**SSR Linkages to Eight Additional Morphological Marker Traits**

Lynn S. Dahleen and Jerome D. Franckowiak

USDA-Agricultural Research Service, PO Box 5677, SU Station, Fargo, ND
58105, and the Dept. Plant Sciences, North Dakota State Univ., Fargo, ND
58105

**Introduction**

More than 1000 morphological markers have been identified in barley
(Franckowiak and Lundqvist 2002). These phenotypic traits were observed
as spontaneous or artificially generated mutants in a wide range of
cultivars over decades of research. Traits identified with alternate
alleles include plant height, spike morphology, and seed size, among
others. Approximately 275 of these markers have been placed on the
morphological marker linkage map of the seven barley chromosomes
(Franckowiak unpublished 2002) and/or on the consensus molecular marker
linkage map (Kleinhofs 2002); approximately 150 to 200 additional
markers have been placed on other maps but the rest have not been
mapped.

Simple sequence repeat (SSR) markers are PCR-amplified regions of two or
three base DNA repeats. Primers were designed to anneal to DNA on either
side of each repeated segment, so size differences in amplification
products are caused by different numbers of repeats in different
genotypes. PCR reactions are easy to set up, do not use any hazardous
chemicals, and only take a few hours for amplification. Products can be
separated on agarose or acrylamide gels and results can be obtained in
one day, which make these molecular markers ideal for short-term
projects. We have worked with high school students in the North Dakota
Governor's School to map morphological traits over the last several
summers. This report describes the location of eight additional
morphological markers through linkage to SSR markers.

**Materials and Methods**

*Mapping population development* A set of mutant lines were selected
from various barley collections based on phenotype and backcrossed four
to six times to 'Bowman' (Table 1). Homozygous backcrossed lines,
BC~n~F~2~ populations, and BC~n~F~2~-derived F~3~ lines were developed
for mapping with molecular markers. Fifty BC~n~F~2~ seeds were sown for
each mutant line in a greenhouse, along with the homozygous mutant
parent and Bowman. The greenhouse was maintained at 21-26^o^C with a 16
h day/8 h night cycle supplied by sodium halide lights. Each plant was
scored as either normal or mutant. Plants were grown to maturity and
harvested. Twelve BC~n~F~3~ seed were sown for each BC~n~F~2~ plant to
identify heterozygous lines. Parent and BC~n~F~2~ seed were sown one per
15 cm clay pot, in a soil-less potting mix supplemented with a slow
release fertilizer (14-14-14). The BC~n~F~3~ seed were sown with six
seed per 15 cm clay pot and scored for the mutant trait at the
appropriate time. Plants were treated with Marathon (Imidaclorprid)
systemic insecticide at approximately the 2-3 leaf stage.

*Trait Analysis* Backcross-derived near-isogenic lines (NILs) and Bowman
were planted in a field near Christchurch (Leeston), New Zealand and
near Aberdeen, ID in 2002 and 2003 for agronomic analysis. Height (cm)
and lodging (1-9 scale) were measured at Christchurch only in both years
at the hard dough stage. Peduncle (cm) and awn length (cm), and the
number of kernels per spike were measured in all four environments. Leaf
length (cm) and width (mm) were measured on the penultimate leaf blade
in all environments except 2002 Christchurch. Rachis internode length
(mm) was measured in all environments except 2003 Aberdeen. Kernel
weight (mg per kernel based on 100 kernels) was measured in all
environments except 2002 Aberdeen. Yield (g/m) was measured at
Christchurch in 2002 and Aberdeen in 2003. Heading date was measured at
Aberdeen in 2002. Lines also were grown in Dundee, Scotland in 2005,
measuring heading date, height, peduncle and awn length, rachis
internode length and number of kernels per spike. Plots were sown in an
augmented block design with Bowman repeated across blocks. Each plot was
a 2-m row with rows spaced 60 cm apart. The seedling rate was
approximately 20 seeds per meter of row. Data were analyzed by Analysis
of Variance using SAS (SAS Institute Inc. 2002) and least squares means
were compared using the General Linear Model procedure. Trait means of
the near-isogenic lines were compared to Bowman means using Least
Significant Difference values.

