Euphytica(2005)142:169–196
DOI:10.1007/s10681-005-1681-5C Springer2005 An introduction to markers,quantitative trait loci(QTL)mapping and marker-assisted selection for crop improvement:The basic concepts
B.C.Y.Collard1,4,?,M.Z.Z.Jahufer2,J.B.Brouwer3&E.C.K.Pang1
1Department of Biotechnology and Environmental Biology,RMIT University,P.O.Box71,Bundoora,Victoria3083, Australia;2AgResearch Ltd.,Grasslands Research Centre,Tennent Drive,Private Bag11008,Palmerston North, New Zealand;3P.O.Box910,Horsham,Victoria,Australia3402;4Present address:Plant Breeding,Genetics and Biotechnology Division,International Rice Research Institute(IRRI),DAPO Box7777,Metro Manila,Philippines; (?author for correspondence:e-mail:bcycollard@https://www.doczj.com/doc/238903763.html,)
Received11July2004;accepted2February2005
Key words:bulked-segregant analysis,DNA markers,linkage map,marker-assisted selection,quantitative trait loci (QTLs),QTL analysis,QTL mapping
Summary
Recognizing the enormous potential of DNA markers in plant breeding,many agricultural research centers and plant breeding institutes have adopted the capacity for marker development and marker-assisted selection(MAS). However,due to rapid developments in marker technology,statistical methodology for identifying quantitative trait loci(QTLs)and the jargon used by molecular biologists,the utility of DNA markers in plant breeding may not be clearly understood by non-molecular biologists.This review provides an introduction to DNA markers and the concept of polymorphism,linkage analysis and map construction,the principles of QTL analysis and how markers may be applied in breeding programs using MAS.This review has been speci?cally written for readers who have only a basic knowledge of molecular biology and/or plant genetics.Its format is therefore ideal for conventional plant breeders,physiologists,pathologists,other plant scientists and students.
Abbreviations:AFLP:ampli?ed fragment length polymorphism;BC:backcross;BSA:bulked-segregant analysis; CIM:composite interval mapping;cM:centiMorgan;DH:doubled haploid;EST:expressed sequence tag;SIM:sim-ple interval mapping;LOD:logarithm of odds;LRS:likelihood ratio statistic;MAS:marker-assisted selection;NIL: near isogenic lines;PCR:polymerase chain reaction;QTL:quantitative trait loci;RAPD:random ampli?ed poly-morphic DNA;RI:recombinant inbred;RFLP:restriction fragment length polymorphism;SSR:simple sequence repeats(microsatellites);SCAR:sequence characterized ampli?ed region;SNP:single nucleotide polymorphism; STS:sequence tagged site
Introduction
Many agriculturally important traits such as yield,qual-ity and some forms of disease resistance are controlled by many genes and are known as quantitative traits(also ‘polygenic,’‘multifactorial’or‘complex’traits).The regions within genomes that contain genes associated with a particular quantitative trait are known as quan-titative trait loci(QTLs).The identi?cation of QTLs based only on conventional phenotypic evaluation is not possible.A major breakthrough in the characteri-zation of quantitative traits that created opportunities to select for QTLs was initiated by the development of DNA(or molecular)markers in the1980s.
One of the main uses of DNA markers in agricul-tural research has been in the construction of linkage maps for diverse crop species.Linkage maps have been utilised for identifying chromosomal regions that contain genes controlling simple traits(controlled by a single gene)and quantitative traits using QTL
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analysis(reviewed by Mohan et al.,1997).The process of constructing linkage maps and conducting QTL analysis–to identify genomic regions associated with traits–is known as QTL mapping(also‘genetic,’‘gene’or‘genome’mapping)(McCouch&Doerge,1995; Mohan et al.,1997;Paterson,1996a,b).DNA markers that are tightly linked to agronomically important genes (called gene‘tagging’)may be used as molecular tools for marker-assisted selection(MAS)in plant breeding (Ribaut&Hoisington,1998).MAS involves using the presence/absence of a marker as a substitute for or to as-sist in phenotypic selection,in a way which may make it more ef?cient,effective,reliable and cost-effective compared to the more conventional plant breeding methodology.The use of DNA markers in plant(and animal)breeding has opened a new realm in agriculture called‘molecular breeding’(Rafalski&Tingey,1993).
DNA markers are widely accepted as potentially valuable tools for crop improvement in rice(Mackill et al.,1999;McCouch&Doerge,1995),wheat(Eagles et al.,2001;Koebner&Summers,2003;Van Sanford et al.,2001),maize(Stuber et al.,1999;Tuberosa et al., 2003),barley(Thomas,2003;Williams,2003),tuber crops(Barone,2004;Fregene et al.,2001;Gebhardt &Valkonen,2001),pulses(Kelly et al.,2003; Muehlbauer et al.,1994;Svetleva et al.,2003;Weeden et al.,1994),oilseeds(Snowdon&Friedt,2004), horticultural crop species(Baird et al.,1996,1997; Mehlenbacher,1995)and pasture species(Jahufer et al.,2002).Some studies suggest that DNA markers will play a vital role in enhancing global food produc-tion by improving the ef?ciency of conventional plant breeding programs(Kasha,1999;Ortiz,1998).Al-though there has been some concern that the outcomes of DNA marker technology as proposed by initial stud-ies may not be as effective as?rst thought,many plant breeding institutions have adopted the capacity for marker development and/or MAS(Eagles et al.,2001; Kelly&Miklas,1998;Lee,1995).An understanding of the basic concepts and methodology of DNA marker development and MAS,including some of the termi-nology used by molecular biologists,will enable plant breeders and researchers working in other relevant disciplines to work together towards a common goal –increasing the ef?ciency of global food production.
A number of excellent reviews have been written about the construction of linkage maps,QTL analy-sis and the application of markers in marker-assisted selection(for example:Haley&Andersson,1997; Jones et al.,1997;Paterson et al.,1991a;Paterson, 1996a,b;Staub et al.,1996;Tanksley,1993;Young,1994).However,the authors of these reviews assumed that the reader had an advanced level of knowledge in molecular biology and plant genetics,with the possi-ble exceptions of the reviews by Paterson(1996a,b) and Jones et al.(1997).Our review has been speci?-cally written for readers with only a basic knowledge of molecular biology and/or plant genetics.It will be a useful reference for conventional plant breeders,physi-ologists,pathologists and other plant scientists,as well as students who are not necessarily engaged in applied molecular biology research but need an understanding of the exciting opportunities offered by this new tech-nology.This review consists of?ve sections:genetic markers,construction of linkage maps,QTL analysis, towards marker-assisted selection and marker-assisted selection.
Section I:Genetic markers
What are genetic markers?
Genetic markers represent genetic differences between individual organisms or species.Generally,they do not represent the target genes themselves but act as‘signs’or‘?ags’.Genetic markers that are located in close proximity to genes(i.e.tightly linked)may be referred to as gene‘tags’.Such markers themselves do not affect the phenotype of the trait of interest because they are located only near or‘linked’to genes controlling the trait.All genetic markers occupy speci?c genomic po-sitions within chromosomes(like genes)called‘loci’(singular‘locus’).
There are three major types of genetic markers: (1)morphological(also‘classical’or‘visible’)mark-ers which themselves are phenotypic traits or charac-ters;(2)biochemical markers,which include allelic variants of enzymes called isozymes;and(3)DNA (or molecular)markers,which reveal sites of varia-tion in DNA(Jones et al.,1997;Winter&Kahl,1995). Morphological markers are usually visually character-ized phenotypic characters such as?ower colour,seed shape,growth habits or pigmentation.Isozyme mark-ers are differences in enzymes that are detected by electrophoresis and speci?c staining.The major dis-advantages of morphological and biochemical mark-ers are that they may be limited in number and are in?uenced by environmental factors or the develop-mental stage of the plant(Winter&Kahl,1995). However,despite these limitations,morphological and biochemical markers have been extremely useful to
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plant breeders(Eagles et al.,2001;Weeden et al., 1994).
DNA markers are the most widely used type of marker predominantly due to their abundance.They arise from different classes of DNA mutations such as substitution mutations(point mutations),rearrange-ments(insertions or deletions)or errors in replication of tandemly repeated DNA(Paterson,1996a).These markers are selectively neutral because they are usu-ally located in non-coding regions of DNA.Unlike morphological and biochemical markers,DNA mark-ers are practically unlimited in number and are not af-fected by environmental factors and/or the develop-mental stage of the plant(Winter&Kahl,1995).Apart from the use of DNA markers in the construction of linkage maps,they have numerous applications in plant breeding such as assessing the level of genetic diver-sity within germplasm and cultivar identity(Baird et al., 1997;Henry,1997;Jahufer et al.,2003;Weising et al., 1995;Winter&Kahl,1995).
DNA markers may be broadly divided into three classes based on the method of their detection:(1) hybridization-based;(2)polymerase chain reaction (PCR)-based and(3)DNA sequence-based(Gupta et al.,1999;Jones et al.,1997;Joshi et al.,1999;Win-ter&Kahl,1995).Essentially,DNA markers may re-veal genetic differences that can be visualised by us-ing a technique called gel electrophoresis and stain-ing with chemicals(ethidium bromide or silver)or detection with radioactive or colourimetric probes. DNA markers are particularly useful if they reveal differences between individuals of the same or dif-ferent species.These markers are called polymorphic markers,whereas markers that do not discriminate between genotypes are called monomorphic markers (Figure1).Polymorphic markers may also be de-scribed as codominant or dominant.This description is based on whether markers can discriminate between homozygotes and heterozygotes(Figure2).Codomi-nant markers indicate differences in size whereas dom-inant markers are either present or absent.Strictly speaking,the different forms of a DNA marker(e.g. different sized bands on gels)are called marker‘al-leles’.Codominant markers may have many differ-ent alleles whereas a dominant marker only has two alleles.
It is beyond the scope of this review to discuss the technical method of how DNA markers are gen-erated.However the advantages and disadvantages of the most commonly used markers are presented in Table
1.Figure1.Diagram representing hypothetical DNA markers between genotypes A,B,C and D.Polymorphic markers are indicated by ar-rows.Markers that do not discriminate between genotypes are called monomorphic markers.(a)Example of SSR markers.The polymor-phic marker reveals size differences for the marker alleles of the four genotypes,and represent a single genetic locus.(b)Examples of markers generated by the RAPD technique.Note that these mark-ers are either present or absent.Often,the sizes of these markers in nucleotide base pairs(bp)are also provided;these sizes are estimated from a molecular weight(MW)DNA ladder.For both polymorphic markers,there are only two different marker alleles.
Section II:Construction of linkage maps
What are linkage maps?
