Genetic analysis and molecular characterization of RFLP DNA markers in barley (Hordeum Vulgare
L.)
by Jeong Sheop Shin
A thesis submitted in partial fulfillment of the requirements of the degree of Doctor of Philosophy in
Crop and Soil Science
Montana State University
© Copyright by Jeong Sheop Shin (1988)
Abstract:
Single or low copy number DNA clones from random genomic DNA libraries using the plasmid vector
pBR322 and the phage EMBL4 were constructed using DNA from barley (Hordeum vulgare L.). This
work was done to provide a relatively large number of genetic markers and to characterize the level of
genetic variation in the barley genome. Selected genomic clones and cDNA clones were used to probe
the barley genome for the presence of restriction fragment length polymorphisms (RFLPs). This
methodology is based upon fragment size differences of defined length that are produced when DNA is
cleaved by restriction endonucleases. A multiple recessive marker stock and a relatively distantly
related cultivar 'Apex' were selected as parents in a cross to map the genomic location of seventeen
RFLP loci. Nine genomic clones and seven cDNA clones produced clear polymorphisms using at least
one restriction endonuclease. The majority of selected genomic clones showed polymorphisms using
two or more restriction endonucleases. This suggests that the variation observed among barley lines is
due to insertion/deletion or rearrangement events rather than point mutations. Utilizing selected single
or low copy clones as probes, it was confirmed that polymorphisms are readily detectable among
cultivars of barley.
Seventeen polymorphic DNA sequences were mapped relative to seventeen previously mapped marker
loci. Genotypes of 34 loci in 100 mapping lines were characterized and described to simplify the
mapping of additional RFLP loci. Twelve of seventeen RFLP loci showed codominant segregation.
Four of the five loci which demonstrated dominance were from genomic clones which hybridized to
several bands in each lane of the Southern blot. The probes and markers utilized in this mapping
project span 680 recombination units of the barley genome, approximately 50 percent of its estimated
recombinational length.
Detailed physical maps of fifteen polymorphic DNA fragments that were mapped in barley were
developed using several restriction endonucleases. All fifteen DNA clones were well characterized by
one or several restriction enzymes. In the Southern blot analysis of double digested genomic DNA
probed with one of these clones, one allele was found to contain about 200 base pair inserted sequences
compared with an alternate allele. The polymorphic region of this clone was sequenced using dideoxy
chain termination reaction.
Polymorphic DNA markers were also utilized to identify barley cultivars. Some cultivars
undifferentiated by hordeins were well discriminated using a subset of the DNA markers. 
GENETIC ANALYSIS AND MOLECULAR CHARACTERIZATION OF 
RFLP DMA MARKERS IN BARLEY (HORDEUM VULGARE L.)
by
Jeong Sheop Shin
A thesis submitted in partial fulfillment 
of the requirements of the degree
of
Doctor of Philosophy 
in
Crop and Soil Science
MONTANA STATE UNIVERSITY 
Bozeman, Montana
\November 1988
£>398
ii
APPROVAL
of a thesis submitted by
Jeong Sheop Shin
This thesis has been read by each member of the thesis committee 
and has been found to be satisfactory regarding content, English usage, 
format, citations, bibliographic style, and consistency, and is ready 
for submission to the College of Graduate Studies.
iJ(<hj~ / 7, / ^  /S'
Date Chairperson, Graduate Committee
Approved for the College of Graduate Studies
Graduate^DeanDate
iii
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment of the 
requirements for a doctoral degree at Montana State University, I agree 
that the Library shall make it available to borrowers under rules of 
the Library. I further agree that copying of this thesis is allowable 
only for scholarly purposes, consistant with "fair use" as prescribed 
in the U.S. Copyright Law. Requests for extensive copying or 
reproduction of this thesis should be referred to University Microfilms 
International, 300 North Zeeb Road, Ann Arbor, Michigan 48106, to whom 
I have granted "the exclusive right to reproduce and distribute copies 
of the dissertation in and from microfilm and the right to reproduce 
and distribute by abstract in any format."
Date
Signature
iv I
ACKNOWLEDGMENTS
I would like to thank Dr. R.W. Wolfe and Dr. S. Muthukrishnan for 
their helping in providing us with the multiple recessive marker stock 
and cDNA clones.
I am especially grateful for and appreciate the ideas and 
enthusiasm of my major advisor, Dr. Tom Blake, who has provided me with 
a positive environment in which to learn and work from start to end. 
Thanks also to Drs. E.A. Hockett, R.L. Ditterline, E.R. Vyse, and V. 
Raboy for helpful suggestions while serving on my graduate committee.
I loved our unselfish laboratory conditions and the heljp and
I
friendship of several coworkers, Drs. Don Lee, Shiaoman Chad, Dave 
Hoffman, Suewiya Pickett, Ms. Mar Sanchez, and Mr. Pat Hensleigh.