*Molecular marker testing* Leaf tissue was harvested from young parent
and BC~n~F~2~ plants grown in the greenhouse and the DNA extracted using
the method of Dahleen et al. (2003). The DNA was then resuspended in 200
μL of modified TE (10 mM Tris-Cl, pH 7.4 and 0.1 mM EDTA). Simple
sequence repeat (SSR) markers (Ramsay et al. 2000) were screened against
the mutant near-isogenic lines and Bowman to identify polymorphisms. The
PCR methods used were described in Dahleen et al. (2003). Amplified
fragments were separated by gel electrophoresis using 4% Super Fine
Resolution (SFR) agarose (Amresco, Solon, OH) in1 X TAE (40 mM
Tris-acetate and 1 mM EDTA). Alternatively, acrylamide gel
electrophoresis was used as described by Wang et al. (2003). The gels
were stained with ethidium bromide, and photographed under UV light.
Markers that detected polymorphisms between Bowman and a mutant near
isogenic line were tested on the corresponding BC~n~F~2~ population. SSR
and mutant trait segregation data were entered into MAPMAKER software
(Lander et al. 1987; Lincoln et al. 1992) to test linkage between the
markers and the mutant trait.

  --------------- -------- ------------ ------------------------- -------------
  Table 1.                                                        
  Morphological                                                   
  markers, Barley                                                 
  Genetic Stock                                                   
  number, their                                                   
  chromosome                                                      
  locations,                                                      
  pedigrees of                                                    
  the mapping                                                     
  populations and                                                 
  the number of                                                   
  backcrosses to                                                  
  Bowman to                                                       
  develop the                                                     
  near-isogenic                                                   
  lines                                                           

  Gene            BGS No.  Chromosome   Pedigree                  Backcrosses

  *cer-zt*.389    BGS437   2H           cer-zt.389/5\*Bowman      4

  *dsp.ah*                 7H           DWS1180 Mut4841/6\*Bowman 5

  *dsp.at*                 3H           Bowman\*5/DWS1220 7117    4

  *dsp.ba*                 7H           DWS1357 UT1713/6\*Bowman  5

  *int-k*.47      BGS546   7H           int-k.47/7\*Bowman        6

  *nec*.50        BGS634   7H           Bowman\*5/nec.50          4
  (*pmr2*)                                                        

  *nec*.54        BGS634   7H           Bowman\*5/nec.54          4
  (*pmr2*)                                                        

  *pyr.ai*                 3H           Bowman\*6/DWS1018         5
  --------------- -------- ------------ ------------------------- -------------

**Results and Discussion**

Linkage between SSR markers and the morphological traits was identified
for each of the eight backcrossed morphological marker lines. All F~2~
populations segregated 3:1 normal:mutant as expected for single
recessive traits. There were no significant differences between Bowman
and near-isogenic lines for lodging, leaf width or heading date.

The eceriferum mutant *cer-zt*.389 (BGS 437) was located approximately
in bin 1 of chromosome 2H, linked to Bmac0134 (Fig. 1). This NIL only
differed from Bowman for kernels per spike, with a significant increase
in *cer-zt*. The intermedium spike mutant *int-k*.47 (BGS 546) was
located in the centromeric region of chromosome 7H, closely linked to
markers Bmag0217 and Bmac0162 in bins 6 to 7. The *int-k*.47 NIL had
significantly reduced height, peduncle length, awn length,
kernels/spike, leaf length, kernel weight, and yield. The two
necroticans mutants (*nec.50* and *nec.54*) are alleles at the premature
ripe 2 (*pmr2*) locus (BGS 634). Analysis of individual populations
indicated both were linked to the same markers on chromosome 7H (Fig.
1). Because they are alleles, a combined linkage analysis was conducted,
which showed tight linkage to markers in bin 5 of 7H (Fig. 2). The
*nec.54* NIL was analyzed in the field and showed reduced yield compared
to Bowman. The pyramidatum mutant *pyr.ai* was linked between markers on
chromosome 3H in bins 5 to 7 (Fig. 1). The NIL was shorter than Bowman,
had shorter rachis internodes, and showed a significant increase in
kernels per spike.