A linkage map may be thought of as a‘road map’of the chromosomes derived from two different parents(Pa-terson,1996a).Linkage maps indicate the position and relative genetic distances between markers along chro-mosomes,which is analogous to signs or landmarks along a highway.The most important use for linkage maps is to identify chromosomal locations contain-ing genes and QTLs associated with traits of interest; such maps may then be referred to as‘QTL’(or‘ge-netic’)maps.‘QTL mapping’is based on the principle that genes and markers segregate via chromosome re-combination(called crossing-over)during meiosis(i.e. sexual reproduction),thus allowing their analysis in the progeny(Paterson,1996a).Genes or markers that are close together or tightly-linked will be transmitted
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Table 1.Advantages and disadvantages of most commonly-used DNA markers for QTL analysis Molecular Codominant (C)marker
or Dominant (D)Advantages Disadvantages
References
Restriction
C
?Robust ?Time-consuming,laborious Beckmann &Soller (1986),fragment length ?Reliable
and expensive
Kochert (1994),Tanksley polymorphism ?Transferable across ?Large amounts of DNA required et al.(1989)
(RFLP)populations
?Limited polymorphism (especially in related lines)Random D
?Quick and simple ?Problems with reproducibility Penner (1996),Welsh &li?ed ?Inexpensive
?Generally not transferable
McClelland (1990),polymorphic ?Multiple loci from a single Williams et al.(1990)
DNA (RAPD)
primer possible ?Small amounts of DNA required Simple sequence C
?Technically simple ?Large amounts of time and McCouch et al.(1997),repeats (SSRs)??Robust and reliable labour required for production Powell et al.(1996),
or ‘microsatellites’
?Transferable between of primers Taramino &Tingey (1996)
populations ?Usually require polyacrylamide
electrophoresis Ampli?ed
D
?Multiple loci ?Large amounts of DNA required V os et al.(1995)
fragment Length
?High levels of
?Complicated methodology
Polymorphism (AFLP)
polymorphism generated
?SSRs
are also known as sequence tagged microsatellite site (STMS)markers (Davierwala et al.,2000;Huettel et al.,1999;Mohapatra et al.,2003;Winter et al.,
1999).
Figure https://www.doczj.com/doc/238903763.html,parison between (a)codominant and (b)dominant markers.Codominant markers can clearly discriminate between homozygotes and heterozygotes whereas dominant markers do not.Genotypes at two marker loci (A and B)are indicated below the gel diagrams.
together from parent to progeny more frequently than genes or markers that are located further apart (Fig-ure 3).In a segregating population,there is a mixture of parental and recombinant genotypes.The frequency of recombinant genotypes can be used to calculate re-combination fractions,which may by used to infer the genetic distance between markers.By analysing the segregation of markers,the relative order and dis-tances between markers can be determined–the lower the frequency of recombination between two markers,
the closer they are situated on a chromosome (con-versely,the higher the frequency of recombination be-tween two markers,the further away they are situated on a chromosome).Markers that have a recombination frequency of 50%are described as ‘unlinked’and as-sumed to be located far apart on the same chromosome or on different chromosomes.For a more detailed ex-planation of genetic linkage,the reader is encouraged to consult basic textbooks on genetics or quantitative genetics (for example,Hartl &Jones,2001;Kearsey &Pooni,1996).Mapping functions are used to convert recombination fractions into map units called centi-Morgans (cM)(discussed later).Linkage maps are con-structed from the analysis of many segregating mark-ers.The three main steps of linkage map construction are:(1)production of a mapping population;(2)iden-ti?cation of polymorphism and (3)linkage analysis of markers.
Mapping populations
The construction of a linkage map requires a segregat-ing plant population (i.e.a population derived from sex-ual reproduction).The parents selected for the mapping population will differ for one or more traits of interest.Population sizes used in preliminary genetic mapping studies generally range from 50to 250individuals
173 Figure3.Diagram indicating cross-over or recombination events between homologous chromosomes that occur during meiosis.Gametes that are produced after meiosis are either parental(P)or recombinant(R).The smaller the distance between two markers,the smaller the chance of recombination occurring between the two markers.Therefore,recombination between markers G and H should occur more frequently than recombination between markers E and F.This can be observed in a segregating mapping population.By analysing the number of recombinants in a population,it could be determined that markers E and F are closer together compared to G and H.
(Mohan et al.,1997),however larger populations are
required for high-resolution mapping.If the map will
be used for QTL studies(which is usually the case),
then an important point to note is that the mapping pop-
ulation must be phenotypically evaluated(i.e.trait data
must be collected)before subsequent QTL mapping.
Generally in self-pollinating species,mapping pop-
ulations originate from parents that are both highly ho-
mozygous(inbred).In cross pollinating species,the sit-
uation is more complicated since most of these species
do not tolerate inbreeding.Many cross pollinating plant
species are also polyploid(contain several sets of chro-
mosome pairs).Mapping populations used for mapping
cross pollinating species may be derived from a cross
between a heterozygous parent and a haploid or ho-
mozygous parent(Wu et al.,1992).For example,in
both the cross pollinating species white clover(Tri-
folium repens L.)and ryegrass(Lolium perenne L.),F1
generation mapping populations were successfully de-
veloped by pair crossing heterozygous parental plants
that were distinctly different for important traits asso-
ciated with plant persistence and seed yield(Barrett
et al.,2004;Forster et al.,2000).
Several different populations may be utilized
for mapping within a given plant species,with each
population type possessing advantages and disadvan-
tages(McCouch&Doerge,1995;Paterson,1996a)
(Figure4).F2populations,derived from F1hybrids,
and backcross(BC)populations,derived by crossing
the F1hybrid to one of the parents,are the simplest
types of mapping populations developed for self
pollinating species.Their main advantages are that
they are easy to construct and require only a short
time to produce.Inbreeding from individual F2plants
allows the construction of recombinant inbred(RI)
lines,which consist of a series of homozygous lines,
each containing a unique combination of chromosomal
segments from the original parents.The length of
time needed for producing RI populations is the major
disadvantage,because usually six to eight generations
are required.Doubled haploid(DH)populations may
be produced by regenerating plants by the induction
of chromosome doubling from pollen grains,however,
the production of DH populations is only possible in
species that are amenable to tissue culture(e.g.cereal
species such as rice,barley and wheat).The major
advantages of RI and DH populations are that they
produce homozygous or‘true-breeding’lines that can
be multiplied and reproduced without genetic change
occurring.This allows for the conduct of replicated
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Figure4.Diagram of main types of mapping populations for self-pollinating species.
trials across different locations and years.Thus both RI and DH populations represent‘eternal’resources for QTL mapping.Furthermore,seed from individual RI or DH lines may be transferred between different laboratories for further linkage analysis and the addition of markers to existing maps,ensuring that all collaborators examine identical material(Paterson, 1996a;Young,1994).
Identi?cation of polymorphism
The second step in the construction of a linkage map is to identify DNA markers that reveal differences be-tween parents(i.e.polymorphic markers).It is critical that suf?cient polymorphism exists between parents in order to construct a linkage map(Young,1994).In gen-eral,cross pollinating species possess higher levels of DNA polymorphism compared to inbreeding species; mapping in inbreeding species generally requires the selection of parents that are distantly related.In many cases,parents that provide adequate polymorphism are selected on the basis of the level of genetic diversity between parents(Anderson et al.,1993;Collard et al., 2003;Joshi&Nguyen,1993;Yu&Nguyen,1994).The choice of DNA markers used for mapping may de-pend on the availability of characterised markers or the appropriateness of particular markers for a particular species.
Once polymorphic markers have been identi?ed, they must be screened across the entire mapping pop-ulation,including the parents(and F1hybrid,if pos-sible).This is known as marker‘genotyping’of the population.Therefore,DNA must be extracted from each individual of the mapping population when DNA markers are used.Examples of DNA markers screened across different populations are shown in Figure5.The expected segregation ratios for codominant and domi-nant markers are presented in Table2.Signi?cant de-viations from expected ratios can be analysed using Table2.Expected segregation ratios for markers in different popu-lation types
Population type Codominant markers Dominant markers
F21:2:1(AA:Aa:aa)3:1(B:bb) Backcross1:1(Cc:cc)1:1(Dd:dd) Recombinant inbred or1:1(EE:ee)1:1(FF:ff)
doubled haploid
175 Figure5.Hypothetical gel photos representing segregating codominant markers(left-hand side)and dominant markers(right-hand side)for typical mapping populations.Codominant markers indicate the complete genotype of a plant.Note that dominant markers cannot discriminate between heterozygotes and one homozygote genotype in F2populations.The segregation ratios of markers can be easily understood by using Punnett squares to derive population genotypes.
chi-square tests.Generally,markers will segregate in a Mendelian fashion although distorted segregation ra-tios may be encountered(Sayed et al.,2002;Xu et al., 1997).
In some polyploid species such as sugarcane, identifying polymorphic markers is more complicated (Ripol et al.,1999).The mapping of diploid relatives of polyploid species can be of great bene?t in developing maps for polyploid species.However,diploid relatives do not exist for all polyploid species(Ripol et al.,1999; Wu et al.,1992).A general method for the mapping of polyploid species is based on the use of single-dose restriction fragments(Wu et al.,1992).
Linkage analysis of markers
The?nal step of the construction of a linkage map in-volves coding data for each DNA marker on each indi-vidual of a population and conducting linkage anal-ysis using computer programs(Figure6).Missing marker data can also be accepted by mapping programs.Although linkage analysis can be performed manu-ally for a few markers,it is not feasible to manually analyze and determine linkages between large numbers of markers that are used to construct maps;computer programs are required for this purpose.Linkage be-tween markers is usually calculated using odds ratios (i.e.the ratio of linkage versus no linkage).This ratio is more conveniently expressed as the logarithm of the ratio,and is called a logarithm of odds(LOD)value or LOD score(Risch,1992).LOD values of>3are typi-cally used to construct linkage maps.A LOD value of 3between two markers indicates that linkage is1000 times more likely(i.e.1000:1)than no linkage(null hypothesis).LOD values may be lowered in order to detect a greater level of linkage or to place additional markers within maps constructed at higher LOD https://www.doczj.com/doc/238903763.html,monly used software programs include Map-maker/EXP(Lander et al.,1987;Lincoln et al.,1993a) and MapManager QTX(Manly et al.,2001),which are freely available from the internet.JoinMap is an-other commonly-used program for constructing link-age maps(Stam,1993).
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Figure6.Construction of a linkage map based on a small recombinant inbred population(20individuals).The?rst parent(P1)is scored as an ‘A’whereas the second parent(P2)is scored as a‘B’.Coding of marker data varies depending on the type of population used.This linkage map was constructed using Map Manager QTX(Manly et al.,2001)using the Haldane mapping function.
