分子标记辅助选择课件.ppt

1、第十七章 分子标记辅助选择第一节 分子标记辅助选择的基本原理第二节 质量性状的标记辅助选择第三节 数量性状的标记辅助选择第四节 分子标记辅助选择的挑战与发展策略 传统的育种主要是根据植株的传统的育种主要是根据植株的表现型表现型进行选进行选择，而环境条件、基因间互作、基因型与环境互作择，而环境条件、基因间互作、基因型与环境互作等多种因素都会影响表型选择效率。等多种因素都会影响表型选择效率。 育种者在长期的实践中不断探索运用育种者在长期的实践中不断探索运用遗传标记遗传标记来提高育种的选择效率与育种预见性。遗传标记包来提高育种的选择效率与育种预见性。遗传标记包括形态学标记、细胞学标记、生化标记与分子

2、标记。括形态学标记、细胞学标记、生化标记与分子标记。 以以DNA多态性为基础的分子标记，目前已在作多态性为基础的分子标记，目前已在作物遗传图谱构建、重要农艺性状基因的标记定位、物遗传图谱构建、重要农艺性状基因的标记定位、种质资源的遗传多样性分析与品种指纹图谱及纯度种质资源的遗传多样性分析与品种指纹图谱及纯度鉴定等方面得到广泛应用，尤其是鉴定等方面得到广泛应用，尤其是分子标记辅助选分子标记辅助选择择（molecular marker-assisted selection ， MAS ）育种更受到人们的重视。）育种更受到人们的重视。1、概念：通过基因定位找到与目标基因紧密连锁的分子标记后，可通过该

3、分子标记间接地对目标性状进行选择。此法称分子标记辅助选择(Molecular Assistant Selection, MAS )。 MAS 是育种中的一个诱人领域, 将给传统的育种研究带来革命性的变化。MAS 主要应用在有利基因的转移和基因的累加等方面。第一节 分子标记辅助选择的基本原理Tester M & Langridge Breeding technologies to increase crop production in a changing world. Science,2010,V 327:818-822RR(1-r)20.9025抗性供体受体RS2r(1-r)0.095SSr

4、20.0025目的基因与标记连锁（交换值为r）亲本中的标记带型F1中的标记带型F2群体中3种标记带型当r=0.05时，根据标记基因型mm选择目的基因型RR，选错的概率约为0.10共 显 性DNA标记 的 辅助 选 择原理mRMSmRMSDefinitions1) Phenotypic selection (PS) based on phenotypic value2) Marker-based selection (MBS) from markers that represent QTL or are linked to QTL3) Marker-assisted selection (MAS

5、) from a combination of phenotypic value and marker information4) Marker-assisted backcrossing (MABC)5) Marker-assisted recurrent selection (MARS)6) Genomic Selection or Genome-wide Selection (GS or GWS) Select for breeding values summed across many markers without estimation of QTL (1) selection wi

6、thout test crossing or a progeny test; (2) selection independent of environments; (3) selection without laborious fieldwork or intensive laboratory work; (4) selection at an earlier breeding stage; (5) selection for multiple genes and/or multiple traits; (6) whole genome selection. Useful if convent

7、ional screening methods are laborious, costly, or environmentally dependent Selections for disease and insect resistance can be made in the absence of the pathogen or pest Greatest potential advantage over phenotypic selection for traits with low penetrance or low heritability May reduce population

8、sizes needed for phenotypic selection May permit selection of individual plants May speed up the breeding process May be effective for early generation testing Selections at the seedling stage can be a great advantage in crops with a long generation time Reduce number of generations in a backcrossin

9、g program by selecting for recovery of the recurrent parent genome as well as genes of interest from the donor parent 2、Most suitable for MAS Pyramid genes for a single trait that could not otherwise be distinguished at the phenotypic level Accumulating multiple quantitative trait loci (QTL) for dis

10、ease resistance may provide a higher level of resistance and/or more durable resistance to changes in the pathogen population MAS may promote deployment of fewer resistance genes Genes for multiple traits of interest may also be combined in one cultivar with relative efficiency Marker technologies p

11、rovide the potential to understand the underlying causes of epistasis and GXE, which could greatly improve selection efficiency Qualitative traits and quantitative traits with high heritability are more amenable to MAS than quantitative traits with low heritability, which is generally the case for p

