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Analysis of Polymorphism from Mitochondrial, Genomic DNA Using Amplified Fragment Length Polymorphism (AFLP) Marker Technique in Hybrid Wheat

作　者: Muhammad Ejaz Khan
导　师: Zhang Gaisheng
学　校: 西北农林科技大学
专　业: 作物遗传育种
关键词: AFLP marker Triticum aestivum Hybrid mtDNA Genetic diversity
分类号: S512.1
类　型: 博士论文
年　份: 2013年
下　载: 15次
引　用: 0次
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内容摘要

The amplified fragment length polymorphism (AFLP) technique is one of absolute informative and cost-effective fingerprinting procedures. It creates polymerase chain reaction (PCR) based multi-locus genotypes supportive in many regions of population genetics.Comparison of two mtDNA extraction methods from yellow etiolated shoots of wheat crop(Triticum aestivum), the isolation of mtDNA is difficult because presence of genomic DNA, RNA and metabolites that interfere with DNA isolation procedures and subsequent applications, such as DNA restriction, amplification and cloning. The method (with alteration) involves inactivation of genomic DNA by DNase I enzyme, RNA by RNase enzyme, contaminant proteins by using Proteinase K and precipitation of polysaccharides in the presence of high salt concentration. The purity of mtDNA was confirmed by PCR amplification of genomic, mitochondrial and chloroplast gene, A260/A280and A260/A230ratios calculated from the Spectrophotometric readings.Produce a good F1hybrid variety in wheat crop; it is necessary to explore a new male-sterile cytoplasm and their maintainer lines. The study was conducted to identify cytoplasmic variation in isonuclear, alloplasmic three male sterile lines Aegilops kotschyi (Ae.kots)-90-110, Aegilops ventricosa (Ae.ven)-90-110, Tirticum sepelta-90-110and their maintainer Line Tirticum asstivum (A)-90-110at molecular level. Comparative analysis of mitochondrial DNA was conducted by using amplified fragment length polymorphism (AFLP) marker.Amplified fragment length polymorphism (AFLP) marker was used to assess genetic diversity in male sterile lines (Hetero-cytoplasm with same nucleus) with esteem of their restorer line in wheat crop. These male sterile lines were judged with thirteen AFLP primer combinations that generated a total of682fragments and113polymorphic fragments. The values of polymorphic information content (PIC), and marker index (MI) demonstrated the utility of the primer combinations used in the present study. UPGMA and Principal coordinate analysis (PCoA) of the genotypic data revealed clustering of accessions as per genetic relationships. The Jaccard’s similarity coefficient values suggested good variability among the male sterile lines indicating their utility in breeding programs. The fallouts of AMOVA divulged large within-group population variations which accounted for77%of the total molecular variance. The among-group population variation, although accounting for23%of the total variation, was statistically significant. Molecular diversity presented in this study will be highly useful for selecting appropriate accessions for plant improvement and hybridization through molecular breeding approaches for evolving suitable conservation strategies.Investigate the behavior of male sterile lines with their maintainer lines and polymorphism application of amplified fragment length polymorphism (AFLP) markers divided into two groups. Out of sixty-four primer pairs five were produced healthy polymorphism. Group I produced27and Group II produced24polymorphic bands. As a whole maximum number of polymorphic bands were produced by the line number2(18) fallowed by line number7(15). In five primer pairs highest number of polymorphic bands was produced by primer pair E1/M4(12) and least by primer pair E1/M2(10). DNA fingerprinting, characterization of germplasm, cytogenetics research and special emphasis has been laid on the genome of bread wheat, highly useful for selecting appropriate accessions for plant improvement and hybridization through molecular breeding approaches for evolving suitable safeguard strategies.