A typical output of a linkage map is shown in Figure7.Linked markers are grouped together into‘linkage groups,’which represent chromosomal segments or entire chromosomes.Referring to the road map analogy,linkage groups represent roads and markers represent signs or landmarks.A dif?culty associated with obtaining an equal number of linkage groups and chromosomes is that the polymorphic markers detected are not necessarily evenly distributed over the chromosome,but clustered in some regions and absent in others(Paterson,1996a).In addition to the non-random distribution of markers,the frequency of recombination is not equal along chromosomes (Hartl&Jones,2001;Young,1994).
The accuracy of measuring the genetic distance and determining marker order is directly related to the number of individuals studied in the mapping popu-lation.Ideally,mapping populations should consist of
177 Figure7.Hypothetical‘framework’linkage map of?ve chromosomes(represented by linkage groups)and26markers.Ideally,a framework map should consist of evenly spaced markers for subsequent QTL analysis.If possible,the framework map should also consist of anchor markers that are present in several maps,so that they can be used to compare regions between maps.
a minimum of50individuals for constructing linkage maps(Young,1994).
Genetic distance and mapping functions
The importance of the distance between genes and markers has been discussed earlier.The greater the distance between markers,the greater the chance of re-combination occurring during meiosis.Distance along a linkage map is measured in terms of the frequency of recombination between genetic markers(Paterson, 1996a).Mapping functions are required to convert recombination fractions into centiMorgans(cM)be-cause recombination frequency and the frequency of crossing-over are not linearly related(Hartl&Jones, 2001;Kearsey&Pooni,1996).When map distances are small(<10cM),the map distance equals the recombi-nation frequency.However,this relationship does not apply for map distances that are greater than10cM (Hartl&Jones,2001).Two commonly used mapping functions are the Kosambi mapping function,which assumes that recombination events in?uence the oc-currence of adjacent recombination events,and the Haldane mapping function,which assumes no interfer-ence between crossover events(Hartl&Jones,2001; Kearsey&Pooni,1996).
It should be noted that distance on a linkage map is not directly related to the physical distance of DNA be-tween genetic markers,but depends on the genome size of the plant species(Paterson,1996a).Furthermore, the relationship between genetic and physical dis-tance varies along a chromosome(Kunzel et al.,2000; Tanksley et al.,1992;Young,1994).For example,there are recombination‘hot spots’and‘cold spots,’which are chromosomal regions in which recombination oc-curs more frequently or less frequently,respectively (Faris et al.,2000;Ma et al.,2001;Yao et al.,2002). Section III:QTL analysis
Principle of QTL analysis
Identifying a gene or QTL within a plant genome is like?nding the proverbial needle in a haystack.How-ever,QTL analysis can be used to divide the haystack
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in manageable piles and systematically search them.In simple terms,QTL analysis is based on the principle of detecting an association between phenotype and the genotype of markers.Markers are used to parti-tion the mapping population into different genotypic groups based on the presence or absence of a particu-lar marker locus and to determine whether signi?cant differences exist between groups with respect to the trait being measured (Tanksley,1993;Young,1996).A signi?cant difference between phenotypic means of the groups (either 2or 3),depending on the marker sys-tem and type of population,indicates that the marker locus being used to partition the mapping population is linked to a QTL controlling the trait (Figure 8).
A logical question that may be asked at this point is ‘why does a signi?cant P value obtained for differences between mean trait values indicate linkage between marker and QTL?’The answer is due to recombination.The closer a marker is from a QTL,the lower the chance of recombination occurring between marker and QTL.Therefore,the QTL and marker will be usually be inher-ited together in the progeny,and the mean of the group with the tightly-linked marker will be signi?cantly
dif-Figure 8.Principle of QTL mapping (adapted from Young,1996).Markers that are linked to a gene or QTL controlling a particular trait (e.g.plant height)will indicate signi?cant differences when the mapping population is partitioned according to the genotype of the marker.Based on the results in this diagram,Marker E is linked to a QTL because there is a signi?cant difference between means.Marker H is unlinked to a QTL because there is no signi?cant difference between means.The closer the marker is to the QTL of interest,the lower the chance for recombination between marker and QTL.
ferent (P <0.05)to the mean of the group without the marker (Figure 9).When a marker is loosely-linked or unlinked to a QTL,there is independent segregation of the marker and QTL.In this situation,there will be no signi?cant difference between means of the genotype groups based on the presence or absence of the loosely-linked marker (Figure 9).Unlinked markers located far apart or on different chromosomes to the QTL are ran-domly inherited with the QTL;therefore,no signi?cant differences between means of the genotype groups will be detected.
Methods to detect QTLs
Three widely-used methods for detecting QTLs are single-marker analysis,simple interval mapping and composite interval mapping (Liu,1998;Tanksley,1993).Single-marker analysis (also ‘single-point anal-ysis’)is the simplest method for detecting QTLs as-sociated with single markers.The statistical methods used for single-marker analysis include t -tests,analysis of variance (ANOV A)and linear regression.Linear
179 Figure9.Diagram indicating tight and loose linkage between marker and QTL.(a)Recombination events(indicated by crosses)occurs between QTL and marker loci.(b)Gametes in population.Markers that are tightly-linked to a QTL(Marker E)are usually inherited with the QTL in the progeny.Markers that are only loosely-linked to a QTL(Marker H)are randomly inherited together.
regression is most commonly used because the coef-?cient of determination(R2)from the marker explains the phenotypic variation arising from the QTL linked to the marker.This method does not require a complete linkage map and can be performed with basic statistical software programs.However,the major disadvantage with this method is that the further a QTL is from a marker,the less likely it will be detected.This is be-cause recombination may occur between the marker and the QTL.This causes the magnitude of the effect of a QTL to be underestimated(Tanksley,1993).The use of a large number of segregating DNA markers covering the entire genome(usually at intervals less than15cM)may minimize both problems(Tanksley, 1993).
The results from single-marker analysis are usually presented in a table,which indicates the chromosome (if known)or linkage group containing the markers, probability values,and the percentage of phenotypic variation explained by the QTL(R2)(Table3).Some-times,the allele size of the marker is also reported. QGene and MapManager QTX are commonly used computer programs to perform single-marker analysis (Manly et al.,2001;Nelson,1997).
The simple interval mapping(SIM)method makes use of linkage maps and analyses intervals between adjacent pairs of linked markers along chromosomes simultaneously,instead of analyzing single markers (Lander&Botstein,1989).The use of linked mark-ers for analysis compensates for recombination be-tween the markers and the QTL,and is considered statistically more powerful compared to single-point analysis(Lander&Botstein,1989;Liu,1998).Many Table3.Single-marker analysis of markers associated
with QTLs using QGene(Nelson,1997)
Chromosome or
Marker linkage group P value R2
E2<0.000191
F20.000158
G20.023026
H20.57012
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researchers have used MapMaker/QTL (Lincoln et al.,1993b)and QGene (Nelson,1997),to conduct SIM.
More recently,composite interval mapping (CIM)has become popular for mapping QTLs.This method combines interval mapping with linear regression and includes additional genetic markers in the statistical model in addition to an adjacent pair of linked markers for interval mapping (Jansen,1993;Jansen &Stam,1994;Zeng,1993,1994).The main advantage of CIM is that it is more precise and effective at mapping QTLs compared to single-point analysis and interval map-ping,especially when linked QTLs are involved.Many researchers have used QTL Cartographer (Basten et al.,1994,2001),MapManager QTX (Manly et al.,2001)and PLABQTL (Utz &Melchinger,1996)to perform CIM.
Understanding interval mapping results
Interval mapping methods produce a pro?le of the likely sites for a QTL between adjacent linked mark-ers.In other words,QTLs are located with respect to a linkage map.The results of the test statistic for SIM and CIM are typically presented using a logarith-mic of odds (LOD)score or likelihood ratio statistic (LRS).There is a direct one-to-one transformation be-tween LOD scores and LRS scores (the conversion
can
Figure 10.Hypothetical output showing a LOD pro?le for chromosome 4.The dotted line represents the signi?cance threshold determined by permutation tests.The output indicates that the most likely position for the QTL is near marker Q (indicated by an arrow).The best ?anking markers for this QTL would be Q and R.
be calculated by:LRS =4.6×LOD)(Liu,1998).These LOD or LRS pro?les are used to identify the most likely position for a QTL in relation to the link-age map,which is the position where the highest LOD value is obtained.A typical output from interval mapping is a graph with markers comprising linkage groups on the x axis and the test statistic on the y axis (Figure 10).
The peak or maximum must also exceed a speci?ed signi?cance level in order for the QTL to be declared as ‘real’(i.e.statistically signi?cant).The determina-tion of signi?cance thresholds is most commonly per-formed using permutation tests (Churchill &Doerge,1994).Brie?y,the phenotypic values of the population are ‘shuf?ed’whilst the marker genotypic values are held constant (i.e.all marker-trait associations are bro-ken)and QTL analysis is performed to assess the level of false positive marker-trait associations (Churchill &Doerge,1994;Hackett,2002;Haley &Andersson,1997).This process is then repeated (e.g.500or 1000times)and signi?cance levels can then be determined based on the level of false positive marker-trait associ-ations.
Before permutation tests were widely accepted as an appropriate method to determine signi?cance thresholds,a LOD score of between 2.0to 3.0(most commonly 3.0)was usually chosen as the signi?cance threshold.
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Reporting and describing QTLs detected from interval mapping
The most common way of reporting QTLs is by in-dicating the most closely linked markers in a table and/or as bars (or oval shapes or arrows)on link-age maps (for example:Beattie et al.,2003;George et al.,2003;Hittalmani et al.,2002;Jampatong et al.,2002;McCouch &Doerge,1995;Pilet-Nayel et al.,2002).The chromosomal regions represented by rect-angles are usually the region that exceeds the signi?-cance threshold (Figure 11).Usually,a pair of markers –the most tightly-linked markers on each side of a QTL –is also reported in a table;these markers are known as ‘?anking’markers.The reason for
reporting
Figure 11.QTL mapping of height and disease resistance traits.Hypothetical QTLs were detected on chromosomes 2,3,4and 5.A major QTL for height was detected on chromosome 2.Two major QTLs for disease resistance were detected on chromosomes 4and 5whereas a minor QTL was detected on chromosome 3(adapted from Hartl &Jones,2001).
?anking markers is that selection based on two mark-ers should be more reliable than selection based on a single marker.The reason for the increased reliability is that there is a much lower chance of recombina-tion between two markers and QTL compared to the chance of recombination between a single marker and QTL.