Finally I wish to express sincere thanks to my father, Sang Don 
Shin, my late mother, Seon Ja Lee, mother, Seung Ja Seong, my wife,
Hyo Mi, daughter, Hee Young, and also to Korean barley researchers for 
their enduring support and encouragement.
TABLE OF CONTENTS
Page
APPROVAL........................................................  ii
STATEMENT OF PERMISSION TO USE...................................  iii
ACKNOWLEDGMENTS....................................   iv
TABLE OF CONTENTS........ ........................... .....;....  v
LIST OF TABLES..................... .........................vii
LIST OF FIGURES................................................  viii
ABSTRACT............................................  x
CHAPTER
1 INTRODUCTION. . . .......................................... I
2 MOLECULAR CLONING AND EVALUATION OF BARLEY GENOMIC
LOW COPY NUMBER DNA CLONES AS GENETIC MARKERS.........  2
Introduction.......................   2
Materials and Methods..........      3
Results and Discussion....................    10
3 A 34 POINT LINKAGE MAP OF THE BARLEY GENOME
INCLUDING 17 RFLP LOCI..................................  15
Introduction...........................  15
Materials and Methods....i..............................  16
Results...................................   19
Discussion..............................................  25
4 PHYSICAL MAPS OF SEVENTEEN INFORMATIVE DNA MARKERS
AND DESCRIPTION OF 100 MAPPING LINES.................... 30
Introduction..........      30
Materials and Methods...................................  31
Results and Discussion..................................  32
5 BARLEY VARIETAL DISCRIMINATION
USING RFLP AND HORDEIN MARKERS..........................  45
Introduction.............................    45
Materials and Methods................................... 46
Results and Discussion..................................  48
V
Page
6 SUMMARY.................................................... 59
REFERENCES......................................................... gj
vi
LIST OF TABLES
Table Page
1 Morphological and biochemical characters evaluated......... 16
2 Informative DNA loci in the barley linkage map............  20
3 Segregations and chi-square goodness-of-fit analysis
for 17 RFLP, 5 isozyme, 2 storage protein,.and 10 
morphological markers in a barley F2 population..........  22
4 Recombination frequencies and standard deviations.........  24-25
5 Genotypes of 34 loci in 100 mapping lines.................  33-35
6 Sequence data of 474 bp from sequence gel of Figure 13.... 43
7 Barley cultivars utilized to identify genetic relationships
using polymorphisms of hordeins and RFLP DNA markers...... 47
8 Data matrix of polymorphic alleles among 21 barley cultivars 54
9 Analysis of allele frequency in a locus and gene diversity
among 21 barley cultivars...... ........................... 55
vii
viii
LIST OF FIGURES
1 Identification of low copy number genomic clones
using EMBL4...................       6
2 • Identification of low copy number genomic clones
using a plasmid vector......... ^......................... 7
3 Digestion of barley genomic DNA with restriction
endonucleases............................................. 9
4 Screening blot of a polymorphic low copy number clone
using a plasmid vector.......    12
5 . Screening blot of a polymorphic low copy DNA clone
using EMBL4..............................................  13
6 Starch gel of 6-phosphogluconate dehydrogenase in an Fz
population...............................................  17
7 SDS-polyacrylamide gel of barley storage proteins.......  18
8 Blot demonstrating Fz segregation.........   21
9 Barley genetic linkage maps of chromosomes I and 2.......  26
10 Barley genetic linkage maps of chromosomes 3 and 5.......  27
11 Barley genetic linkage map of chromosome 7...............  28
12 Restriction maps of 15 informative DNA clones which have
been mapped in barley chromosomes........................  37-41
13 Autoradiograph of double digested genomic DNA blot
hybridized with probe pxMSU 21........  42
14 Sequence of Bam HI and Sst I double digested pxMSU 2.1
fragment that identified polymorphism.......I............  44
15 Polymorphic allelic patterns of pxKSU 21.....   49
16 Polymorphic allelic patterns of pxKSU 32.........    50
17 Polymorphic allelic patterns of pxMSU 21.................  51
18 Polymorphic allelic patterns of pxMSU 11...........    51
19 Polymorphic allelic patterns of pxKSU 11.................  52
Figure Page
Figure Page
20 Polymorphic allelic patterns of pxKSU 71.................  52
21 Polymorphic allelic patterns of pxKSU 31............. . 53
22 Dendrograph of 21 barley cultivars using the genetic
distances given by computer program, PAUP................  57
ix
XABSTRACT
Single or low copy number DNA clones from random genomic DNA 
libraries using the plasmid vector pBR322 and the phage EMBL4 were 
constructed using DNA from barley (Hordeum vulgare L.). This work was 
done to provide a relatively large number of genetic markers and to 
characterize the level of genetic variation in the barley genome. 
