Germplasm
Twenty-two accessions of Hordeum vulgare ssp spontaneum (ssp
spontaneum) thirty-two accessions of Hordeum vulgare ssp vulgare
(ssp vulgare) representing mapping population parents and germplasm
of interest to the Oregon State University breeding program,
and ninety-six elite lines and varieties from the Busch Agricultural
Resources, Inc. (BARI) barley improvement program were selected
for this study. The ssp spontaneum accessions represent different
areas of geographic origin. The genotypes in the ssp vulgare
group represent mapping population parents and lines of interest
to our breeding program and vary in terms of growth habit,
end-use, row type, and geographic origin. The BARI lines included
86 six-row and 10 two-row spring malting barley genotypes.
Of the BARI six-rows, 82 are elite breeding lines and 4 are
parents and/or malting quality standards. Of the BARI two-rows,
7 are elite breeding lines and 2 are parents and/or malting
quality standards.
SSR genotyping
Fifty-five SSRs of know map location, and distributed throughout
the seven linkage groups, were assayed on the 147 accessions
using fluorescent-tagged primers. Forty-seven of the SSR primer
sets were developed and mapped by Ramsay et al. (2000), six
by Liu et al. (1996) and two by Becker and Heun (1995). The
reverse primers were labeled with FAM, TET, NED or HEX fluorescent
dye. DNA amplifications were performed using either an MJ Research
PTC-100 or MWG Biotech Primus 96 Plus thermal cycler. PCR reactions
were carried out in a 10 µl reaction mix containing 37.5
ng of template DNA, 1x PCR buffer, 0.025 units Taq DNA polymerase,
(Qiagen), 0.2 nM dNTPs and 0.1 picomoles of forward and reverse
primers. Information on primer sequences, and PCR amplification
conditions for each set of primers, are available at http://www.scri.sari.ac.uk/ssr
(Ramsay et al. 2000), in Liu et al. (1996) and in Becker and
Heun (1995).
PCR amplified fragments from differentially labeled SSR primers
and with nonoverlaping fragment sizes were simultaneously analyzed
in the same gel lane and separated on an ABI Prism 377 DNA
Sequencer at the Oregon State University (Central Service Lab)
or on an ABI Prism 3700 DNA Sequencer at OMIC, Inc., Portland,
OR. Gene Scan® and Genotyper® Software (Applied Biosystems,
Perkin Elmer, Forster City, CA) were used for automated data
collection and to determine the allele sizes in base pairs,
based on the internal standard.
The ABI Prism systems give allele sizes in numbers of base
pairs (bp) with to two decimal places. We rounded fractional
numbers to the nearest integer. Alleles with fractional values >0.51
were rounded upward, and alleles with fractional values <0.50
were rounded downward. Thus, for a given locus, alleles differing
by one bp were declared different.
Only the primer pairs yielding a single amplicon were used
for the subsequent analyses. This led to different numbers
of SSRs scored in each of the three sets of germplasm. Fifty-three
of the SSRs were single copy in the ssp spontaneum and ssp
vulgare mapping population parent accessions and two were multicopy;
accordingly 53 SSRs were assayed in these two sets of germplasm.
Of the 55 SSRs, four were multicopy in the BARI germplasm.
For seven of the primer sets, no amplification products were
obtained after two attempts. Accordingly, 44 SSRs were assayed
in the BARI germplasm. Forty-two of the 44 SSRs assayed in
the BARI germplasm were also scored in the ssp spontaneum and
ssp vulgare sets and these 42 SSRs, common to the three groups
of germplasm, were used for a diversity analyses, which has
been submitted for publication.
Eighteen of the 42 SSRs analyses were assayed on ssp spontaneum,
ssp. vulgare, and BARI accessions with the ABI3700. For the
remaining 24 SSRs, allele sizes for the BARI lines were assayed
on the ABI 3700 and the allele sizes for the ssp spontaneum/ssp
vulgare germplasm arrays were assayed on the ABI 377. In order
to integrate ABI Prism 377 and ABI Prism 3700 data, the allele
size of cultivar Morex was used as a reference. This cultivar
was run for every SSR on both platforms. SSR allele sizes for
each BARI accession obtained from the ABI Prism 3700 were adjusted
to ABI Prism 377 allele sizes based on the number of bp differences
observed for Morex. Morex alleles for the same SSR locus were
consistently two or three bp pair smaller on the ABI Prism
3700. On either platform, however, repeated assessment of the
same SSR gave highly reproducible allele sizes.
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