It should also be noted that QTLs can only be de-tected for traits that segregate between the parents used to construct the mapping population.Therefore,in or-der to maximize the data obtained from a QTL mapping study,several criteria may be used for phenotypic eval-uation of a single trait (Flandez-Galvez et al.,2003a;Paterson et al.,1988;Pilet-Nayel et al.,2002).QTLs that are detected in common regions (based on different
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criteria for a single trait)are likely to be important QTLs for controlling the trait.
Mapping populations may also be constructed based on parents that segregate for multiple traits.This is advantageous because QTLs controlling the differ-ent traits can be located on a single map(Beattie et al., 2003;Khairallah et al.,1998;Marquez-Cedillo et al., 2001;Serquen et al.,1997;Tar’an et al.,2002).How-ever,for many parental genotypes used to construct mapping populations,this is not always possible,be-cause the parents may only segregate for one trait of interest.Furthermore,the same set of lines of the map-ping population used for phenotypic evaluation must be available for marker genotyping,and subsequent QTL analysis,which may be dif?cult with completely or semi-destructive bioassays(e.g.screening for resis-tance to necrotrophic fungal pathogens).
In general terms,an individual QTL may also be described as‘major’or‘minor’.This de?nition is based on the proportion of the phenotypic variation explained by a QTL(based on the R2value):major QTLs will ac-count for a relatively large amount(e.g.>10%)and minor QTLs will usually account for<10%.Some-times,major QTLs may refer to QTLs that are stable across environments whereas minor QTLs may refer to QTLs that may be environmentally sensitive,especially for QTLs that are associated with disease resistance(Li et al.,2001;Lindhout,2002;Pilet-Nayel et al.,2002).
In more formal terms,QTLs may be classi?ed as:(1)suggestive;(2)signi?cant;and(3)highly-signi?cant(Lander&Kruglyak,1995).Lander& Kruglyak(1995)proposed this classi?cation in order to “avoid a?ood of false positive claims”and also ensure that“true hints of linkage”were not missed.Signi?cant and highly-signi?cant QTLs were given signi?cance levels of5and0.1%,respectively,whereas a sugges-tive QTL is one that would be expected to occur once at random in a QTL mapping study(in other words, there is a warning regarding the reliability of sugges-tive QTLs).The mapping program MapManager QTX reports QTL mapping results with this classi?cation (Manly et al.,2001).
Con?dence intervals for QTLs
Although the most likely position of a QTL is the map position at which the highest LOD or LRS score is detected,QTLs actually occur within con?dence inter-vals.There are several ways in which con?dence inter-vals can be calculated.The simplest is the‘one-LOD support interval,’which is determined by?nding the region on both sides of a QTL peak that corresponds to a decrease of1LOD score(Hackett,2002;Lander& Botstein,1989).‘Bootstrapping’–a statistical method for resampling–is another method to determine the con-?dence interval of QTLs(Liu,1998;Visscher et al., 1996),and can be easily applied within some mapping software programs such as MapManager QTX(Manly et al.,2001).
Number of markers and marker spacing
There is no absolute value for the number of DNA markers required for a genetic map,since the num-ber of markers varies with the number and length of chromosomes in the organism.For detection of QTLs, a relatively sparse‘framework’(or‘skeletal’or‘scaf-fold’)map consisting of evenly spaced markers is ad-equate,and preliminary genetic mapping studies gen-erally contain between100and200markers(Mohan et al.,1997).However,this depends on the genome size of the species;more markers are required for mapping in species with large genomes.Darvasi et al.(1993)re-ported that the power of detecting a QTL was virtually the same for a marker spacing of10cM as for an in-?nite number of markers,and only slightly decreased for marker spacing of20or even50cM.
Making comparisons between maps
All linkage maps are unique and are a product of the mapping population(derived from two speci?c par-ents)and the types of markers used.Even if the same set of markers is used to construct linkage maps,there is no guarantee that all of the markers will be poly-morphic between different populations.Therefore,in order to correlate information from one map to another, common markers are https://www.doczj.com/doc/238903763.html,mon markers that are highly polymorphic in mapping populations are called‘anchor’(also‘core’markers).Anchor mark-ers are typically SSRs or RFLPs(Ablett et al.,2003; Flandez-Galvez et al.,2003b;Gardiner et al.,1993). Speci?c groups of anchor markers,that are located in close proximity to each other in speci?c genomic re-gions,may be referred to as‘bins’.Bins are used to integrate maps,and are de?ned as‘10–20cM regions along chromosomes;the boundaries of each are de?ned by a set of core RFLP markers’(Polacco et al.,2002).If common anchor markers have been incorporated into different maps,they can be aligned together to produce ‘consensus’maps(Ablett et al.,2003;Karakousis et al., 2003).Consensus maps are produced by combining
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or merging different maps,constructed from differ-ent genotypes,together.Such consensus maps can be extremely useful for ef?ciently constructing new maps (with evenly spaced markers)or targeted(or localized) mapping.For example,a consensus map can indicate which markers are located in a speci?c region contain-ing a QTL,and thus be used to identify more tightly linked markers.
The study of similarities and differences of markers and genes within and between species,genera or higher taxonomic divisions is referred to as comparative map-ping(Paterson et al.,1991a).It involves analysing the extent of the conservation between maps of the order in which markers occur(i.e.collinear markers);con-served marker order is referred to as‘synteny’.Com-parative mapping may assist in the construction of new linkage maps(or localized maps of speci?c genomic regions)and in predicting the locations of QTLs in dif-ferent mapping populations(Young,1994).Previous linkage maps may provide an indication of which mark-ers are polymorphic,as well as provide an indication of linkage groups and the order of markers within linkage groups.Furthermore,comparative mapping may reveal evolutionary relationships between taxa.
Factors in?uencing the detection of QTLs
There are many factors that in?uence the detection of QTLs segregating in a population(Asins,2002; Tanksley,1993).The main ones are genetic proper-ties of QTLs that control traits,environmental effects, population size and experimental error.
Genetic properties of QTLs controlling traits in-clude the magnitude of the effect of individual QTLs. Only QTLs with suf?ciently large phenotypic effects will be detected;QTLs with small effects may fall be-low the signi?cance threshold of detection.Another genetic property is the distance between linked QTLs. QTLs that are closely-linked(approximately20cM or less)will usually be detected as a single QTL in typical population sizes(<500)(Tanksley,1993).
Environmental effects may have a profound in?u-ence on the expression of quantitative traits.Experi-ments that are replicated across sites and over time(e.g. different seasons and years)may enable the researcher to investigate environmental in?uences on QTLs affect-ing trait(s)of interest(George et al.,2003;Hittalmani et al.,2002;Jampatong et al.,2002;Lindhout,2002; Paterson et al.,1991b;Price&Courtois,1999).As dis-cussed previously,RI or DH populations are ideal for these purposes.
The most important experimental design factor is the size of the population used in the mapping study. The larger the population,the more accurate the map-ping study and the more likely it is to allow detection of QTLs with smaller effects(Haley&Andersson,1997; Tanksley,1993).An increase in population size pro-vides gains in statistical power,estimates of gene ef-fects and con?dence intervals of the locations of QTLs (Beavis,1998;Darvasi et al.,1993).
The main sources of experimental error are mis-takes in marker genotyping and errors in phenotypic evaluation.Genotyping errors and missing data can affect the order and distance between markers within linkage maps(Hackett,2002).The accuracy of pheno-typic evaluation is of the utmost importance for the ac-curacy of QTL mapping.A reliable QTL map can only be produced from reliable phenotypic data.Replicated phenotypic measurements or the use of clones(via cut-tings)can be used to improve the accuracy of QTL map-ping by reducing background‘noise’(Danesh et al., 1994;Haley&Andersson,1997).Some thorough stud-ies include those where phenotypic evaluations have been conducted in both?eld and glasshouse trials, for ascochyta blight resistance in chickpea(Flandez-Galvez et al.,2003a),bacterial brown spot in common bean(Jung et al.,2003),and downy mildew resistance in pearl millet(Jones et al.,2002).
Con?rmation of QTL mapping
Ideally,due to the factors described above,QTL map-ping studies should be independently con?rmed or ver-i?ed(Lander&Kruglyak,1995).Such con?rmation studies(referred to as‘replication studies’by Lander &Kruglyak,1995)may involve independent popula-tions constructed from the same parental genotypes or closely-related genotypes used in the primary QTL mapping study.Sometimes,larger population sizes may be used.Furthermore,some recent studies have proposed that QTL positions and effects should be eval-uated in independent populations,because QTL map-ping based on typical population sizes results in a low power of QTL detection and a large bias of QTL ef-fects(Melchinger et al.,1998;Utz et al.,2000).Un-fortunately,due to constraints such as lack of research funding and time,and possibly a lack of understanding of the need to con?rm results,QTL mapping studies are rarely con?rmed.Some notable exceptions are the con-?rmation of QTLs associated with root-knot nematode resistance(Li et al.,2001)and bud blight resistance in soybean(Fasoula et al.,2003).
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Another approach used to con?rm QTLs has been to use a speci?c type of population called near isogenic lines(NILs).NILs are created by crossing a donor parent(e.g.wild parent possessing a speci?c trait of interest)to a recurrent parent(e.g.an elite cultivar). The F1hybrid is then backcrossed to the recurrent par-ent to produce a?rst backcross generation(BC1).The BC1is then repeatedly backcrossed to the recurrent parent for a number of generations(e.g.7).The?nal BC7will contain practically all of the recurrent parent genome except for the small chromosomal region con-taining a gene or QTL of interest.Homozygous F2lines can be obtained by sel?ng the BC7plant.It should be noted that in order to produce a NIL containing a target gene,the gene has to be selected for during each round of backcrossing.By genotyping NILs with important markers,and comparing mean trait values of particular NIL lines with the recurrent parent,the effects of QTLs can be con?rmed.Examples of studies utilizing NILs to con?rm QTLs include agronomic traits in tomato (Bernacchi et al.,1998),leaf rust resistance in barley (Van Berloo et al.,2001),nematode resistance in soy-bean(Glover et al.,2004)and phosphorus uptake in rice(Wissuwa&Ae,2001).
Short cuts for gene tagging
The construction of linkage maps and QTL analysis takes a considerable amount of time and effort,and may be very expensive.Therefore,alternative methods that can save time and money would be extremely use-ful,especially if resources are limited.Two‘short-cut’methods used to identify markers that tag QTLs are bulked segregant analysis(BSA)and selective geno-typing.Both methods require mapping populations.
