图位克隆及重组单株

1假设现在给你的一个课题是：克隆孟德尔试验中控制紫花和白花的基因。课件下载地址链接：密码：z1ub2第一步构建群体：F2群体？BC1群体？3开发分子标记基于DNA-DNA杂交的DNA标记RFLP、VNTR基于PCR技术的DNA标记RAPD、DAF、AP-PCR、ISSR、SSR、SCAR、STS基于限制性酶切和PCR的DNA标记AFLP、CAPS基于单个核苷酸多态性的DNA标记SNP4Screeningthesegregatingpopulation（indel（插入/缺失）标记）筛选隔离种群P1P1P2PWF2ProgeniesP1P2-+-+-+-+++-+-++++-++-+-+P+--++++-++---+++-+-+++++P+-+-+-+++-+-++++-++-+-++P5Mappingrawdata记录原始数据123456789……99100M1101011001……01M2000110010……11M3011001101……01M4110111011……01M5101010001……01M6101010001……01M7101000001……01M..001001101……011:hybrid杂交0:parents亲本ProgeniesMarker6Mappingwhengenome/linkagemapavailable基因组/连锁图谱的映射选用遗传作图软件完成遗传图MAPMAKER/EXP软件7Whereisthegenecontrollingfloweringcolorinpea?控制豌豆开花颜色的基因在哪里8Linkagegroup:M17,M26,M45,M56,M5,M6,

M7,M76,M21,M91,M126

LOD=2354M17M45M5M6M7M21M56M76M126M26M91Whereisthegenecontrollingfloweringcolorinpea?9Mappingrawdata123456789……99100M1101011001……01M2000110010……11M3011001101……01M4110111011……01M5101010001……01M6101010001……01M71（紫）0（白）

1（紫）

0（白）

00001……01M..001001101……011:hybrid0:parentsProgeniesMarker1010Linkagegroup:Order1:M17,M26,M45,M56,M5,M6,

M7,M76,M21,M91,M126

LOD=2354M17M45M5M6M7M21M56M76M126M26M91M7isthephenotypic（表型）marker11111111MarkerProgeny

M5M6M761(紫)1112（白）000……33（紫）00142（白）10058（紫）11065（白）11079（紫）011

binantsWhereisthephenotypicmarker?121212MarkerProgeny

M5M6M76M71(紫)111紫

2（白）000白……33（紫）001紫42（白）100白58（紫）110紫

65（白）110白79（紫）011紫

binantsWhereisthephenotypicmarker?131313MarkerProgeny

M5M6M761(紫)1112（白）000……33（紫）00142（白）10058（紫）11065（白）11079（紫）011

binantsWhereisthephenotypicmarker?M710101011414MarkerProgeny

M5M6M7M761(紫)11112（白）0000……33（紫）001142（白）

100058（紫）111065（白）110079（紫）0111

binantsM5M7M76M6Whereisthephenotypicmarker?15151515MarkerProgeny

M5M6M761(紫)1112（白）000……33（紫）00142（白）10058（紫）11060（紫）11065（白）11079（紫）011

binantsWhereisthephenotypicmarker?M7101011111616Linkagegroup:Order1:M17,M26,M45,M56,M5,M6,

M7,M76,M21,M91,M126

LOD=2354M17M45M5M6M7M21M56M76M126M26M91Confirmtheorderofmarkersinalinkagegroup确定连锁群标记的顺序Order2:M17,M26,M45,M56,M6,M5,M7,M76,M21,M91,M126

LOD=23541717TheorderofM5,M6,M7,M76MarkerProgeny

M5M6M7M761(紫)11112（白）0000……33（紫）001142（白）

100058（紫）111065（白）110079（紫）1011

binantsM5M7M76M61818TheorderofM5,M6,M7,M76MarkerProgeny

M6M5M7M761111120000……330011420

100581110651100790111

binantsM6M7M76M519Whereisthegenecontrollingfloweringcolorinpea?mm1m2MM1M2Mm1M2MM1m2mM1m2P1P2Targetgene/trait20Co-segregatingMarkermayBefarawayfromPurpleFlowerGene(dependsonpopulationsize)mm1m2MM1M2Mm1M2MM1m2mM1m2Analyzebothco-segregatingandflankingmarkers!21Inolddays:map-basedcloningMREMCloningMScreeningMSequencingATG……TGAComplementationtestChangeofphenotype22PhysicalMapMM1M2MMMM2M123PhysicalMapMM1M2MM2M1Isitpossiblethatyourtargetgeneisinthesamecloneasamarkerthatdoesnotco-segregatewithit?24Whydoweneedalargesegregatingpopulation?TonarrowdownyourtargetgeneMM1M2M3M4M5M625FineMappingwhengenome/linkagemapavailableTraitM1M2MM1M2M3M4M5M6Increasepopulationsize,morebinant26分组混合分析法（Bulkedsegregantanalysis,BSA）+DNA/RNA-seq(二代测序)基于性状表现型的BSA法根据目标性状的表现型对分离群体进行分组混合。将个体或株系依据相对表型差异分成两组，形成各自的DNA混合池。这两个DNA池之间除了在目标基因座所在的染色体区域存在差异外，来自基因组其他部分的DNA组成完全相同，都是该作图群体基因库的一个随机样本。27BSA+RNA-SEQorDNA-SEQCompleteSequencingof100BC1Individuals……P1P2P1P2-+--+++--+--+++-+++--+……---++-++-+---+----PoolingExtremePenotypes-+

+++++

+

++

+++

++++

++

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-----……P1P2P1P2Phenotype29基于标记基因型的BSA法根据目标基因两侧的分子标记的基因型对分离群体进行分组混合适合于目标基因已定位在分子连锁图上，需要进一步寻找与目标基因更为紧密的分子标记的情况30ABA1B1P1A2B2P2分离群体（如F2）A1B1/A1B1A2B2/A2B2依据基因型A1B1/A1B1和A2B2/A2B2构建DNA混合池，它们只是在目标区段上存在差异，目标区段以外的遗传背景相同寻找两DNA混合池之间的多态型，有可能在目标区段上找到与目标基因紧密连锁的分子标记。31TTT32PrincipleofBSA-RNA-seq

GTTATTTA

GC

TA

TTTATTTAWhite

pool

Loc1Loc2Loc3Loc4Position