12、henotypic selection as well. Epistasis (or effect of genetic background) and genotype by environment interactions (GXE) can confound progress from MAS just as they do in conventional selection schemes. Efforts to improve the precision of QTL estimation through increased replication and multilocation

13、al testing will also increase the efficiency of phenotypic selection, thereby reducing the gains that may be attained through MAS. Catch 22: If phenotypes are poor indicators of genotypes, you cannot map QTL for use in MAS If phenotypic data are good, you dont need MASParadox of MAS1)How tightly it

14、is linked to genes controlling important traits.2) The relative importance of those genes in determining the phenotype.3)The consistency of linkage disequilibrium between the marker and QTL4) The frequency of the QTL (MAS will be more beneficial when the QTL is in low frequency)3. The utility of a m

15、arker depends on Requirements for wide-scale application of MAS Validation of QTL in breeding materials Multiple markers in vicinity of QTL desirable Simple, quick, inexpensive protocols for tissue sampling, DNA extraction, genotyping, and data collection Efficient data tracking, management, and int

16、egration with phenotypic data Decision support tools for breeders optimal design of selection strategies accurate selection of genotypes大多数情况下，质量性状无需借助于分子标记，但采用分子标记辅助选择可提高选择效率： 表现型测定难度大或费用太高； 表现型只能在个体发育后期才能测量，而育种实践中希望在早期选择； 除目标性状外，还需对遗传背景进行选择； 质量-数量性状的选择。第二节 质量性状的标记辅助选择1、标记辅助选择的基本方法1）前景选择（foreground

17、 selection） 对目标基因的选择单标记 可靠性：取决于标记与目标基因间连锁的紧密程度。如，标记座位M/m与目标基因座位Q/q连锁，重组率为r，则在F2根据标记基因型M/M获得目标基因型Q/Q的概率为 p=（1-r）2。 选择正确率随重组率增加而迅速降低。 如要求正确率在90%以上，则标记与目标基因间的重 组率必须5%。 Genotypes of F2 plants for Xa-21 gene as determined by a PCR marker and progeny testsPCR analysisF3 progency testAccuracy（%）GenotypeNo.

18、 of plantsGenotypeNo. of plantsRR34RR3191.2Rr3Rr28RR485.7Rr24The distance between marker pTA248 and Xa-21 gene is about 1cM (Ronld and Tanksley, 1991). The 9% inaccuracy may reflect the error in phenotyping or the variation in recombination frequency from mapping population to breeding population. 如

19、要求选到1株目标基因型的概率为P，则必须选择带 有目标基因型M/M的植株的最少数目为： n=log（1-P）2/log（1-r）2 即使重组率高达0.3，也只要选择7株具有基因型M/M的植株，就有99%的把握能保证其中有1株为目标基因型；而如果不用标记辅助选择（相当于标记与目标基因间无连锁，即r=0.5），则至少需要16株。 双边标记 可靠性将大大提高。 如标记M1/m1和M2/m2各位于目标基因座Q/q的一侧，与目标基因间的重组率分别为r1和r2，则： F1的基因型为M1QM2/m1qm2 F1 产生的标记基因型为M1M2的配子有两种类型， 包含目标等位基因的M1QM2和包含非目标等位基因的

20、双交换M1qM2，由于双交换发生频率很低，因此，在后代中通过同时跟踪M1和M2来选择目标等位基因Q，正确率必然很高。 在无干扰时，F2代通过选择标记基因型M1M2/ M1M2而获得目标基因型Q/Q的概率为：p= （1-r1）2 （1-r2）2/（1-r1）2 （1-r2）+r1r2 在两标记间的图距固定的情况下， r1 = r2时p最小（即目标基因位于两标记间的中点）。 在实际情况下，单交换间一般总是存在相互干扰的，双交换的概率更小，因此双标记选择的正确率比理论期望值更高。 Marker-assisted identification of Pi-z5 gene in a segregatin

21、g population with flanking markersPredictionProgeny testAccuracy ofRG456RG64RRRrrrpredictionRR(31)aRR(25)a25100%Rr624RR44Rr53Rr4414397.70%rr5Rr1rr25rr2424100%a: Number of plants predicted to carry the indicated genotype. The distance between RG456 and RG64 is about 14.4cM.2）背景选择（background selection