BSA is a method used to detect markers located in speci?c chromosomal regions(Michelmore et al., 1991).Brie?y,two pools or‘bulks’of DNA samples are combined from10–20individual plants from a seg-regating population;these two bulks should differ for a trait of interest(e.g.resistant vs.susceptible to a par-ticular disease).By making DNA bulks,all loci are randomised,except for the region containing the gene of interest.Markers are screened across the two bulks. Polymorphic markers may represent markers that are linked to a gene or QTL of interest(Figure12).The entire population is then genotyped with these poly-morphic markers and a localized linkage map may be generated.This enables QTL analysis to be performed and the position of a QTL to be determined(Ford et al., 1999).
BSA is generally used to tag genes controlling sim-ple traits,but the method may also be used to iden-tify markers linked to major QTLs(Wang&Paterson, 1994).‘High-throughput’or‘high-volume’marker techniques such as RAPD or AFLP,that can gener-ate multiple markers from a single DNA preparation, are generally preferred for BSA.
Selective genotyping(also known as‘distribution extreme analysis’or‘trait-based marker analysis’)in-volves selecting individuals from a population that rep-resent the phenotypic extremes or tails of the trait being analysed(Foolad&Jones,1993;Lander&Botstein, 1989;Zhang et al.,2003).Linkage map construction and QTL analysis is performed using only the individ-uals with extreme phenotypes(Figure13).By geno-typing a subsample of the population,the costs of a mapping study can be signi?cantly reduced.Selective genotyping is typically used when growing and phe-notyping individuals in a mapping population is easier and/or cheaper than genotyping using DNA marker as-says.The disadvantages of this method are that it is not ef?cient in determining the effects of QTLs and that only one trait can be tested at a time(because the individuals selected for extreme phenotypic values will usually not represent extreme phenotypic values for other traits)(Tanksley,1993).Furthermore,single-point analysis cannot be used for QTL detection,be-cause the phenotypic effects would be grossly overesti-mated;interval mapping methods must be used(Lander &Botstein,1989).
Section IV:Towards marker-assisted selection Marker-assisted selection(MAS)is a method whereby a phenotype is selected on the the genotype of a marker (see Section V).However,the markers identi?ed in preliminary genetic mapping studies are seldom suit-able for marker-assisted selection without further test-ing and possibly further development.Markers that are not adequately tested before use in MAS programs may not be reliable for predicting phenotype,and will there-fore be useless.Generally,the steps required for the de-velopment of markers for use in MAS includes:high-resolution mapping,validation of markers and possibly marker conversion(discussed below).
High-resolution mapping of QTLs
The preliminary aim of QTL mapping is to pro-duce a comprehensive‘framework’(also‘skeletal’or ‘scaffold’linkage map)that covers all chromosomes
185 Figure12.The preparation of DNA bulks for a simple disease resistance trait(a)and a quantitative quality trait(?ower colour)(b).In both cases,two bulks(B1and B2)are made from individuals displaying extreme phenotypic scores.(c)Polymorphic markers(indicated by arrows) that are identi?ed between bulks may represent markers that are linked to genes or QTLs controlling the traits.Such markers are then used to genotype the entire mapping population and QTL analysis performed.(Adapted from Langridge et al.,2001;Tanksley et al.,1995.)
evenly in order to identify markers?anking those QTLs that control traits of interest.There are several more steps required,because even the closest markers?ank-ing a QTL may not be tightly linked to a gene of interest (Michelmore,1995).This means that recombination can occur between a marker and QTL,thus reducing the reliability and usefulness of the marker.By using larger population sizes and a greater number of markers,more tightly-linked markers can be identi?ed;this process is termed‘high-resolution mapping’(also‘?ne map-ping’).Therefore,high-resolution mapping of QTLs may be used to develop reliable markers for marker-assisted selection(at least<5cM but ideally<1cM away from the gene)and also to discriminate between a single gene or several linked genes(Michelmore,1995; Mohan et al.,1997).
There is no universal number for the appropriate population size required for high-resolution mapping.However,population sizes that have been used for high-resolution mapping have consisted of>1000indi-viduals to resolve QTLs to distances between?anking markers of<1cM(Blair et al.,2003;Chunwongse et al.,1997;Li et al.,2003).
The mapping of additional markers may saturate framework maps.High-throughput marker techniques that generate multiple loci per primer combination(e.g. AFLP)are usually preferred for increasing marker density(Figure14).Bulked-segregant analysis may also be used to identify additional markers linked to speci?c chromosomal regions(Campbell et al., 2001;Giovannoni et al.,1991).However,the extent to which framework maps can be saturated depends on the size of the population used to construct the map.In many cases,the sizes of segregating pop-ulations being used are too small to permit high-resolution mapping,since smaller populations have
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Figure13.Selective genotyping.The entire population is phenotypically evaluated for a particular trait(e.g.disease resistance).However,only individuals representing the phenotypic extremes from the mapping population are selected for marker genotyping,and subsequent linkage and QTL analysis.
fewer recombinants than larger populations(Tanksley, 1993).
High-resolution maps of speci?c chromosomal re-gions may also be constructed by using NILs(Blair et al.,2003).Markers that are polymorphic between NILs and the recurrent parent should represent markers that are linked to the target gene and can be incorpo-rated into a high-resolution map.
Validation of markers
Generally,markers should be validated by testing their effectiveness in determining the target phenotype in independent populations and different genetic back-grounds,which is referred to as‘marker validation’(Cakir et al.,2003;Collins et al.,2003;Jung et al., 1999;Langridge et al.,2001;Li et al.,2001;Sharp et al.,2001).In other words,marker validation involves testing the reliability of markers to predict phenotype. This indicates whether or not a marker could be used in routine screening for MAS(Ogbonnaya et al.,2001; Sharp et al.,2001).
Markers should also be validated by testing for the presence of the marker on a range of cultivars and other important genotypes(Sharp et al.,2001;Spielmeyer et al.,2003).Some studies have warned of the dan-ger of assuming that marker-QTL linkages will remain in different genetic backgrounds or in different test-ing environments,especially for complex traits such as yield(Reyna&Sneller,2001).Even when a single gene controls a particular trait,there is no guarantee that DNA markers identi?ed in one population will be use-ful in different populations,especially when the popu-lations originate from distantly related germplasm(Yu et al.,2000).For markers to be most useful in breeding programs,they should reveal polymorphism in differ-ent populations derived from a wide range of different parental genotypes(Langridge et al.,2001).
Marker conversion
There are two instances where markers may need to be converted into other types of markers:when there are problems of reproducibility(e.g.RAPDs)and when the marker technique is complicated,time-consuming or expensive(e.g.RFLPs or AFLPs).The problem of reproducibility may be overcome by the development
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Figure14.High-resolution linkage mapping.Additional markers have been utilised to‘?ll in the gaps’between the anchor mark-ers.The identi?cation of additional markers in the vicinity of QTLs (e.g.between Q and R on chromosome4)could be useful for MAS.
A bulked-segregant analysis approach can be applied to target spe-ci?c chromosomal regions as shown on chromosome5(between V and W).
of sequence characterised ampli?ed regions(SCARs) or sequence-tagged sites(STSs)derived by cloning and sequencing speci?c RAPD markers(Jung et al.,1999; Paran&Michelmore,1993).SCAR markers are ro-bust and reliable.They detect a single locus and may be codominant(Paran&Michelmore,1993).RFLP and AFLP markers may also be converted into SCAR or STS markers(Lehmensiek et al.,2001;Shan et al., 1999).The use of PCR-based markers that are con-verted from RFLP or AFLP markers is technically sim-pler,less time-consuming and cheaper.STS markers may also be transferable to related species(Brondani et al.,2003;Lem&Lallemand,2003).
Section V:Marker-assisted selection
Selecting plants in a segregating progeny that contain appropriate combinations of genes is a critical compo-nent of plant breeding(Ribaut&Betran,1999;Weeden et al.,1994).Moreover,plant breeders typically work with hundreds or even thousands of populations,which often contain large numbers(Ribaut&Betran,1999; Witcombe&Virk,2001).‘Marker-assisted selection’(also‘marker-assisted breeding’or‘marker-aided se-lection’)may greatly increase the ef?ciency and effec-tiveness in plant breeding compared to conventional breeding methods.Once markers that are tightly linked to genes or QTLs of interest have been identi?ed,prior to?eld evaluation of large numbers of plants,breeders may use speci?c DNA marker alleles as a diagnostic tool to identify plants carrying the genes or QTLs(Fig-ure15)(Michelmore,1995;Ribaut et al.,1997;Young, 1996).The advantages of MAS include:
?time saving from the substitution of complex?eld trials(that need to be conducted at particular times of year or at speci?c locations,or are technically complicated)with molecular tests;?elimination of unreliable phenotypic evaluation as-sociated with?eld trials due to environmental ef-fects;
?selection of genotypes at seedling stage;?gene‘pyramiding’or combining multiple genes si-multaneously;
?avoid the transfer of undesirable or deleterious genes (‘linkage drag’;this is of particular relevance when the introgression of genes from wild species is in-volved).
?selecting for traits with low heritability ?testing for speci?c traits where phenotypic evalua-tion is not feasible(e.g.quarantine restrictions may prevent exotic pathogens to be used for screening). Selection of QTLs for MAS
One commonly asked question is that“since quanti-tative traits are controlled by at least several QTLs, how many QTLs are typically selected for MAS?”Theoretically,all markers that are tightly linked to QTLs could be used for MAS.However,due to the cost of utilizing several QTLs,only markers that are tightly linked to no more than three QTLs are typically used(Ribaut&Betran,1999),although there have been reports of up to5QTLs being introgressed into tomato via MAS(Lecomte et al.,2004).Even selecting for a single QTL via MAS can be bene?cial in plant breeding;such a QTL should account for the largest proportion of phenotypic variance for the trait (Ribaut&Betran,1999;Tanksley,1993).Furthermore, all QTLs selected for MAS should be stable across
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Figure 15.MAS scheme for early generation selection in a typical breeding program for disease resistance (adapted from Ribaut &Hoisington,1998;Ribaut &Betran,1999).A susceptible (S)parent is crossed with a resistant (R)parent and the F 1plant is self-pollinated to produce a F 2population.In this diagram,a robust marker has been developed for a major QTL controlling disease resistance (indicated by the arrow).By using a marker to assist selection,plant breeders may substitute large ?eld trials and eliminate many unwanted genotypes (indicated by crosses)and retain only those plants possessing the desirable genotypes (indicated by arrows).Note that 75%of plants may be eliminated after one cycle of MAS.This is important because plant breeders typically use very large populations (e.g.2000F 2plants)derived from a single cross and may use populations derived from hundreds or even thousands of crosses in a single year.
environments (Hittalmani et al.,2002;Ribaut &Betran,1999).