Selected genomic clones and cDNA clones were used to probe the barley 
genome for the presence of restriction fragment length polymorphisms 
(RFLPs). This methodology is based upon fragment size differences of 
defined length that are produced when DNA is cleaved by restriction 
endonucleases. A multiple recessive marker stock and a relatively 
distantly related cultivar 'Apex' were selected as parents in a cross 
to map the genomic location of seventeen RFLP loci. Nine genomic 
clones and seven cDNA clones produced clear polymorphisms using at 
least one restriction endonuclease. The majority of selected genomic 
clones showed polymorphisms using two or more restriction 
endonucleases. This suggests that the variation observed among barley 
lines is due to insertion/deletion or rearrangement events rather than 
point mutations. Utilizing selected single or low copy clones as 
probes, it was confirmed that polymorphisms are readily detectable 
among cultivars of barley.
Seventeen polymorphic DNA sequences were mapped relative to 
seventeen previously mapped marker loci. Genotypes of 34 loci in 100 
mapping lines were characterized and described to simplify the mapping 
of additional RFLP loci. Twelve of seventeen RFLP loci showed 
codominant segregation. Four of the five loci which demonstrated 
dominance were from genomic clones which hybridized to several bands in 
each lane of the Southern blot. The probes and markers utilized in 
this mapping project span 680 recombination units of the barley genome, 
approximately 50 percent of its estimated recombinational length.
Detailed physical maps of fifteen polymorphic DNA fragments that 
were mapped in barley were developed using several restriction 
endonucleases. All fifteen DNA clones were well characterized by one 
or several restriction enzymes. In the Southern blot analysis of 
double digested genomic DNA probed with one of these clones, one allele 
was found to contain about 200 base pair inserted sequences compared 
with an alternate allele. The polymorphic region of this clone was 
sequenced using dideoxy chain termination reaction.
Polymorphic DNA markers were also utilized to identify barley 
cultivars. Some cultivars undifferentiated by hordeins were well 
discriminated using a subset of the DNA markers.
ICHAPTER I 
INTRODUCTION
The lack of available genetic markers in cultivated genotypes has 
limited the development of saturated genetic linkage maps in plant 
species. Analysis of restriction fragment length polymorphisms (RFLPs) 
will provide a relatively unlimited number of genetic markers and 
permits the construction of detailed genetic linkage maps in eukaryotic 
species. The studies reported here focussed on the recombinational 
location of selected RFLP DMA markers relative to previously mapped 
marker loci in the nuclear genome of barley (Hordeum vulgare L.).
The goals of the first part of this investigation were to screen 
single or low copy number DNA probes selected from random genomic DNA 
libraries and to identify genomic RFLPs. The objectives of the second 
part of this study were to utilize the selected barley DNA clones as 
genetic markers and to locate them in barley chromosomes relative to 
previously mapped morphological and biochemical markers. In the third 
part of this study, the genotypes of thirty-four loci in 100 mapping 
lines were described and their application as a template to simplify 
the mapping of additional RFLP loci was discussed. Fifteen out of 
seventeen mapped DNA clones were also characterized by restriction 
mapping analysis and the polymorphic region of one interested 
polymorphic probe was sequenced. The objective of the final part of 
the study was to determine the relative utility of RFLP markers in 
cultivar identification.
2CHAPTER 2
MOLECULAR CLONING AND EVALUATION OF BARLEY GENOMIC 
LOW COPY NUMBER DNA CLONES AS GENETIC MARKERS
Introduction
Restriction fragment length polymorphism (RFLP) analysis probes 
specific regions of the genome for the presence of variation at the DNA 
level (Grodzicker et al., 1974; Botstein et al., 1980). RFLPs were 
first identified in temperature sensitive mutations of adenoviruses 
(Grodzicker et al., 1974). This methodology is based on DNA fragment 
size differences of defined length that are produced by cleavage of DNA 
with restriction endonucleases and that are identified by Southern 
(1975) blot analysis. The use of RFLPs was proposed as a new source of 
genetic markers for the human genome in 1980 (Botstein et al., 1980; 
Bishop and Skolnick, 1980). These studies demonstrated the basic 
principle of using random single copy DNA probes to detect DNA sequence 
polymorphisms among different genotypes. Gusella et al. (1983) 
identified polymorphic DNA marker loci associated with the mutant 
allele causing Huntington's disease using this technology.
In basic plant genetics as well as in plant breeding, RFLPs have 
been suggested as potent tools (Tanksley, 1983; Beckman and Seller, 
1983; Burr et al, 1983; Seller and Beckman, 1983; Evola et al., 1986; 
Helentjaris et al., 1985; Landry and Michelmore, 1987). The promising 
potential for this technology in plants is based on the practically 
unlimited amount of variability at the DNA level in their genomes.
