作物育种学_孙其信_分子标记

SCAR sequence characterized amplified region.
Target DNA fragment cloning and sequencing
Design primers about 18~24bp according to the sequence of two flank regions
提莫菲维小麦G祖染色体特异性RAPD 标记和SCAR标记
PCR-based markers
AFLPs: amplified fragment length polymorphisms A combination of PCR and RFLP Informative fingerprints of amplified fragments
Requires: - target DNA - thermostable DNA polymerase (Taq) - oligonucleotide primer(s) (7-30nt) - dNTPs + Mg++
RAPD-PCR Randomly amplified polymorphic PCR
分
子
标 记
Sequencing based markers新型的分子标记 （SNP标记、EST标记 ）
2）原理
DNA-based markers
A
B
C
D
E
F
RFLPs: origins of polymorphisms
A = base pattern B = insertion C = deletion D = new restriction site within probe sequence E = loss of restriction site
Requires no probe ,cloning or sequencing, Is PCR-based and may detect several loci simultaneously. Short (about 10 base pair) random primer sequences, and generally results in a presence/absence polymorphism. Small material ,easy, inexpensive and fast,
Single base mutation – Insertion / deletion
– Rearrangement
Advantages of Molecular Marker
1) Available in very large numbers 2) Not affected by the environment 3) Can be scored at any stage 4) No interlocus interactions
decr. length
probe site
F = new restriction site outside probe region
RE
RE
3）程序
二、分子标记的原理和遗传特性
(一)RFLP标记
1.RFLP标记的原理 植物基因组DNA上的 碱基替换、插入、缺失 或重复等，造成某种限 制性内切酶酶切位点的 增加或丧失，从而产生 限制性片断长度多态 性。
DNA提取 基 用DNA限制性内切酶消化 本
凝胶电泳分离，转移到滤膜上
布 Southern杂交 骤 放射性自显影或酶学检测
2.RFLP标记的特点 （1）遍布于整个基因组， 数量 几乎是无限的； （2）无表型效应， 不受发育阶段器官特异性限制； （3）共显性， 可区分纯合子和 杂合子； （4）结果稳定、可靠； （5）DNA需要量大，检测技术繁杂， 难以用于大规模的育种实践中
RAPD-PCR Randomly amplified polymorphic PCR
The short primers, however, may be easily affected by annealing conditions and results may not always be consistently reproducible. This marker technology is not appropriate for use in comparative mapping.
- a readily detectable sequence of DNA or protein whose inheritance can be monitored
To be useful molecular markers must possess certain characteristics: polymorphic reproducible preferably display co-dominant inheritance (both forms detectable in heterozygotes) fast and inexpensive to detect
分子标记的特点： （1）直接以DNA的形式表现，表现稳定 （2）数量多 （3）多态性高 （4）表现为中性，不影响目标性状的表达； （5）许多标记表现为共显性的特点，能区别 纯合体和杂合体。 （6）成本不太高
Molecular Marker
Based on variation in the DNA sequence
To use DNA you need a roadmap
Chapter 17 molecular marker assisted selection breeding
1 Introduction
DNA
RNA Protein Phenotype
表型
2 Molecular Markers
What are they?
It is useful for phylogenetic studies (because a single RAPD primer may detect more than one loci).
PCR-based markers
RAPDs:
random amplified polymorphic DNA markers
PCR-based markers
RAPDs
Issues:
Some reproducibility problems
Same band on gel = same DNA fragment? One band on gel = one DNA fragment?
PCR-based markers
Sequence-tagged sites as markers
Microsatellite-based markers (SSR: short sequence repeats) SCARs: sequence-characterized amplified regions CAPS: cleaved amplified polymorphic sequences
PCR amplification ，detect the polymorphism
More robust and not as random as RAPDs
PCR-based markers
SCARs
Informative RAPD-generated fragment is sequenced - no longer anonymous Sequence-specific primers (22-30 nt) designed to target this locus More reproducible (long specific primers) Now a co-dominant marker; detectable in both homozygotes and heterozygotes
PCR-based markers
AFLPs
1. Digest genomic DNA with restriction enzymes 2. Ligate commercial adaptors (defined sequences) to both ends of the fragments 3. Carry out PCR on the adaptor-ligated mixture, using primers that target the adaptor, but that vary in the base(s) at the 3’ end of the primer
AFLP Amplified Fragment length Polymorhisms
PCR-based marker system involving amplification of DNA after cleavage with restriction enzymes. Requires no cloning or sequencing. It operates on much the same principle as a RAPD, but the primer consists of a longer (~ 15 bp) fixed portion and a short (2-4 bp) random portion. The long fixed portion gives the primer stability and the short random portion means it will amplify many loci - one may get over 100 loci amplified with a single AFLP primer. Polymorphism is detected as the presence/absence of a band. The amplification is not completely random (as in the case of RAPDs) nor is it based on a known host DNA sequence (as in the case of SSRs). Rather, it is based on selective amplification of subsets of tagged fragments.