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Plant materials and DNA extraction
A total of 62 traditional Ethiopian highland maize accessions were used for this study (Table 4.1). Previously, a representative subset of 180 of the 287 maize accessions collected from different highland regions in Ethiopia was analyzed for 15 morphological and agronomic traits (Chapter 3). Principal component and cluster analyses grouped these 180 accessions into four main clusters. The 62 accessions were chosen from the four clusters to represent the different agroecologies of Ethiopia and the range of morphological and agronomic variation observed in the field. For each of the 62 accessions, genomic DNA was extracted from leaf discs, harvested from 15 three-week old plants (one 10-mm leaf disc per plant). For two accessions, individual DNA samples were also isolated from the 15 plants used for bulked sampling. DNA was extracted using the QIAGEN DNeasy plant Mini Kit, (QIAGEN, GmbH, Hilden) and homogenization was performed using the FP-120 FastPrep instrument (QBiogene, Carlsbad, CA, USA; Myburg et al., 2001). DNA quantity and quality was determined on 0.8% (w/v) agarose gel electrophoresis using known quantities of lambda DNA as a concentration standard.
AFLP analysis
AFLP template preparation was performed using AFLP template preparation kits from LI-COR Biosciences (LI-COR, Lincoln, NE, USA) according to the manufacturers’ instructions, except that 10 μl diluted R/L mix, 2.0 mM MgCl2 and 1.0 U Taq polymerase were used in the preamplification step. Polymerase chain reactions (PCRs) were performed using a BIO-RAD iCycler (Version 3.021, BIO-RAD Laboratories, Inc.). The preselective amplification cycle profile was as follows:
incubation for 10 s at 72°C, followed by 30 cycles of denaturation for 10 s at 94°C, annealing for 30 s at 56°C, and extension for 1 min at 72°C with a 1 s per cycle increasing extension time. Selective amplification was performed on 1:20 diluted (in SABAX water) preselective amplification products with the following cycling profile:
13 cycles of 2 min at 94°C, 30 s at 65°C (reduced by 0.7°C per cycle), and 1 min at 72°C; followed by 20 cycles of 10 s at 94°C, 30 s at 56°C, and 1 min (extended 1 s per cycle) at 72°C. The preselective and selective amplification primer pairs all had two and three-nucleotide extensions at the 3’ end, respectively. In all reactions only the EcoRI primers were 5’labelled with infrared dyes (IRDye 700 or IRDye 800, LICOR). Initially, eight accessions were chosen to test the amplification successes of different primer combinations. The polymorphism rates and the total number of scorable fragments were evaluated in these eight accessions with 32 primer combinations. Eight primer combinations (Table 4.3) with the highest polymorphism rates and large numbers of clearly scorable fragments were selected to analyze the full set of 62 accessions.
Gel electrophoresis and scoring
An equal volume of loading solution (LI-COR) was added to each selective amplification reaction. Samples were denatured at 95°C for 3 min and placed on ice for 10 min before loading. A volume of 0.8 μl was loaded with an 8-channel syringe (Hamilton, Reno, Nevada) onto 25-cm 8% Long Ranger gels (BMA, Rockland, ME, USA). Electrophoresis and detection of AFLP fragments were performed on LI-COR IR2 (model 4200S) automated DNA analyzers. The electrophoresis parameters were set to 1500V, 40 mA, 40 W, 50°C, and a scan speed of 3. The run-time was set to 4 h and gel images were saved as TIF files for further analysis. The gel images were scored using a binary scoring system that recorded the presence and absence of bands as 1 and 0, respectively. Semi-automated scoring was performed with SAGAMX (Version 3.2, LI-COR) and followed by manual editing to make adjustments to the automated score where necessary. A locus was scored as polymorphic when the frequency of the most common allele (band present or absent) was less than 0.97 (absent or present in at least two individuals). Bands with the same mobility were considered as identical products (Waugh et al., 1997), receiving equal values regardless of their fluorescence intensity.
CHAPTER 1
GENERAL INTRODUCTION
1.1 Maize breeding in Ethiopia: Historical overview
1.2 Importance of maize in the highlands of Ethiopia
1.3 Objectives and outline of the study
CHAPTER 2
LITERATURE REVIEW
2.1 Maize is an important crop for genetic analysis
2.2 Methods for assessing genetic variation
2.3 Use of pooled DNA samples in the study of genetic variation
2.4 Correlation between phenotypic and molecular markers distance
2.5 Statistical measures for assessing genetic diversity
2.6 Gene mapping/tagging
2.7 Marker-assisted selection and breeding
2.8 Conclusions
CHAPTER 3
PHENOTYPIC DIVERSITY FOR MORPHOLOGICAL AND AGRONOMIC TRAITS IN TRADITIONAL ETHIOPIAN HIGHLAND MAIZE ACCESSIONS
3.1 ABSTRACT
3.2 INTRODUCTION
3.3 MATERIALS AND METHODS
3.4 RESULTS
3.5 DISCUSSION
CHAPTER 4
BULKED-AFLP ANALYSIS OF GENETIC DIVERSITY AMONG TRADITIONAL ETHIOPIAN HIGHLAND MAIZE ACCESSIONS
4.1 ABSTRACT
4.2 INTRODUCTION
4.3 MATERIALS AND METHODS
4.4 RESULTS
4.5 DISCUSSION
CHAPTER 5
ANALYSIS OF GENETIC DIVERSITY OF ETHIOPIAN HIGHLAND MAIZE ACCESSIONS USING SSR MARKERS
5.1 ABSTRACT
5.2 INTRODUCTION
5.3 MATERIALS AND METHODS
5.4 RESULTS
5.5 DISCUSSION
CHAPTER 6
A COMPARATIVE STUDY OF MOLECULAR AND MORPHOLOGICAL METHODS OF DESCRIBING GENETIC RELATIONSHIPS IN MAIZE
6.1 ABSTRACT
6.2 INTRODUCTION
6.3 MATERIALS AND METHODS
6.4 RESULTS
6.5 DISCUSSION
CHAPTER 7
ASSOCIATION OF SIMPLE SEQUENCE REPEATS WITH QUANTITATIVE TRAITS IN ETHIOPIAN HIGHLAND MAIZE ACCESSIONS AND THE EFFECT OF ADMIXTURE
7.1 ABSTRACT
7.2 INTRODUCTION
7.3 MATERIALS AND METHODS
7.4 RESULTS
7.6 DISCUSSION
CHAPTER 8
CONCLUSIONS AND RECOMMENDATIONS
Use of bulked leaf samples for genetic diversity studies
REFRENCES