22、） 对除了目标基因之外的其他部分（遗传背景）的选择 在F2，根据两个相邻标记，可推测出它们之间的染色体区段的来源和组成。 参考完整的分子遗传图谱，检测所有标记基因型，最后绘制出图示基因型（graphic genotype）。 122222222222122223211222222222212222333312333322111A品种B品种的F2代一个个体的图示基因型，共5对染色体，白色表示来自A品种的区段，黑色表示来自B品种的区段，红色表示发生了交换的区段；竖杠表示标记所在位置。2 2 2、标记辅助选择在育种上的应用1）多基因聚合（gene pyramiding） 将分散在不同品种中的有用基因

23、聚合到一个基因组中 A gene-pyramidingscheme cumulating six target genes. （Servin等，2004）3个抗稻瘟病基因在染色体上的位置例子1：通过分子标记辅助选择方法聚合水稻抗稻瘟病基因（Zheng el al. 1995）C101LACC101A51 C101LACC101PKT F1F1F2 150株F2 150株用相邻标记进行选择 Pi-1Pi-2Pi-1Pi-410个Pi-1和Pi-2纯合的植株10个Pi-1和Pi-4纯合的植株F2 150株标记辅助选择 F13个抗病基因纯合的植株三个近等基因系例2：N. Huang et al （1

24、997）Pyramiding of bacterial blight resistance genes in rice:marker-assisted selection using RFLP and PCR DNA marker-assisted selection was used to pyramid four bacterial blight resistance genes, Xa-4, xa-5, xa-13 and Xa-21. Breeding lines with two, three and four resistance genes were developed and

25、tested for resistance to the bacterial blight pathogen (Xanthomonas oryzae pv. oryzae). The pyramid lines showed a wider spectrum and a higher level of resistance than lines with only a single gene. 近等基因系接种株数标记分析株数2）分子标记辅助回交育种（gene transfer，MAB） 将供体亲本中的有用基因转移或渗入到受体亲本的遗传背景中，从而达到改良受体亲本个别性状的目的。在每一代都需通过

26、对供体等位基因的选择使目标基因座位保持杂合，同时提高目标座位以外的其他染色体区域的受体基因组（Recipient genome content，RGC）比例。 Marker-assisted backcrossing Foreground selection：markers can be used in combination with or to replace screening for the target gene or QTL. Recombinant selection: markers can be used to select BC progeny with the targe

27、t gene and recombination events between the target locus and linked flanking markers. Background selection: Markers unlinked to the target locus can be used to select BC progeny with the greatest proportion of recurrent parent genome.(1) 前景选择（正选择） 保证从每一回交世代选出的作为下一轮回交亲本的个体都包含目的基因。 通过标记基因型的选择来控制目的座位基因

28、型，可通过TCR值来评定效果。 Target Control Rate (TCR)： 对任一回交后代个体，当其标记基因型为杂合的“供/受”时，其目标座位基因型也为“供/受”的概率。换句话说，一个个体在标记为期望基因型，而目标座位不是期望基因型的概率为（100-TCR）。Target Control Rate = TCR(%) = Pr (TD /TR) | (MD /MR) 100 = Pr (TD MD /TR MR) / Pr (MD /MR) 100 TD 和 TR为目标座位上供体和受体的等位基因； MD和MR为标记座位上供体和受体的等位基因； Pr(X/Y) 表示基因型X/Y的概率，|

29、 表示条件； It is clearly seen that control of the target by a single marker is not satisfactory in most cases. The marker must be as close as 1 cM to the target to keep the risk of losing the target below 5% over five BC generations. Even with a single marker at 1 cM, the risk of losing the target is cl

30、ose to 10% in BC10. For greater distances of a single marker, the risk becomes rapidly too high. Target controlled by single marker （Hospital，2003）单标记 当目标座位为两侧标记界定时，即使标记相距较远，效果也是很好的，如两侧标记分别相距10cM时，其控制效果与单标记距离在1cM的效果相近。很显然，这种结果的原因在于当有两侧标记时，要打破标记与目标的连锁需要进行双交换；而单标记情况下，只需要发生单交换。Target controlled by two