Cost/bene?t analysis of MAS
The cost of using ‘tools’in breeding programs is a ma-jor consideration.The cost of using MAS compared to conventional plant breeding varies considerably be-tween studies.Dreher et al.(2003)indicated that the cost-effectiveness needs to be considered on a case by case basis.Factors that in?uence the cost of utilizing markers include:inheritance of the trait,method of phe-notypic evaluation,?eld/glasshouse and labour costs,and the cost of resources.
In some cases,phenotypic screening is cheaper compared to marker-assisted selection (Bohn et al.,2001;Dreher et al.,2003).However,in other cases,phenotypic screening may require time-consuming and expensive assays,and the use of markers will then be preferable.Some studies involving markers for disease resistance have shown that once markers have been de-veloped for MAS,it is cheaper than conventional meth-ods (Yu et al.,2000).In other situations,phenotypic evaluation may be time-consuming and/or dif?cult and therefore using markers may be cheaper and preferable (Dreher et al.,2003;Young,1999;Yu et al.,2000).An important consideration for MAS,often not reported,is that while markers may be cheaper to use,there is a
large initial cost in their development.An estimate for the cost to develop a single marker was AUD $100,040(Langridge et al.,2001).Marker-assisted backcrossing
Using conventional breeding methods,it typically takes 6–8backcrosses to fully recover the recurrent parent genome.The theoretical proportion of the recurrent parent genome after n generations of backcrossing is given by:(2n +1?1)/2n +1(where n =number of back-crosses;assuming an in?nite population size).The percentages of recurrent parent recovery after each backcross generation are presented in Table 4.The percentages shown in Table 4are only achieved with large populations;the percentages are usually lower in
Table 4.Percentage of recurrent parent genome after backcrossing Recurrent
Generation parent genome (%)BC175.0BC287.5BC393.8BC496.9BC598.4BC6
99.2
一、一般现在时： 1. The Browns ______ a nice car and Brown's brother _______ a nice jeep. A. have / have B. has / has C. have / has D. has / have 2. If their house ______ not like ours, what _______ it look like? A. is / is B. is / does C. does / does D. does / is 3. - ______ you think he will come? - If it _______ tomorrow, he will not come. A. Do / rains B. Are / rains C. Do / will rain D. Are / will rain 4. The little child ______ not even know that the moon _______ around the earth. A. do / move B. do / moves C. does / moves D. did / moved 5. Many a student _ A. are / goes 【练习答案】 二、现在进行时： ______ fond of films, but a good student seldom _______ to the cinema B. is / goes C. are / go D. is / go 1.C 2.B 3.A 4.C 5.B is / am / are + 现在分词 1. How can you ______ If you are not ______ ? A. listening / hearing B. hear / listening C. be listening / heard D. be hearing / listening to 2. ___________________________________________ The girl even won't have her lunch before she ____________________________________ her homework. A. will finish B. is finishing C. had finished D. finishes 3. Those who have applied for the post( 职位 ) _______ in the office.( 此题超前 ) A. are being interviewed B. are interviewing C. interviewing 4. The old scientist _______ to do more for the country. A. is wishing B. has been wishing C. wishes 5. If he ______, don't wake him up. 练习答案】 1.B 2.D 3.A 4.C 5.B 三、一般过去时 1. Yesterday I _______ (think) that you were not in Beijing. 2. Alice usually ______ (sit) in the front of the classroom, but she ________ (sit) at the back this morning. 3. He ______ (tell) the news to us three days ago. 4. He ______ (begin) to teach Chinese in 1990. 5. she would not telephone me if she ______ (have) no time. 测试精编 II ： 1. They ______ the trip until the rain stopped. A. continued B. didn't continue C. hadn't continued 2. The local peasants gave the soldiers clothes and food without which they ______ of hunger and cold.(without 在这里表条件，你知道吗 ?) A. would die B. will die C. would be dead D. would have died 3. It was not until then that I came to know that the earth _______ around the sun. D. to be interviewing D. has been wished A. still sleeps B. is still sleeping C. still has been sleeping D. will be sleeping still D. would continue
Lesson 4 An exciting trip 词汇学习 1.区分： （1）exciting adj. 令人兴奋的； The news is exciting.这新闻让人兴奋exciting boy 令人兴奋的男孩. （2）excited adj. 兴奋的-ed: 自己感到/ -ing:令人感到 I am excited. 我很兴奋 （3）excite v. 激动（这类动词的宾语一定是人，让后面的人感到……） The news excited me. 这个消息使我兴奋。 类似用法：interesting adj. 令人感到有趣的；interested adj. 感到有意思的interesting man, The man is interesting. interest v.对……感兴趣The book interests me.那本书让我感到很有趣 2. receive v. 接受, 收到 vt. 接到，收到，得到When did you receive that letter?你什么时候收到那封信的？receive 是“收到”，指的是一个被动的动作，主观上接受与否不清楚。 receive/have a letter from sb. accept 同意接收 This morning I received a bunch of flowers from a boy, but I didn't accept it. 今早我收到一束一个男生送的花，但我没有接受 区分：take则是主动的“拿”、“取” I received a beautiful pen from my uncle. 我送叔叔那拿了一支漂亮的笔。 My brother took it from me yesterday. 我哥哥昨天从我这拿的。 take 也可以作收到take the exam 接受考试；take advice 接受建议 3.firm （1）n. 商行, 公司law firm法律公司 （2）adj 结实的，稳固的 The car is very firm. 区分 firm中小型公司；company n. 正规公司；business商行 4.a great number of +可数名词复数= a great many +可数名词复数
第一节．单项填空，从A,B,C,D四个选项中选出可以填入空白处的最佳选项。（共计15小题，每小题1分，满分15分） 21.---Would you mind if I turned on the TV and watched CCTV news? ---___________. As a m atter of fact, I also like watching CCTV news. A. No, you can’t B. Yes, I do C. No, go ahead D. OK, no problem 22.She talked ______ she saw the accident. But in fact she only heard of it from others. A. so that B. as though C. even though D. once 23.The traffic in our county is very busy, for some main streets _______. A.have been rebuilt B.rebuilt C.are being rebuilt D.are rebuilding 24.Nobody knows what happened _____ her _____ the morning of May 1. A. for; in B . with; on C. to; on D. to; in 25.---I missed the first part of the film.It was really a pity. ---You ______home half an hour earlier. A.should go B.must have gone C.should leave D.should have left 26. The (H1N1) flu ________quickly in Lixin last month. A. spread B. spreads C. traveled D. travels 27. With so much homework _____, Tom has to stay at home. A. to do B. to be done C. done D. doing 28.---- Your daughter looks shy. ----After all, it is the first time that she ____ a speech to the public. A.had made B. has made C. is making D. makes 29.---- I hear Jane has gone to the Holy Island for her holiday. ---- Oh, how nice! Do you know when she ____? A. was leaving B. had left C. has left D. left
新概念英语第二册Lesson67语法知识点 表示“能力”的情态助动词及其他有关的动词： can/ could, be able to 与 manage to (1)在第43课的语法中，我们学习了表示“能力”的 can/could。用将来时的句子中表示“能力”时通常用 will be able to, 在表示成功地完成过去某个动作时用was able to而不用could： I can't remember where I've seen him. 我想不起来我在什么地方见过他。 I can sing some songs, but I can't play the piano. 我会唱一些歌，但我不会弹钢琴。 He thought he could pass the exam easily, but he failed. 他以为他能轻易地通过考试，结果他没通过。 After Byrd had ordered his men to throw out two heavy food sacks, the plane was then able to rise. 在伯德命令他的助手们扔掉两个沉重的食物袋之后，飞机才可以上升了。 Jane can't swim yet. She'll be able to swim in a few months time. 简不会游泳。再过几个月她就会游泳了。 (2)表示“能力”的另一种方式是用 manage to。在表示成功地完成过去某个动作时，它经常代替 be able to。与 be able to相比，它更强调“虽然困难很大，但仍能……”这种含义。试比较： He finished the job yesterday. 他昨天把工作做完了。(陈述一个事实) He was able to finish the job yesterday. 他昨天把工作做完了。(表示可能) He managed to finish the job yesterday.
新概念英语综合测试题 班级：姓名： 听力部分 1.听音，选出你所听到的单词。10 1. ( ) A. wash B. wait C. shave D. sleep 2. ( ) A. June B. July C. September D. December 3. ( ) A. south B. north C. east D. west 4. ( ) A. season B. set C. summer D. snow 5. ( ) A. climate B. country C. chicken D. steak 6. ( ) A. mince B. meat C. sweet D. butter 7. ( ) A. bean B. pear C. pea D. peach 8. ( ) A. tomato B. potato C. pleasant D. spring 9. ( ) A. Spain B. Sweden C. Holland D. England 10( ) A. beer B. banana C. blackboard D. butter 2.为你所听到的单词选择正确的汉语意思。10 1. ( ) A. 甜的 B. 新鲜的 C. 上等的 D. 成熟的 2. ( ) A. 香蕉 B. 果酱 C. 苹果 D. 果酒 3. ( ) A. 饼干 B. 蛋糕 C. 水壶 D. 茶壶 4. ( ) A. 举起 B. 喜欢 C. 想 D. 展出 5. ( ) A. 后面 B. 找到 C. 沸腾 D. 当然 6. ( ) A. 面包 B. 咖啡 C. 肥皂 D. 烟丝 7. ( ) A. 前面 B. 后面 C. 左边 D. 右边 8. ( ) A. 花瓶 B. 茶壶 C. 盘子 D. 瓷器 9. ( ) A. 努力的 B. 工作 C. 书架 D. 锤子 10. ( ) A. 建筑物 B. 公园 C. 山谷 D. 村庄 3. 为你所听到的问题选择正确的答案。10 1. ( ) A. Yes, it is. B. Yes, she is. 2. ( ) A. I’m a keyboard operator. B. She’s my teacher. 3. ( ) A. I’m fine. B. She’s OK. 4. ( ) A. I’m blue. B. It’s blue.