3Recently RFLPs were utilized to saturate the genetic linkage maps 
in maize and tomato (Relentjaris et al., 1986), in tomato (Berriatzky 
and Tanksley, 1986) and in lettuce (Landry et al., 1987).
The objectives of this study were to select single or low copy 
number DMA clones from barley random genomic DNA libraries using the 
plasmid vector pBR322 and the phage EMBL4, and to use these clones to 
identify RFLPs in the barley genome. In order to detect these RFLPs, 
selected single copy DNA probes were hybridized to Southern blots 
containing restriction endonuclease-digested barley DNA from a multiple 
recessive marker stock and the European 2-rowed cultivar 'Apex*.
Materials and Methods
Plant DNA Extraction
Leaf and stem tissues of barley seedlings were freeze-dried in a 
VirTis freezedryer for 3-4 days. Total plant DNA was extracted from 
the lyophilized tissue using modifications of the method of Murray and 
Thompson (1980) suggested by Saghai-Maroof et al. (1984).
Buffers and Abbreviations
Stock solutions and working solutions utilized in this study were 
prepared as followed:
20 x SSPE : 3.6 M NaCl, 0.2 M sodium phosphate (pH 7.0),
0.2 M EDTA
20 x SSC : 3 M NaCl, 0.3 M trisodium citrate
10 % Blotto : 10 % non-fat powdered milk, 0.2 % sodium azide
45 x TBE (Tris-Borate) Buffer : Tris base 54 g, boric acid 27.5 g, 
0.5 M EDTA (pH 8.0) 20 ml, water up to I L 
Gel loading Dye Solution : 0.25 % bromophenol blue, 0.25 % xylene 
cyanol, 40 % (w/v) sucrose
10 x Ligation Buffer : 0.66 M Tris^HCl (pH 7.5), 50 mM MgCl ,
50 mM dithiothreitol, 10 mM ATP 
10 x Nick-translation Buffer : 0.5 M Tris-HCl (pH 7.2), 0.1 M 
MgSO-j, I mM dithiothreitol, 500 ug/ml bovine serum 
albumin (BSA)
Prehybridization Solution (nitrocellulose) : 5 x SSC, 5 x
Denhardt's reagent, 20 mM Na-phosphate (pH 6.5),
50 % formamide, 100 ug/ml denatured salmon sperm DNA 
Hybridization Solution (nitrocellulose) : 5 x SSC, I x Denhardt1s 
reagent, 20 mM Na-phosphate (pH 6.5), 50 % formamide, 
100 ug/ml denatured salmon sperm DNA 
I x Denhardt's Solution : 0.02 % Ficoll 400, 0.02 % polyvinyl­
pyrrolidone, 0.02 % BSA
Library Construction and Evaluation
Barley genomic DNA libraries were constructed in the phage vector 
EMBL4 (Frischauf et al., 1983) and the plasmid vector pBR322 using 
total DNA from the barley Cultivars 'Betzes1 and 'Traill', 
respectively. Phage and plasmid clones were randomly selected and 
amplified using the methods of Maniatis et al. (1982). Plasmid DNA was 
isolated from E^ coli hosts using the mini-prep procedure of Birboim
and Doly (1979). Phage clones were randomly picked from the library
5and cloned DNAs were prepared following the procedure of Maniatis et 
al. (1982). Isolated phage DNA was digested with restriction 
endonucleases, Eco RI, Hind III, Bam HI or Sal I. For plasmid clones, 
prescreening was first performed by colony hybridization (Grunstein and 
Hogness, 1975). Selected plasmids were digested with Bam HI and 
electrophoresed in 0.8 %, H O  x 135 mm horizontal agarose gel using I x 
TBE buffer at 2 V/cm overnight.
Gels were then stained with ethidium bromide and photographed 
under UV light. Restriction fragments were transferred to either 
nitrocellulose (Southern, 1975) or Zeta-probe nylon membrane (Reed and 
Mann, 1985). Nitrocellulose filters were baked at 80 °C in a vacuum 
oven for 2 hours after transfer was completed. Filters were hybridized 
with total DNA from 'Betzes' barley which had been radioactively 
labeled by nick-translation (Rigby et al., 1977). Single and low copy 
number barley inserts were identified as those which bound low or 
undetectable amounts of the total barley DNA probe (Figures I and 2). 
Selected phage fragments were subcloned into the plasmid vector pBR322. 
Selected clones from the plasmid library were utilized directly.
Genomic Blot Preparation
The parents utilized in the mapping study were a multiple 
recessive marker stock (MMS) developed by Dr. R.9. Wolfe (1984) 
(discussed in Chapter 3) and the European 2-rowed cultivar 'Apex'. 
Isolated DNA from these lines was quantified by fluorometry using the 
DNA specific fluorescent dye Hoechst 33258. Fifteen ug aliquots of DNA 
were digested with the restriction endonucleases Bam HI, Hind III, Eco
6Figure I. Identification of low copy number genomic clones using 
EMBL4. A: Gel of 14 EMBL4 clones containing random barley fragments 
digested with Hind III. B: Autoradiograph of blot of A probed with 
total nick-translated barley DMA.