全文目录

ABSTRACT  5-10
1. INTRODUCTION  10-20
  1.1 Hybrid Wheat  10-11
  1.2 Cytoplasmic male sterility (CMS)  11-12
  1.3 PCR-based molecular markers  12
  1.4 Random amplified polymorphic DNA (RAPD)  12-13
  1.5 DNA amplification fingerprinting (DAF)  13
  1.6 Sequence-tagged site (STS)  13-14
  1.7 Directed search (amplification of low copy DNA)  14
  1.8 Sequence characterized amplified regions (SCARs)  14
  1.9 Amplified fragment length polymorphisms (AFLPs)  14-15
  1.10 Simple sequence repeats (SSRs) or microsatellite  15-16
  1.11 Sequencing and DNA chip based markers  16
  1.12 Comparison of different types of DNA markers  16-17
  1.13 Applications of molecular markers  17-18
  1.14 Fingerprinting and variety identification  18
  1.15 Genetic diversity studies  18-19
  1.16 Genetic loyalty and germplasm characterization  19-20
2. COMPARISON OF SMALL SCALE METHODS FOR THE RAPID ANDEFFICIENT EXTRACTION OF MITOCHONDRIAL DNA FROM WHEAT CROPSUITABLE FOR DOWN-STREAM PROCESS  20-29
  2.1 Introduction  20
  2.2 Material and Method  20-24
    2.2.1 Plant Material  20-21
    2.2.2 Mitochondrial DNA isolation  21
    2.2.3 Method-Ⅰ  21-22
    2.2.4 Method-Ⅱ  22-23
    2.2.5 Agarose gel electrophoreses  23
    2.2.6 DNA quality assessment  23
    2.2.7 PCR amplification  23
    2.2.8 AFLP Marker Analyzes  23-24
  2.3 Results and Discussion  24-29
3. ANALYSIS OF MITOCHONDRIAL DNA USING AFLP MARKER, FROM MALESTERILE AND THEIR MAINTAINER LINES WITH ISONUCLEUS,ALLOCYTOPLASM IN WHEAT CROP  29-36
  3.1 Introduction  29-30
  3.2 Material and method  30-32
    3.2.1 DNA isolation  30
    3.2.2 Amplified fragment length polymorphisms (AFLP) analysis  30-31
    3.2.3 Mitochondrial DNA purity identification  31-32
  3.3 Results  32-34
  3.4 Discussion  34-36
4. ANALYSIS OF GENETIC DIVERSITY IDENTIFIED BY AMPLIFIEDFRAGMENT LENGTH POLYMORPHISM MARKER IN HYBRID WHEAT  36-48
  4.1 Introduction  36-37
  4.2 Material and Method  37-41
    4.2.1 DNA isolation  38
    4.2.2 Amplified Fragment Length Polymorphism (AFLP) Marker  38-40
    4.2.3 Statistical analysis  40-41
    4.2.4 AMOVA analysis  41
  4.3 Results  41-44
  4.4 Discussion  44-48
5. SCRUTINIZE THE BEHAVIOR OF MALE STERILE LINES WITH THEIRMAINTAINER LINE AND POLYMORPHISM USING AFLP MARKER IN HYBRIDWHEAT  48-57
  5.1 Introduction  48-49
  5.2 Material and method  49-51
    5.2.1 Plant material  49
    5.2.2 Isolation of genomic DNA  49-50
    5.2.3 Amplified fragment length polymorphism analysis marker (AFLP) analysis  50
    5.2.4 Digestion and ligation  50
    5.2.5 Non selective amplification  50
    5.2.6 Selective amplification  50-51
    5.2.7 Analysis of the selective PCR product  51
  5.3 Results  51-55
  5.4 Discussion  55-57
6. CONCLUSIONS  57-58
7. REFERENCES  58-71
8. ACKNOWLEDGEMENTS  71-72
9. Author brief introduction  72

Analysis of Polymorphism from Mitochondrial, Genomic DNA Using Amplified Fragment Length Polymorphism (AFLP) Marker Technique in Hybrid Wheat

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