biāo)記SNP_Pp01_45712702,位于桃基因組Pp01_45.71處,等位基因?yàn)镚和T。基于親本(‘96-5-1’‘10-7’)重測(cè)序數(shù)據(jù),在精細(xì)定位區(qū)間內(nèi)開(kāi)發(fā)緊密連鎖的In Del標(biāo)記KYYZ_Pp01_45799758,位于Pp01_45.79處。對(duì)驗(yàn)證群體92個(gè)單株進(jìn)行PCR擴(kuò)增,經(jīng)聚丙烯酰胺凝膠電泳檢測(cè)表明,僅1個(gè)單株基因型與表型不符,分子鑒定準(zhǔn)確率為98.91%�！窘Y(jié)論】利用親本深度測(cè)序與SNP標(biāo)記技術(shù)相結(jié)合的方法,精細(xì)定位了控制桃抗蚜性狀的基因,將抗蚜基因縮小到物理區(qū)間460 Kb內(nèi),共包含56個(gè)轉(zhuǎn)錄本,包括52個(gè)候選基因。
[Abstract]:[objective] to explore the SNP loci and candidate genes closely linked to aphid resistance traits in peach, so as to lay a foundation for the breeding of peach resistance molecular marker assisted selection. In order to further reveal the genetic basis and molecular mechanism of aphid resistance in peach, [methods] the aphid resistant peach was used as the female parent, and the cultivar 'Zhong Nectarine 13' was used as the male parent. F _ 1 segregation population was obtained by hybridization for gene mapping. F _ 1 segregated population was used as the material to verify the accuracy of locus loci by crossing 10-7 (sensitive aphids) with 96-5-1 (aphids resistant) as the female parent. Referring to the Prunus persicaGenome v2.0.a1), the SNP marker based on Sanger sequencing was developed, and the Sanger sequence was amplified by PCR amplification in the parent Prunus 01-77-3 'Nectarine 13' and four progenies of Prunus persicaGenome v2.0.a1). After obtaining candidate SNP, the population validation was expanded. To achieve the preliminary location of the resistance gene. The complete genome depth sequencing was carried out with coverage of about 70 脳, and SNP markers based on Sanger sequencing and HRM analysis were developed in the initial location interval. In order to obtain the molecular markers closely linked to the aphid-resistant gene, 141 seedlings were genotyped by screening the polymorphic markers. The in Del loci closely linked to aphid resistance traits were developed within the fine mapping interval to verify whether in Del markers were linked to aphids resistance phenotypes. Finally, the candidate genes were analyzed with reference to the loci of peach genome. [results] the harm of aphids to the single shoot of F1 progenies was observed by artificial inoculation. The results showed that the ratio of aphids resistance to aphids to aphid resistance to aphids was close to 1 ~ (-1) P = 0.556, 蠂 ~ (2) = 0.348, which was in accordance with Mendelian inheritance rule. Based on the results of Sanger sequencing, the aphid resistance gene was preliminarily located between the genomic Pp01_38011783 and Pp01_47231340 of peach, and the physical distance was about 9.22 Mb. Clean data 17.109 GB was generated by deep sequencing of parent genome, with an average coverage of 75.19 脳. A total of 29 pairs of SNP markers based on HRM and Sanger sequencing were developed in the initial Pp01 mapping region. Eleven pairs of closely linked markers were obtained after screening. The genotyping results showed that, The marker closely linked to aphid resistance gene is located at 45.66Mb and 46.12Mb in the peach genomic Pp01. The alleles T and C Pp011are G and C respectively, and the genetic distance is 1.4c MN 2.1c; the marker completely linked to aphid resistance gene is Pp01Mb. The SNPs Pp01s, located at the peach genome Pp01_45.71, The alleles were G and T. Based on the resequenced data of the parent, the closely linked in Del marker KYYZPp0145799758 was developed in the fine mapping region, which is located at Pp01_45.79. PCR amplification was carried out on 92 individual plants in a validated population. The results of polyacrylamide gel electrophoresis showed that only one individual genotype did not match the phenotype. The accuracy of molecular identification was 98.91. [conclusion] by combining parental deep sequencing with SNP marker technique, the gene controlling aphid resistance in peach was mapped carefully, and the gene was reduced to a physical interval of 460Kb, containing 56 transcripts. There are 52 candidate genes.
【作者單位】： 中國(guó)農(nóng)業(yè)科學(xué)院鄭州果樹(shù)研究所/國(guó)家桃葡萄改良中心/農(nóng)業(yè)部果樹(shù)育種技術(shù)重點(diǎn)實(shí)驗(yàn)室;
【基金】：國(guó)家自然科學(xué)基金(31470679) 中國(guó)農(nóng)業(yè)科學(xué)院科技創(chuàng)新工程(CAAS-ASTIP-2016-ZFRI) 河南省科技計(jì)劃項(xiàng)目(152102110110)
【分類(lèi)號(hào)】：S436.621.21
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本文編號(hào)：1785771