Estimated molecular weights in kilobase pairs listed at left. 
Arrows indicate low copy number fragments tested for identification of 
polymorphisms between 'Apex' and 'MMS'.
7Al
Kbp
23.1»
9.4»
6.6»
4.4»
2.3» 
2 0»
Figure 2. Identification of low copy number genomic clones using a 
plasmid vector. A: Gel of 12 plasmid vector pBR322 clones containing 
random barley DNA fragments digested with Bam HI. B: Autoradiograph 
of blot of A probed with nick-translated total barley genomic DNA.
Estimated molecular weights in kilobase pairs listed at left. 
Arrows indicate single or low copy number barley DNA fragments.
a
8RI, Eco RV, and Dra I, separated by gel electrophoresis (Figure 3), and 
transferred to Zeta-probe nylon membrane as indicated above.
Probe Labeling
Approximately 0.1 ug of cloned DNA fragment or total barley DNA 
was labeled with 32P dNTPs using nick-translation (Rigby et al.,
1977) . The labeled DNA probes were separated from unincorporated 
nucleotides using centrifuged Sephadex G-50 I cc columns.
Alternatively, agarose gel slices containing the fragment of interest 
were labeled by primer extension (Feinberg and Vogelstein, 1984) and 
utilized without removing the unincorporated nucleotides. Prior to 
hybridization, the labeled probes were mixed with 0.2 ml of 0.2 N NaOH 
and denatured by heating to IOO0C for 10 minutes.
Hybridization
Nitrocellulose filters were prehybridized and hybridized at 420C 
using 50 % formamide according to the method of Spruill et al. (1981). 
Zeta-probe nylon membrane was prehybridized and hybridized in 15-20 ml 
of 1.5 x SSPE, 1.0 % SDS and 0.5 % Blotto solution at 680C in a water 
incubator with gentle shaking for 4-24 hours (Reed and Mann, 1985).
The carrier salmon sperm DNA (5 mg) and radioactive-labeled probe were 
denatured immediately before adding it to the hybridization solution.
Hashing and Autoradiography
Three washes for 15 minutes at room temperature in 300 ml of 2 x 
SSC/0.1 % SDS, 0.5 x SSC/0.1 % SDS and 0.1 x SSC/0.1 % SDS solutions
9BH1 HS E1 ES D1
Figure 3. Digestion of barley genomic DNA with restriction 
endonucleases. Fifteen micrograms of total genomic DNA from the lines 
'MMS' and 'Apex' was digested with five different restriction 
endonucleases. Letters indicate following DNA samples and restriction 
endonucleases; M: multiple recessive marker stock. A: Apex. BH1: Bam 
HI. H3: Hind III. El:Eco RI. ES: Eco RV. Dl: Dra I.
10
successively were followed by two or three final washes with prewarmed 
solutions of 0.1 x SSC/1.0 % SDS at 65°C.
Filters were wrapped in plastic wrap and placed adjacent to a 
sheet of X-ray film in an exposure cassette with one or two 
intensifying screens. Autoradiography was performed at -700C for 5-7 
days.
Filters were reused by removing the hybridization probe. Two 
methods were used with equal success. Filters were either washed two 
times of washing in an initially boiling solution of 0.1 x SSC/0.5 % 
SDS which was allowed to cool over a 20 minute period or washed in 0.2 
N NaOH for 20 minutes followed by 0.5 M Tris-HCl (pH 7.5)/0.1 x SSC/ . 
0.1 % SDS wash solution. Both methods completely removed the 
hybridization probe.
Results and Discussion 
Low Copy Number Clone Selection
In order to determine which clones contained single or low copy 
number sequences, each clone was hybridized to the 32P-Iabeled total 
plant DNA (Figures I and 2). The duration of autoradiography and the 
specific activity of the total DNA probe were critical factors in 
recognizing repeat-free fragments. Bands showing very faint or no 
hybridization signal after 5 days autoradiography were selected as 
potential unique or low copy number sequences.
Fifty phage containing unique barley DNA inserts were screened.
In the library using plasmid pBR322, 141 colonies out of 1361 total 
transformed cells (about 10 %) were observed as Ampr Tets showing
11
recombinants. Of the 141 pBR322 recombinants, 39 single or low copy 
number probes (29 single and 10 low copy number clones) were identified 
and advanced to further screening.
Restriction Fragment Length Polymorphisms
The selected single or low copy number DNA probes were hybridized 
to Southern blots containing five different restriction endonuclease- 
digested barley genomic DNAs from a multiple recessive marker stock 
(MMS) and the European two-rowed cultivar 'Apex' (Figure 3). Utilizing 
these selected clones, it was confirmed that.polymorphisms are readily 
detectable in barley genome. Eight single or low copy number probes 
using from the pBR322 library and six fragments cloned into EMBL4 were 
screened. Nine genomic clones of these were identified which produced 
clear, consistent results in Southern blot analysis (detailed in 
Chapter 3).
