Analysis of genetic diversity identified by amplified fragment length polymorphism marker in hybrid wheat.

Amplified fragment length polymorphism markers were used to assess genetic diversity in 10 male sterile wheat crop lines (hetero-cytoplasm with the same nucleus) in relation to a restorer wheat line. These male sterile lines were evaluated using 64 amplified fragment length polymorphism primer combinations, and 13 primers produced polymorphic bands, generating a total 682 fragments. Of the 682 fragments, 113 were polymorphic. The polymorphic information content and marker index values demonstrated the utility of the primer combinations used in the present study. Unweighted pair group method with arithmetic mean and principal coordinate analysis of the genotypic data revealed clustering of accessions based on genetic relationships, and accessions were separated into 2 groups with their restorer line. Jaccard's similarity coefficient values suggested good variability among the male sterile lines, indicating their utility in breeding programs. The fallouts of analysis of molecular variance showed large within-group population variation, accounting for 77% of variation, while among-group comparison accounted for 23% of the total molecular variation, which was statistically significant. The molecular diversity observed in this study will be useful for selecting appropriate accessions for plant improvement and hybridization through molecular-breeding approaches and for developing suitable conservation strategies.

Examination of polymorphism in hybrid wheat by using amplified fragment length polymorphism marker.

To investigate the polymorphism of two groups of male sterile lines and their maintainer lines, we used amplified fragment length polymorphism markers. Of the 64 primer pairs, five reproducibly demonstrated polymorphism. Group I had 27 polymorphic bands, and Group II had 24 polymorphic bands. The maximum number of polymorphic bands was produced by line 2 (18 polymorphic bands) followed by line 7 (15 polymorphic bands). Of the five primer pairs, the highest number of polymorphic bands was produced by primer pair E1/M4, and the smallest number of polymorphic bands was produced by primer pair E1/M2. DNA fingerprinting, germplasm characterization, and cytogenetic research have especially been emphasized in studies of bread wheat; these methods are highly useful to select appropriate accessions for plant improvement and hybridization through molecular breeding approaches to evolve suitable safeguard strategies.

Comparison of small scale methods for the rapid and efficient extraction of mitochondrial DNA from wheat crop suitable for down-stream processes.

We evaluated and compared 2 mitochondrial DNA (mtDNA) extraction methods in terms of DNA quality and success of subsequent polymerase chain reaction (PCR) amplifications from yellow etiolated shoots of wheat crop (Triticum aestivum). mtDNA ex-traction is difficult because the presence of metabolites interfere with DNA isolation procedures and downstream applications such as DNA restriction, amplification, and cloning. The method (with modification) involved 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 a high salt concentration. The DNase I and RNA enzyme ratio was adjusted to 10:8 mL. The purity of mtDNA was confirmed by PCR amplification of genomic, mitochondrial, and chloroplast (rbcL) gene. The mitochondrial COXIII gene of 400 bp was amplified; the b-actin and chloroplast genes were not amplified. A260/A280 (1.89) and A260/A230 (2.07) ratios were calculated using a spectrophotometer. The isolated mtDNA was amenable to amplification and restriction digestion. The technique is fast, reproducible, and suitable for PCR-based markers.

Analysis of mitochondrial DNA using amplified fragment length polymorphism markers of isonuclear allocytoplasmic male sterile wheat accessions and their maintainer lines.

To produce a good F1 hybrid variety wheat crop, it is necessary to explore novel cytoplasmic male sterility (CMS) lines and their maintainer line. This study aimed to identify cytoplasmic variation in three isonuclear-alloplasmic male sterile lines Aegilops kotschyi (Ae.kots) -90-110, Aegilops ventricosa (Ae.ven) -90-110, and Triticum spelta (T.spelta) -90-110 and their maintainer line, A-90-110, at the molecular level. Mitochondrial DNA (mtDNA) was isolated using a combination of centrifugation and density gradient ultracentrifugation, sucrose sedimentation, lysis with sodium dodecyl sulfate (SDS), potassium proteinase, and phenol/chloroform extraction methods. To detect mtDNA purity, specific primers were designed for nuclear (ß-actin) and mitochondrial (COXIII) genes. Results indicated that the mtDNA was pure, and therefore suitable for polymerase chain reaction (PCR) and genetic analysis. Comparative analysis of mtDNA was conducted using amplified fragment length polymorphism (AFLP) markers. Reproducible polymorphisms were detected between the Aegilops and Triticum species and the male sterile lines. Four specific primers were screened from 64 AFLP marker primers, which provided the molecular basis for further studies investigating specific cytoplasmic male sterility characteristics.