Lesson 44 B 1to see 2 working 3 ironing 4 to leave 5 to argue/ arguing 6 to come 7 seeing 8 knocking 9 waiting 10 to rain/ raining 11 w orking 12 taking Lesson 45 2．难点练习答案 1 back 2 robbed… stole 3 back 4 stole 5 robbed Lesson 46 B1 with 2 to…for 3 with 4 for 5 to 6 at 7 to 8 to 9 for 10 to…at 11 at 12 with 13 with 14 to…for 15 for 16 for 17 with 18 for 19 at 20 with 21 to 22 with 23 at 24 to 25 with…to 26 to…with27 for 28 to…to 29 for 30 to 31 for 32 to 33 to…with 34 for 35 to 36 to 37 at 38 at 39 with 40 for Lesson 47 C 1 will have finished 2 broke 3 were 4 could D 1 mustn't 2 needn't Lesson 48 A 1 which 2 denied 3 fetched 4 too 5 jobs 6 One…a…who 7 past 8 next 9 watching 10 continually 11 remarked 12 robbed D 1 made 2 do 3 make 4 do 5 Do 6 make 7 made8 does E1 out 2 up 3 up 4 up…away 5 up 6 out 7 back 8 up with 9 up with Lesson 49 Lesson 50 B‘Let's eat here,’I said to my wife. ‘I(would) prefer to have a drink first,’she answered. ‘That's a good idea,’I said. I picked up the menu.‘I don't understand a thing.’I said.‘It's all in Spanish.’
新概念二册语法总结-标准化文件发布号：（9556-EUATWK-MWUB-WUNN-INNUL-DDQTY-KII
新概念二册语法总结（新东方） 1. 简单句的结构: 主语+谓语+宾语+状语（地点状语+方式状语+时间状语）时间状语还可以放在句首 2. 一般现在时，现在进行时感叹句：what+名词+主语+谓语， how+形容词/副词+主语+谓语 频度副词：位于主语和谓语之间，常见频度副词及其程度的深浅： always, often, usually, frequently, sometimes, hardly, rarely, seldom, never I always buy CDs on Sundays. 3. 一般过去时直接宾语和间接宾语： 主语＋及物动词＋间接宾语＋直接宾语直接宾语是及物动词的直接对象，间接宾语是及物动词的动作所涉及的人或事务，也可以说间接宾语表示动作是对谁做的，或者是为谁做的。所以间接宾语要用名词或者宾格代词来担当。 He gives me a book. me间接宾语, a book直接宾语 直接宾语和间接宾语的位置调换时要加一个介词to或for，to表示动作对谁而做， for表示动作为谁而做。 主语＋及物动词＋直接宾语＋介词+间接宾语Give the book to me. Send a letter to him. I bought a coat for my mother. 4. 现在完成时，注意频度及时间副词的位置 receive/take 5. 复习：一般过去时与现在完成时的区别in the way/on the way/in this way/by the way/in a way/get out of my way/Don’t stand in my way./by the way/no way spare/to spare 6. 冠词用法（一）1．不定冠词用于修饰单数可数名词，当一个单词的第一个发音为元音时要用冠词an, 如果是辅音用a即可。 2．不定冠词还可以用来修饰一类事物，有时候也可以省略： A cat is a lovely animal. Cat is a lovely animal. 3．不可数名词加冠词表示一类事物：Apple is a fruit. 4．如果特制某人,某物或上文提过的人或物时要用定冠词the 5． Some表示一些，可以修饰可数名词及不可数名词。 6．在姓名，城市，地名，过名，月份，节日，星期前不加冠词，季节前也一般不加，特指时加。 put on/take off/look for/look after/knock at/knock sth. off/knock off/knock %off/knock over/knock out 7. 过去进行时，时间状语短语：在短语中动词后面的介词或副词叫小品词，有些小品词既可以紧跟在动词后面也可以跟在动词的宾语后面，而有些则只能紧跟着动词使用。 8. 形容词的比较级与最高级单音节词的比较级最高级：直接加-er, -est: small-smaller, smallest 以e结尾的加-r, -st: large-larger, largest 以辅音字母加y结尾的词变y为i加-er, -est: busy-busier, busiest
难点练习答案 1. What a wonderful garden (this is)! 2. What a surprise ( this is )! 3. What a lot of troube he is causing! 4. What wonderful actors ( they are )! 5. What a hard-working woman ( she is )! 6. What a tall building ( it is)! 7. What a terrible film ( it is )! 8. What a clever boy you are! 9. What a pretty girl ( she is )! 10. What a strange guy ( he is )! 多项选择题 1. c 2. d 3. c 4. c 5. a 6.b 7. b 8. a 9. d 10. c 11. d 12. b 第三课 P21 练习 B. 参照课文原文
C. died, left, spent, bought, went, stayed, listened, lent, kept, lost P22 难点练习答案 1.He paid some money to the shopkeeper. 2.He handed the prize to me. 3.The waiter brought the man a bottle of beer. 4.He sold me all his books. 5.The shop assistant found me some curtain material. 6.He did a big favor for me. 7.She showed her new hat to her husband. 8.She promised the finder a reward. 9.He gave some advice to his son. 10.His uncle left some money to / for him. 11.He is teaching us English. 12.I bought you this bunch of flowers. 13.Bring me that book please. 14.He offered a cigarette to me.
课文语法要点 Lesson1 简单陈述句的语序 Lesson2 一般现在时和现在进行时 Lesson3 现在完成时；一般过去时与一般现在时；直接宾语与间接宾语 Lesson4 现在完成时与一般过去时的区别（1）；现在完成时与现在进行时的区别；同位语 Lesson5 带way的一些短语；不定冠词和定冠词；现在完成时与一般过去时的区别（2） Lesson6 短语动词；过去进行时（1）；a，the，some的用法 Lesson7 短语动词中的小品词；表示时间的介词；过去进行时(2) Lesson8 形容词和副词的比较级和最高级 Lesson9 被动语态；引导时间状语的介词in，on，at，during，till，与until；否定句的两种形式：not any与no Lesson10 现在进行时与过去进行时的被动语态；双重所有格 Lesson11 动词+名次/代词+带to的不定式 Lesson12 一般将来时 Lesson13 将来进行时 Lesson14 过去完成时 Lesson15 直接引语和间接引语 Lesson16 条件句（1） Lesson17 Must，have to，与 have got to的用法 Lesson18 实义动词have的用法 Lesson19 表示许可的can和may Lesson20 介词与动名词 Lesson21 情态动词在被动语态中的应用 Lesson22 一些常见的动词词组 Lesson23 There is 和 it is的用法 Lesson24 无语法重点注重复习 Lesson25 并列句的语序 Lesson26 一般现在时与现在进行时的动词使用 Lesson27 表示过去习惯动作的used to 和一般过去时的特殊用法 Lesson28 现在完成时与since；关系从句及关系代词 Lesson29 现在完成时与一般过去时的区别 Lesson30 定冠词the的用法；some与any的区别 Lesson31 过去进行时与一般过去时；use to的用法 Lesson32 比较结构表示法；Little和few的用法 Lesson33 用于表示目的和方向的介词和副词 Lesson34 被动语态用法补充 Lesson35 复习，本课无新的语法点 Lesson36 Will和be going to Lesson37 一般将来完成时；将来完成进行时 Lesson38 过去完成时与从属连词when，before，after，until Lesson39 间接引语中的say，tell，ask；间接一般疑问句；间接疑问词疑问句Lesson40 条件句（2）
综合能力部 “新”意解读“概”之英语理“念”全晰 以学员为中心加上严格的教学质量,名师打造全新英语学习体系,听、说、读、写、译综合英语实力全面提高。大学四、六级、考研、口语、国 外各项考试；中高考、成人、企业培训等各类英语基础及提高课 程。 杜立明:新东方口语三剑客，新东方教育集团优秀讲师，人称英语怪才，托福650，GRE词汇满分，单词量30000，授课风诙谐幽默、激情洋溢。 王翔：重庆新东方语音专家，自创JS语音学习法，立竿见影改变你的语音状态。以独特的逻辑方式让你了解口语学习全过程。互联网专家，精通网 络编程、FLASH，开发过许多英语学习网站。Flash游戏《红色小屋》、 《鼠标大师》曾风靡全国。PS：巴基斯坦网络第一人 洪虹：Tesol证书获得者，英语专业八级，原正大软件集团英语特聘培训师。 没有唇枪舌剑的基因，没有高谈阔论的细胞，没有一落地就注定的幽默 感，唯有朴实和真诚伴我一路走来，却也受益非浅。单纯的教书，单纯 的生活，单纯的学习是单纯的我最向往的美好生活。 肖坤雪：英语教育学硕士，专业八级，周游列国，擅长口笔译。英美文化文学研究方向，高校7年英语教学经验。历年全国高校英语研讨会与会者。曾 入选英领事New Castle University 英语教师项目。长年从事重庆中 小学英语教师培训工作。著有英语培训教材《双语音乐教学》、《英美 文学作品百篇诵读》。 闭薇娜：经济学硕士，经济产业规划投资方向。英专语八级，资深明星教师，曾任英国领事馆教育推广助理，西门子中国商务助理，上课风格轻松，激 情飞扬。 何春梅：英语语言文学硕士，翻译理论与实践方向，英语专八高分，曾在多家国内外语培训机构任职，多年从事英语教学工作，授课深入浅出，幽默风 趣。 张帆：重庆新东方资深教师。英文教育专业毕业，曾经担任国际导游。擅长各年龄段英文听说教学。深受学生好评。于条件状语从句中用一般现在时 表现将来时，以莫须有的心情开心琢磨任性的现世。迷恋于声色世界， 在新东方留连。 郑瑞珺：英语八级，法语四级。曾参加国际模拟联合国大会，获得最佳代表，并组织四川外语学院模联大会。先后参加过布雷顿森林体系会议、20国
新概念英语第一册练习题（一） Lesson 1 Excuse me! 请大家将你的答案写在后面吧 Lesson 1 Excuse me! 对不起！ A About you Copy this dialogue. Add your own name at the end. 抄写这段对话，. 在结尾处加上你的名字。Sue: Excuse me. ______ John: Yes ______ Sue: What's your name ______ John: Pardon ______ Sue: What's your name ______ John: My name is John. ______ Sue: What's your name ______ You: My name is...... ______ B Vocabulary： Write the correct words in the questions．. 用正确的词完成以下问句。 book car coat dress house√. pen pencil shirt wathc 1 Is this your h______ 6 Is this your c______ 2 Is this your w______ 7 Is this your c______ 3 Is this your sh ______ 8 Is this your d______ 4 Is this your b______ 9 Is this your p______ 5 Is this your p______ 10 Is this your s ______ C Numbers: Write the numbers in figures．. 用阿拉伯数字表示以下数词。 three 3 ten______ one______ four______ six______ five______ eight_________ seven______ two______ nine______ 新概念英语第一册练习题（二） Lesson 2 Is this your.... 这是你的....吗？ A Structure Write questions with the words．用所给的词写出问句。 Handbag Is this your handbag？ 1 book ______ 2 car ______ 3 coat ______ 4 dress ______ 5 house ______ 6 pen ______ 7 pencil ______ 8 shirt ______ 9 skirt ______ 10 watch ______