Some probes displayed different sizes of hybridization bands and 
the other showed presence vs. absence of detectable bands within two 
parental lines. Differences among higher molecular weight DNA 
fragments were quite difficult to identify. Most of the clones 
. evaluated identified polymorphisms using several restriction 
endonucleases. The simplest explanation for this observation is that 
the variation observed among barley lines is due to rearrangements or 
insertion/deletion events, rather than point mutations.
The autoradiograph using the probe pxMSU 21 demonstrated clear 
polymorphisms using Bam HI and Hind III (Figure 4). Using Hind III,
12
M A 
BHI
M A 
H3
M A 
E1 ME5A V
Figure 4. Screening blot of a polymorphic low copy number DNA clone 
using a plasmid vector. Fifteen micrograms of DNA from the lines 'MMS' 
and 'Apex' was digested with five different DNA restriction 
endonucleases and transferred to nylon membrane. Estimated molecular 
weights in kilobase pairs listed at left. Letters indicate the 
following DNA samples and restriction endonucleases; M: multiple 
recessive marker stock, A: Apex, BHl: Bam HI, H3: Hind III, El: Eco RI, 
ES: Eco RV, Dl: Dra I.
Blot was hybridized with nick translated pxMSU 21. Parental 
variation was identified with both Bam HI and Hind III.
13
Figure 5. Screening blot of a polymorphic low copy DNA clone using 
EMBL4. Fifteen micrograms of DNA from the lines 'MMS' and 'Apex' was 
digested with five different DNA restriction endonucleases and 
transferred to nylon membrane. Lambda molecular weight marker lanes of 
left and right ends are completely identical to the cloning vector and 
showed darkened hybridization signal. Letters indicate the following 
DNA samples and restriction enzymes; M: MMS, A: Apex, BHl: Bam HI, H3: 
Hind TH, El: Eco RI, ES: Eco RV, Dl: Dra I. Genomic DNA blot was 
hybridized with nick translated pxMSU 72. The clearest parental 
variation was identified with Hind III.
14
pxMSU 72 showed an approximately 3.4 Kbp size difference between 1MMS' 
and 'Apex' (Figure 5). These restriction endonucleases were utilized 
in a subsequent mapping study.
15
CHAPTER 3
A 34 POINT LINKAGE MAP OF THE BARLEY GENOME 
INCLUDING 17 RFLP LOCI
Introduction
Increasing the number of informative marker loci in crop species 
will improve our ability to both understand the genetic basis for 
complex characters and aid in the identification and characterization 
of germplasm (Beckmann and Seller, 1983). In many important 
agricultural species the lack of available genetic markers has limited 
the development of saturated genetic linkage maps.
Analysis of restriction fragment length polymorphisms (RFLPs) 
provides relatively unlimited numbers of genetic markers and permits 
the construction of detailed genetic linkage maps. After the use of 
RFLPs as genetic loci was proposed in human genetic linkage map 
(Botstein et al., 1980; Bishop and Skolnick, 1980), this approach was 
extensively utilized to saturate the genetic linkage maps in humans 
(Gusella, 1986), in maize and tomato (Helentjaris et al., 1986), in 
tomato (Bernatzky and Tanksley, 1986) and lettuce (Landry at al., 
1987).
In this chapter, the recombinational location of seventeen cloned 
DNA sequences relative to seventeen previously mapped marker loci is 
described. Nine low copy number sequences which identified RFLPs in 
cultivated barley were selected as described in Chapter.2. The 
recombinational locations of seven cDNA clones and a clone of the
16
barley and wheat ribosomal gene clusters also were identified relative 
to ten previously mapped morphological marker loci, five previously 
mapped isozyme loci and two storage protein loci.
Materials and Methods
Plant Materials
The parents utilized in this mapping study were a multiple 
recessive marker stock (MMS) and the European 2-rowed cultivar 'Apex'. 
One hundred F2 plants and twelve F3 progeny from each F2 plant were 
evaluated for the ten morphological characters listed in Table I.
Table I. Morphological and biochemical characters evaluated.