31、flanking markers （Hospital，2003） 双标记 回交1 回交2 回交3 回交6传统回交育种 75.0 87.7 93.3 99.0 标记辅助 85.5 98.0 100.0回交育种回交育种中轮回基因组百分比Young and Tanksley，1989，计算机模拟 (2) 背景选择（负选择） 加快遗传背景恢复成轮回亲本基因组的速度，以缩短育种年限. 指有利基因和不利基因间的连锁，使回交育种在导入有利基因的同时也带入了不利基因，常常造成性状改良后的新品种与原目标不一致. 传统的回交育种中，无法鉴别目标基因附近所发生的遗传重组，只能靠碰巧来选择消除了连锁累赘的个体。大致需

32、要100代才能基本消除连锁累赘的目的。避免或减轻连锁累赘(linkage drag)的问题。 用高密度的分子标记连锁图就有可能直接选择到在目标基因附近发生了遗传重组的个体。只要在BC1和BC2中进行标记辅助选择，即可得到含有目标基因的供体染色体片段长度不大于2cM的植株，从而只需两个回交世代就可达到基本消除连锁累赘的目的。传统回交育种标记辅助回交育种年F1BC1BC2BC3BC20BC100年0.51 1.5黑色代表供体基因组；白色代表轮回基因组 （Tanksley et al. 1989）0.51 1.5210.5 50.5Efficiency of a typical marker-ass

33、isted Efficiency of a typical marker-assisted backcross schemebackcross scheme（HospitalHospital，20032003） GenerationpopulationHomozygosity at selected markers(%)RGC%No selectionCarrier chromosomeNon-carrier chromosomeBC17038.460.67975BC210073.687.492.287.5BC31509398.89893.7BC43001001009996.9 Target-

34、marker distance on the carrier chromosome was 2 cM. Each non-carrier chromosome was controlled by three markers.Example: Markerassisted transfer of Xa21: strategyMinghui 63IRBB 21(Xa21)Minghui 63 F1Minghui 63 BC1F1Minghui 63 BC2F1BC3F1BC3F2(Xa21)Selection for recombination on either side of the Xa21

35、 locus Selection for recombination on the other side of the Xa21 locus Selection for Minghui 63 backgroundMinghui 63 (Xa21) Selection for Xa21 homozygotesChen Sheng Zhang QifaMH63(Xa21)MH63(CK) 数量性状的遗传基础 主效基因-微效基因 多个微效基因作用 数量性状的分子标记辅助选择 主效基因的辅助选择 微效基因的辅助选择 全基因组选择 第三节第三节 数量性状的标记辅助选择数量性状的标记辅助选择 1.主效基因

36、的分子标记选择主效基因的分子标记选择 性状分解; 性状数据获得; 分子标记定位; 利用近等基因系建立精细定位群体; 精细定位和基因效应验证; 分子标记的验证; 分子标记辅助育种.M. Wissuwa M. Yano N. Ae,Mapping of QTLs for phosphorus-deficiency tolerance in rice (Oryza sativa L.) Theor Appl Genet (1998) 97: 777783 A success story in MAS on rice Pi efficiencyM. Wissuwa J. Wegner N. Ae M.

37、 Yano,Substitution mapping of Pup1: a major QTL increasing phosphorus uptake of rice from a phosphorus-deficient soil.Theor Appl Genet (2002) 105:890897Graphical genotypes for the Kasalath segmenton chromosome 12 of 160 selected F3 individuals of six F2 families, as well as the meansand standard dev

38、iationsfor tiller numbers of genotypic classes within familiesSequence comparison of phosphorus uptake 1 (Pup1) loci and flanking regions in three rice varieties. A Nucmer alignment of the unmasked Kasalath sequence assembled from three BAC clones with the corresponding sequences in genotypes Nippon

39、bare (japonica reference genome) and 93-11 (indica reference genome) is illustrated.Sigrid Heuer, et al. Comparative sequence analyses of the major quantitative trait locusPhosphorus uptake1(Pup1) reveal a complexgenetic structure. Plant Biotechnology Journal(2009)7, pp. 456471Chin JH, et al., Devel

