Different DNA analysis techniques are availablefor genome analysis in cereals, such as restrictionfragment length polymorphisms (RFLP), random-amplified polymorphic DNA (RAPD), simplesequence repeats (SSR), expressed sequence tags(EST), sequence characterized amplified regions(SCAR) and amplified fragment length polymor-phisms (AFLP). Simple sequence repeats, alsoknown as microsatellites, are tandem repeat motifscomposed of one to six nucleotides, which areubiquitous,abundantandhighlypolymorphicinalleukaryotic genomes (Tautz and Renz 1984). SSRsin particular have been reported to be useful toanalyze the structure of germplasm collections, be-cause they are codominant, multiallelic and chro-mosomespecific(Huangetal.2002).SSRsgenerallydetect high levels of polymorphism (Ro ¨der et al.1998), can detect heterogeneity and heterozygotes,andareamenabletoautomatedanalysis.Therefore,SSR primers were used in this study to investigatethe genetic diversity of emmer wheat collected fromeleven different geographical regions.
Materials and methods
Plant materials
A total of 73 accessions of
Triticum dicoccon
Schrank germplasm held at the institute of Plant Genetics and Crop Plant Research (IPK),Gatersleben, Germany were used. The accessionswere collected from 11 different countries of NearEast, Middle East, Central Asia, Europe andNorth Africa. Besides, the hexaploid
T. aestivum
varieties ‘Chinese Spring’ and ‘Aztec’ were in-cluded as standards, Lists of the accession andcountry of origin are presented in Table 1.
DNA extraction and PCR amplification
For each accession, nine kernels were pooled andDNA was isolated following the procedure in Ful-ton et al. (1995) with extraction buffer described inPlaschke et al. (1995). PCR amplifications wereperformed as described by Ro ¨der et al. (1998;unpublished). PCR reaction contained 50–100 ngtemplate DNA, 250 nM cy5-labelled forward pri-mer, 250 nM unlabelled reverse primer, 0.2 mMdNTPs, 2.5
l
L PCR buffer (10
·
), 1.5 mM MgCl
2
,1 U
Taq
DNA polymerase in a total volume of 25
l
L, Fragment detection was performed accord-ing to Ro ¨der et al. 1998). Fragments were detectedby an Automated Laser Fluorescence (ALF) se-quencer (Amersham Biosciences) and fragment si-zes were calculated using the computer programFragment Analyser 1.02 (Amersham Biosciences)by comparison with internal size standards. In thecase of weak or no fragment products, PCRamplifications were repeated to exclude failed PCRreaction as the cause of the null allele.
Microsatellite loci
The microsatellites used for analysis are listed inTable 2. Twenty-eight Gatersleben Wheat Micro-satellites (GWM) and one microsatellite from apseudogliadine gene, Taglgap, representing two
Table 1.
Country of origin, sample size (
n
), and IPK accession numbers of plant materials used in the study.Country
n
IPK Accession NumbersArmenia 9 Tri 9542, Tri 9543, Tri 11167, Tri 16207, Tri 17698, Tri 18039, Tri 18041, Tri 18042, and Tri 18043Azerbaijan 7 Tri 18092, Tri 18093, Tri 18094, Tri 18095, Tri 18097, Tri 18098, and Tri 18099Georgia 6 Tri 13158, Tri 16608, Tri 16609, Tri 18193, Tri 18197, and Tri 18206Iran 11 Tri 6141, Tri 6158, Tri 7305, Tri 18080, Tri 18081, Tri 18082, Tri 5861, Tri 18084, Tri 18085,Tri 18087, and Tri 18089Israel 4 Tri 3424, Tri 16877, Tri 16879, and Tri 16880Italy 6 Tri 16723, Tri 16726, Tri 16798, Tri 16803, Tri 16807, and Tri 18243Morocco 6 Tri 2884, Tri 4313, Tri 17429, Tri 18167, Tri 18168, and Tri 18169Slovakia 9 Tri 9867, Tri 9868, Tri 10063, Tri 10064, Tri 10066, Tri 10069, Tri 10070, Tri 10071, and Tri 10317Spain 6 Tri 16885, Tri 18114, Tri 18115, Tri 18156, Tri 18161, and Tri 18164Turkey 5 Tri 584, Tri 17023, Tri 17038, Tri 17040, and Tri 17058Yemen 4 Tri 28004, Tri 28027, Tri 28072, and Tri 18073Total 73
545
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