Locus Designation Phenotype Chromosomal Location
WX waxy endosperm I
n naked caryopsis I
lk2 short awn I
V six rowed 2
wst, ,B white stripe 2
al albino lemma 3
i fertile laterals 4
O orange lemma base and nodes 6
S short rachilla hairs 7
r smooth awn 7
Per I peroxidase 2
Per 2 peroxidase 2
Est I esterase 3
Est 2 esterase 3
Pgd 2 6-phosphogluconate dehydrogenase 5
Hor I C hordeins 5
Hbr 2 B hordeins 5
DMA Markers and biochemical loci
Nine low copy number random genomic DMA clones which identified 
clear polymorphisms were utilized in analysis of F2 progeny from the
17
cross described above. Seven cDNA clones selected from an endosperm 
cDNA library (Muthukrishnan et al., 1983) and a clone of the barley and 
wheat ribosomal gene clusters (Gerlach and Bedrock, 1979; Saghai-Maroof 
et al., 1984) were also utilized. One hundred Fz plants and six Fs 
progeny each were characterized for polymorphisms esterase, peroxidase 
and 6-phosphogluconate dehydrogenase isozymes (Table I and Figure 5) 
using the methods described in Benito et al. (1988). Six Fs seeds from 
each Fz plant were evaluated for B and C hordein polymorphisms (Figure 
6) according to the methods of Blake et al. (1982).
MA MA
Figure 6. Starch gel of 6-phosphogluconate dehydrogenase in an Fz 
population. Arrow indicates the segregating Pgd-2 locus in chromosome 
5. Two samples of each left and right margin are two parents; M: MMS, 
A: Apex. Single band of higher and lower molecular weight are 
homozygous for allele from 'MMS' and 'Apex', respectively. Triple 
bands indicate heterozygous type individual.
18
Figure 7. SDS-polyacrylamide gel of barley storage proteins. Six Fs 
seeds from each Fz plant were evaluated for B and C hordein 
polymorphisms designated Hor 2 locus and Hor I locus in barley 
chromosome 5, respectively. But D hordeins designated Hor 3 locus in 
the same chromosome did not show clear polymorphisms in these parents. 
Estimated molecular weights in kilodalton listed at right. Letters 
indicate as following; B: B hordeins, C: C hordeins, D: D hordeins.
19
DMA Extraction, Southern Blotting and Hybridization
DMA isolation, Southern blotting, labeling, hybridization, and 
autoradiography procedures used were identical to those detailed in 
Chapter 2.
Linkage Analysis
Recombination analyses were performed using Linkage-1 (Suiter et 
al., 1983) packaged for maximum likelihood linkage analysis (Allard, 
1956). While gametic frequencies for isozymic and storage protein loci 
did not differ significantly from chi-square expectations, differencies 
in one class of homozygote indicated that loci associated with the 
morphological marker locus v and three RFLP loci appeared to have 
modified gametic or zygotic viability (Table 3). This has been 
observed frequently in the past (Heun 1987), but a clear understanding 
of the mechanism underlying the bias in segregation data is required 
before a correction can be applied to the data.
Results
Evaluation of RFLP DMA Markers
Nine genomic clones and seven cDNA clones were identified which 
produced clear, consistent results in Southern blot analysis of 
segregating progeny from mapping cross, MMS x Apex (Table 2)
(Figure 8). The ribosomal clone, pTA71, also proved useful in this 
analysis.
This approach to maximizing the chance of observing significant 
linkages revolved around the use of previously mapped isozyme, storage
20
protein and morphological marker loci (Wolfe, 1984; Benito et al., 
1988).
Table 2. Informative DNA loci in the barley linkage map
Clone
name
Vector Insertion 
site(s)
Size
(Kbp)
Cloner
Polymorphism. 
Enzyme MMS Apex
Lab name
pMSU 11 . pBR322 . BHl 3.0 JS H3 6.0 8.6 BS134
pMSU 12 pBR322 BHl 3.0 JS H3 6.6 - BS20
pKSU 11 pBR322 Pl 0.6 SM H3 6.1 8.3 MC44
pMSU 21 pBR322 BHl 3.0 JS BHl 3.2 3.4 BS113
pMSU 22 pBR322 BH1/S1 1.0 SC H3 9.3 11.2 BC146
pKSU 21 pBR322 Pl O.S SM ES 11.6 6.4 MCll
pKSU 31 pBR322 Pl 0.B SM H3 5.5 - MC22
pKSU 32 pBR322 Pl 0.4 SM ES 5.1 2.2 MCS
pMSU BI pBR322 BHl 3.0 JS BHl 20.8 32.8 BS16
pMSU 71 pBR322 BHl 4.0 JS H3 3.8 
I 9
BS118
pMSU 72 pBR322 BH1/S1 2.0 SC H3 6.6 3.2 BC107
pMSU 73 pBR322 BHl B.O JS BHl , 5.1 9.4 BS95
pMSU 74 pBR322 BH1/S1 1.0 SC H3 10.0 7.4 BC196
pKSU 71 pBR322 Pl O.S SM BHl 11.3 6.6 MC26
pKSU 72 pBR322 Pl 0.2 SM H3 6.3 4.7 MC75
pKSU 73 pBR322 Pl 0.2 SM Dl 2.9 3.6 MC24
pTA71(Rrn2) pAC184 El 9.0 WG Tl 1.0 PTA71
Abbreviation used in Table: Pl=Pst I, H3=1Hind III, BHl=Bam HI,
EB=Eco RV, Sl=Sal I, Tl=Taq I, Dl=Dra I, El=Eco RI; JS=J.S. Shin, 
SM=S. Muthukrishnan, SC=S. Chao, WG=W. Gerlach.