40、opment and application of gene-based markers for the major rice QTL Phosphorus uptake 1. Theor Appl Genet (2010) 120:10731086Chin JH, et al., Development and application of gene-based markers for the major rice QTL Phosphorus uptake 1. Theor Appl Genet (2010) 120:10731086Pup1 haplotype of 58 rice ge

41、notypes. The Pup1 haplotype was determined by genomic PCR using seven Pup1 gene-based markers (K41K59) and two closely flanking markers (RM28073, Ba76H14_7154).Pup1 haplotype survey. Rice accessions genotyped with the Pup1 markers were grouped according to their preferred cropping systems and variet

42、al group (indica, japonica, aus) (a). In (b) the same genotypes were grouped according to their classification as modern or traditional varieties.Chin JH, et al., Development and application of gene-based markers for the major rice QTL Phosphorus uptake 1. Theor Appl Genet (2010) 120:10731086Root el

43、ongation and reduction in shoot length under P deficiency in hydroponics culture solution. A set of contrasting Pup1 near-isogenic lines with the Pup1 locus (NILC443, NIL6-4, and NIL14-4) and sister lines without the Pup1 locus (NIL6-3, NIL14-6) were grown in hydropnics solution with 0 and 100 lM P,

44、 respectivelyChin JH, et al., Developing Rice with High Yield under Phosphorus Deficiency: Pup1 Sequence to Application. Plant Physiology, 2011, Vol. 156, pp. 12021216Pup1 gene models and positions of markers. Sequence comparisons of marker ampliconsPCR amplicons of Pup1 gene-specific markers in rep

45、resentative rice varieties.Pup1 haplotype in diverse rice genotypesPup1 core markersField evaluation and selection of IR64-Pup1 and IR74-Pup1 breeding lines 数量性状的标记辅助选择意义很大，但困难也很大，目前多局限在理论研究上，实际成功例子很少： （1）只有少数数量性状的全部QTL被精细定位出来，无法对数量性状进行全面的标记辅助选择； （2）育种过程中同时对许多目标QTL进行选择相当困难； （3）上位性效应对选择效果影响很大，常使选择达不到

46、预期目的。2.微效基因的辅助选择1）基因型选择：即对每个目标QTL利用其两侧相邻标记或单个紧密连锁的标记进行选择，其原理和方法与质量性状相似。 2）关键问题：The uncertainty of the true target location，i.e. the exact location of the target is often not known. 3）计算TCR值：假定QTL检测已经完成，QTL的期望（最可能）位置已经确定，同时已给出置信区间，而真实的目标是位于期望QTL附近的某一点，遵循一个均值为0、方差为置信区间长度的正态分布。对每一个可能的真实位置都计算TCR值，然后平均 （

47、Hospital and Charcosset，1997）。4）不完全基因型选择，即对那些已定位的QTL进行基因型选择。在选择目标QTL的同时，同样也可以利用分子标记进行背景选择，加快遗传背景恢复成轮回亲本的速度。分子标记在水稻产量遗传改良上的应用 通过分子技术在野生稻中，发现两个增产QTL，每个QTL具有比对照品种增产18%的效应（ Xiao 等，1995） 。已将其中的一个QTL转移到栽培稻中，用这种携带野生稻增产QTL水稻配组的杂交稻，2003年在浏阳作双季晚稻百亩示范，亩产700公斤。J23A/Q611 Shen et al. (2005) Used BNL1521 and BNL29

48、61 markers for QTLfs21 selection, and BNL1122 for QTLfs22 selection. The fiber strength of the selected individual plants showed the great improvement. 分子标记在棉花纤维强度遗传改良上的应用 Now it may be feasible to ask how best to use markers to make genetic gainsdiscovery of SNPs and other high density polymorphic

49、markershigh throughput genotyping has reduced costscosts for phenotyping continue to increase May be possible to select an array of markers without establishing significant associations with traits3 全基因组选择(Genome-wide selection )Genome-wide selection1.In a training population (both genotypic and phe

50、notypic data available), fit a large number of markers as random effects in a linear model to estimate all genetic effects simultaneously for a quantitative trait. The aim is to capture all of the additive genetic variance due to alleles with both large and small effects on the trait.2.In a breeding