Lesson1. 1. (b) 2. (c) 3. (b) 4. (d) 5. (c) 6. (a) 7. (d) 8. (b) 9. (a) 10. (c) 11. (c) 12. (c) Lesson2. 1. (c) 2. (d) 3. (c) 4. (c) 5. (a) 6.(b) 7. (b) 8. (a) 9. (d) 10. (c) 11. (d) 12. (b) Lesson3. 1. (c) 2. (a) 3. (c) 4. (a) 5. (d) 6. (b) 7. (c) 8. (c) 9. (b) 10. (a) 11. (b) 12. (b) Lesson4. 1. (d) 2. (b) 3. (a) 4. (b) 5. (b) 6. (a) 7. (c) 8. (b) 9. (c) 10. (a) 11. (c) 12. (c) Lesson5. 1. (c) 2. (a) 3. (d) 4. (b) 5. (c) 6. (d) 7. (a) 8. (b) 9. (c) 10. (b) 11. (a) 12. (d) Lesson6. 1. (d) 2. (a) 3. (c) 4. (d) 5. (d) 6. (a) 7. (d) 8. (a) 9. (b) 10. (a) 11. (d) 12. (a) Lesson7. 1. (b) 2. (c) 3. (c) 4. (d) 5. (a) 6. (c) 7. (d) 8. (a) 9. (c) 10. (b) 11. (a) 12. (b) Lesson8. 1. (d) 2. (b) 3. (b) 4. (a) 5. (c) 6. (c) 7. (b) 8. (b) 9. (a) 10. (d) 11. (b) 12. (b) Lesson9. 1. (b) 2. (b) 3. (d) 4. (a) 5. (a) 6. (b) 7. (b) 8. (d) 9. (b) 10. (b) 11. (d) 12. (c) Lesson10. 1. (a) 2. (d) 3. (d) 4. (c) 5. (b) 6. (c) 7. (a) 8. (c) 9. (a) 10. (c) 11. (c) 12. (a) Lesson11. 1. (b) 2. (b) 3. (b) 4. (a) 5. (b) 6. (c) 7. (c) 8. (a) 9. (c) 10. (c) 11. (b) 12. (d) Lesson12. 1. (c) 2. (c) 3. (a) 4. (d) 5. (d) 6. (a) 7. (d) 8. (a) 9. (c) 10. (d) 11. (a) 12.(a) Lesson13. 1. (b) 2. (d) 3. (b) 4. (c) 5. (a) 6. (b) 7. (b) 8. (c) 9. (a) 10. (a) 11. (a) 12. (d) Lesson14. 1. (b) 2. (c) 3. (a) 4. (c) 5. (d) 6. (b) 7. (c) 8. (b) 9. (c) 10. (b) 11. (b) 12. (b) Lesson15. 1. (d) 2. (b) 3. (c) 4. (b) 5. (c) 6. (d) 7. (a) 8. (d) 9. (c) 10. (c) 11. (c) 12. (b) Lesson16. 1. (a) 2. (a) 3. (d) 4. (a) 5. (b) 6. (a) 7. (d) 8. (a) 9. (d) 10. (d) 11. (d) 12. (a) Lesson17. 1. (d) 2. (b) 3. (b) 4. (d) 5. (c) 6. (c) 7. (b) 8. (a) 9. (a) 10. (c) 11. (a) 12. (d) Lesson18. 1. (b) 2. (d) 3. (b) 4. (d) 5. (b) 6. (c) 7. (d) 8. (c) 9. (a) 10. (c) 11. (c) 12. (b) Lesson19. 1. (a) 2. (d) 3. (c) 4. (c) 5. (d) 6. (b) 7. (c) 8. (b) 9. (c) 10. (a) 11. (c) 12. (c) Lesson20. 1. (b) 2. (c) 3. (b) 4. (b) 5. (c) 6. (b) 7. (c) 8. (a) 9. (c) 10.(c) 11. (d) 12. (a) Lesson21. 1. (c) 2. (d) 3. (c) 4. (d) 5. (a) 6. (c) 7. (b) 8. (b) 9. (a) 10. (d) 11. (c) 12. (c)
1. 被动语态（二） will/must/can/would/could/have to be done have/has been done must have been done drive 用法 home/house 2. 介词用法：见书 3. 复习 there be句型 it做为虚主语可以用来表示时间，天气，温度，距离，人或指代一个句子或不定式。 4. Summary of Unit two 5. 并列句 我们可以将几个简单句连在一起,组成并列句,常用以下连词: and, but, so, yet, or, both…and, either…or, neither…nor, not only…but …as well, not only…but also 主谓一致: 当主语由and , both…and 连接时,通常用复数谓语. Both the girls and the boy are his friends. 当主语由neither… nor, either…or, not only…but also或or 连接时,谓语动词与nor, or, but also后面的一致,在英语语法中，这被称为就
近一致. Neither he nor I am going to the airport. Not only Mary but also her parents have gone abroad. 6. 一般现在时的复习,状态动词,标点符号的使用. 以下一些动词很少用与进行时态: appear(看起来), appreciate(鉴赏), believe(相信), feel(感觉到), forget(忘记), hear(听见), know(知道), like(喜欢), look like(看起来像), notice(注意到), remember(记得), resemble(形似), see(看到), think(认为), understand(理解), have(拥有),love(爱), seem(看起来), show(显示), mind(介意), sound(听起来), hate(讨厌), detest(憎恨), desire(意欲), 引号: 引号位于一行之上,应该在句尾其他标点符号, 如逗号,句号,问好之外. 引语的第一个词以大写字母开头. 在said, asked, 等词后面用逗号,只有当它们位于句尾时才在它们后面用句号. 当said, asked等词置于引语之间时,句子的后半部分以小写开头. 当一个新的说话人开始讲话时, 要另起一个段落. 7. 复习一般过去时/不规则过去式和过去分词 常用不规则动词过去式和过去分词在英语里约有300多个 put up(搭建), put out(扑灭), put on(穿上), put sb. up(为某人安排住宿), put up with(容忍), put down(抄写), put off(推迟), put away(放好，放到一边去)
<<新概念英语1>> 1——5课 原谅我（宾格）是的 be动词现在时第三人称单数这你的，你们的 （女用）手提包原谅，请再说一遍它感谢你（们）非常地钢笔铅笔 书手表上衣，外衣连衣裙裙子衬衣小汽车房子伞 请这里我的票号码五对不起的先生衣帽存放处 一套衣服学校老师儿子女儿先生好早晨小姐新的学生法国的，法国人德国的，德国人美好的遇见日本的，日本人 韩国的，韩国人中国的，中国人也 新概念英语1 6——10课 牌号瑞典的英国的美国的意大利的沃尔沃标致梅赛德斯 丰田大宇迷你福特菲亚特我 be动词现在时第一人称单数 be动词现在时复数 名字什么国籍工作电脑键盘操作人员工程师警察女警察 出租汽车司机空中小姐邮递员护士机械师理发师家庭妇女 送牛奶的人喂（表示问候）喂，嗨怎样今天身体好美好的谢谢再见 见胖的女人瘦的高的矮的脏的干净的热的冷的
新概念英语1 11——15课 谁的蓝色的大概白色的抓住父亲母亲女衬衫姐，妹 领带兄，弟他的她的颜色绿色来楼上漂亮的，时髦的帽子相同的可爱的，秀美动人的箱子地毯狗海关官员 女孩，姑娘丹麦的，丹麦人朋友挪威的，挪威人护照棕色的旅游者 新概念英语1 16——20课 俄罗斯的，俄罗斯人荷兰的，荷兰人这些（this的复数）红色的灰色的黄色的黑色的橘黄色的雇员勤奋的推销员男人办公室助手事情 孩子们（child的复数）累，疲乏男孩渴妈妈（儿语）坐下好，可以 冰淇淋大的小的开着的关着的轻的重的长的鞋子 祖父，外祖父祖母，外祖母 新概念英语1 21——25课 给一个哪一个空的满的大的小的尖的，锋利的小的 大的钝的盒子，箱子杯子茶杯瓶子罐头刀子叉子勺子在……之上架子，搁板课桌桌子盘子食橱香烟电视机地板 梳妆台杂志床报纸立体声音响夫人厨房电冰箱右边 带电的，可通电的左边炉子，炊具中间（属于）……的房间杯子
学霸英语新概念第三册语法详解和总结（带习题和答案） 第一章英语从句 Subordination 英语从句主要有定语从句，状语从句和名词性从句（主语从句，宾语从句，表语从句，同位语从句） 一．定语从句 定语从句：由关系代词who, whom, whose, that, which; 关系副词when, where, why 引导。 （下面十个句子请读5遍并脱口译出！） 1. The death notices tell us about people who have died during the week. 2. The man (whom) you spoke to just now is my friend. 3. The building whose lights are on is beautiful. 4. Please find a place which we can have a private talk in. 5. The knee is the joint where the thighbone meets the large bone of the lower leg. 6. He still remembers the day when he went to school. 7. It is no need telling us the reason why you didn't finish it in time. 8. He has three sons, two of whom died in the war. 9. Mr. Smith, whose wife is a clerk, teaches us English. 10. In the Sunday paper there are comics, which children enjoy. 只能用that和who引导的定语从句 A．all, nothing, anything, a few, one做先行词指物时 B．先行词前有形容词最高级修饰时，后面常跟that而不是which. C．先行词前有the only, the first, the last, the next, the very等词修饰时，引导词只能用that。 D．当先行词是anyone, anybody, everyone, everybody, someone, somebody时，后面要用who或whom； ●All that glitters is not gold. 闪光的并非都是金子。 as 引导定语从句 as 引导的定语从句有两种形式： A．引导限制性语从句。 在此类定语从句中，as常与主语中作为其先行词的such, the same或as联用构成，“such... as”，“the same... as”和“as ...as”句型，可代替先行词。 例如：We hope to get such a tool as he is using. 我们希望得到他正在用的那种工具。 B．as 引导非限制性定语从句时，作用与which相同，as作为关系代词代替整个主句。 （这是语法考试的一个考点。） ［注意区别］： as 引导的从句用于句首、句中或句后，而which引导的定语从句不能放在句首。 例如：As is reported, a foreign delegation will visit the city. 据报道，一个外国代表团将访问这个城市。 二．状语从句 超级作文联接词及词组，全部拿下！ ①原因：because, since, now that（既然）as, for, this reason.... ②结果：so that, so, therefore, consequently, so as to, as a result .... ③时间：after, before, when, while, as, until, as soon as, since, by the time, once, lately, presently, shortly after, currently, at present, nowadays ... ④条件：if, only if., once, unless, in the event (that), in case (that), provided that, on the condition that, etc.