Three additional dense spike mutants were mapped, *dsp.ah*, *dsp.at*,
and *dsp.ba*. The first two were linked to markers on chromosome 7H near
the centromere while *dsp.ba* was located near the centromere on the
short arm of chromosome 3H (Fig. 1). Previously located dense spike loci
include *dsp1* on chromosome 7HS, *dsp9* on chromosome 6HL, and *dsp10*
on chromosome 3HS. Agronomic trait performance of these three lines was
similar to Bowman for most traits. The *dsp.ah* mutant showed
significantly reduced height, peduncle length and rachis internode
length, and lower yields but showed a significant increase in the number
of kernels per spike. The *dsp.at* mutant was shorter and had reduced
peduncle and rachis internode lengths, while *dsp.ba* only showed a
decrease in rachis internode length compared to Bowman. These
differences in the *dsp* mutants indicate it is unlikely they are
alleles, and they are unlikely to be alleles of *dsp1*, *ert-a*, *ert-d*
or *ert-m*, mutants with similar dense spikes.

In summary, we have placed eight additional morphological mutants on the
barley molecular linkage map. One (*cer-zt*) mapped to chromosome 2H,
two (*dsp.ba* and *pyr.ai*) mapped to chromosome 3H, and five (*dsp.ah*,
*dsp.at*, *int-k*.47, *nec*.50 and *nec*.54) mapped to chromosome 7H.

![](media/image1.wmf){width="6.495833333333334in"
height="4.207638888888889in"}

![](media/image2.wmf){width="4.288194444444445in"
height="2.8722222222222222in"}

Figure 1. Linkage maps showing positions of morphological markers in
relation to SSR markers mapped in segregating populations of 50 F~2~
plants for each trait. Cumulative linkage distances are on the left of
the vertical bar and marker names are on the right. Approximate
chromosomal bin locations for the SSRs are in parentheses.

![](media/image3.wmf){width="1.8208333333333333in"
height="2.582638888888889in"}

Figure 2. Linkage map showing the position of the *nec*.50 and *nec*.54
when the trait and marker data were combined for these alleles at the
*pmr2* locus (BGS 634).

**References**

Dahleen LS, Franckowiak JD and Vander Wal LJ. 2003. Exposing students
and teachers to plant molecular genetics with short-term barley gene
mapping projects. J Natural Resour Life Sci Educ 32:61-64.

Franckowiak JD and Lundqvist U. 2002. Current list of new and revised
barley genetic stock descriptions \[Online\]. Available at
http://wheat.pw.usda.gov/ggpages/bgn/32/index.html (verified 18 May
2006). Barley Genet Newsl 32:47--137.

Kleinhofs A. 2002. Integrating molecular and morphological/physiological
marker maps \[Online\]. Available at
http://wheat.pw.usda.gov/ggpages/bgn/32/index.html (verified 18 May
2006). Barley Genet. Newsl. 32:152--159.

Lander E, Green P, Abrahamson J, Barlow A, Daley M, Lincoln S, and
Newburg L. 1987. MAPMAKER: an interactive computer package for
constructing primary genetic linkage maps of experimental and natural
populations. Genomics 1:174-181.

Lincoln S, Daly M and Lander E. 1992. Constructing genetic maps with
MAPMAKER/EXP 3.0. Whitehead Inst. Tech. Rep. 3^rd^ ed. Whitehead Inst.,
Cambridge, MA.

Ramsay L, Macaulay M, Ivanissevich S, MacLean K, Cardle L, Fuller J,
Edwards KJ. Tuvesson S, Morgante M, Massari A, Maestri E, Marmiroli N,
Sjakste T, Ganal M, Powell W, and Waugh R. 2000. A simple sequence
repeat-based linkage map of barley. Genetics 156:1997-2005.

Wang D, Shi J, Carlson SR, Cregan PB, Ward RW, and Diers BW. 2003. A
low-cost, high-throughput polyacrylamide gel electrophoresis system for
genotyping with microsatellite DNA markers. Crop Sci 43:1828-1832.