Twelve of seventeen RFLP loci showed codominant segregation. Four 
of the five loci which demonstrated as dominant allele (presence vs. 
absence of a band) were from genomic clones which hybridized to several 
bands in each lane of the Southern blot. Either comigration of bands 
or varying numbers of hybridizing sequences between genotypes could 
explain this result. Careful experiments to precisely determine 
sequence copy number will be required to distinguish between these 
hypotheses.
21
Figure 8. Blot demonstrating Fz segregation. DNA from 10 Fz plants 
was digested with Hind III, electrophoresed, blotted and probed with 
pxMSU 72. Simple Mendelian segregation was observed for the 
polymorphisms indicated by arrows. Lanes labeled '11' are homozygous 
for the allele from 'MMS', lanes labeled '12' are heterozygous, and 
lanes labeled '22' indicate homozygosity for the allele from 'Apex'.
22
Table 3. Segregations and chi-square goodness-of-fit analysis for 17 
RFLP, 5 isozyme, 2 storage protein, and 10 morphological 
markers in a barley Fz population.
Locus
Homozygous
MMS Heterozygous
Homozygous
Apex Chi-square
WX 16 56 28 3.745
n 21 48 30 1.359
lk2 22 43 34 4.025
V 15 46 38 10.732*
al 21 43 35 4.945
i 16 61 22 5.399
O 25 45 29 0.894
S 29 41 29 2.429
r 24 54 21 0.518
wst,,B 25 44 31 1.825
Estl 25 41 34 4.345
Est2 30 40 30 3.425
Perl 20 52 28 1.105
Per2 14 58 28 5.785
Pgd 2 28 50 22 0.550
Horl 17 42 33 5.156
Hor2 - 23 44 25 0.158
xMSU 11 22 37 17 0.454
xMSU 12 45 — 17 0.086
XKSU 11 11 24 27 10.202*
xMSU 21 25 40 18 0.946
xMSU 22 14 37 5 7.759*
xKSU 21 22 42 22 0.006
xKSU 31 — 53 — 18 0.005
xKSU 32 17 48 16 2.278
xMSU 51 29 —  55 — 3.571
xMSU 71 — 74 — 17 1.615
xMSU 72 22 49 21 0.245
xMSU 73 22 50 23 0.153
xMSU 74 27 24 18 7.862*
xKSU 71 27 47 15 2.949
XKSU 72 21 38 16 0.433
xKSU 73 24 45 18 0.649
Rrn2 68 18 0.558
* indicates Chi-square value greater than would be expected by chance 
at the 0.05 level of significance.
23
ji. •. 
.
Mapping of RFLP Loci
Using a multiple marker stock (MMS) as one parent in linkage 
analysis provided seventeen ’benchmark* loci which had been previously 
mapped. Figures 9, 10, 11 and Table 4 display a thirty-four point 
linkage map which utilized ten morphological markers, 5 isozyme loci 
and 2 hordeins as reference points in map construction. With the 
exception of chromosome four and six, significant linkages were found 
between marker loci and biochemical and RFLP loci. In the case of 
chromosome six, the previously located Rrnl locus was not found to vary 
among the parents of this population with the enzymes evaluated. Eight 
of the sixteen clones identified polymorphisms with multiple 
restriction endonucleases. The simplest explanation for this 
observation is that the variation observed among barley lines is due to 
rearrangements or insertion/deletion events rather than point 
mutations.
Nomenclature
The clones and loci they identify are named following 
recommendations of several researchers compiled by Dr. Gary Hart (Hart, 
pers. comm.). The initial three letters indicate the clone source, the 
next two or three digits indicate the clone number, and in this project 
the first digit was assigned to the chromosome number to which the 
clone has been initially mapped. As none of clones have a known 
function, the locus names differ from the clone names only by the 
prefix 1X' to indicate that they are RFLP loci.
'i
24
Table 4. Recombination frequencies and standard deviations.
Chromosome
Number Linked loci
No. of 
progeny
Recombination frequency and 
S.D.
I wx - xKSU 11 62 34.88 +/- 5.60
I xKSU 11 - n 61 38.12 +/- 5.91
I n - lk2 99 17.51 W -  2.86
I lk2 -• xMSU 12 62 28.89 +/- 6.64
I xMSU 12 - xMSU 11 53 27.51 +/- 7.03
I wx - n 99 44.78 +/- 4.95
I
B
11 - lk2 61 38.02 +/- 5.90
I n - xMSU 12 62 36.25 +/- 7.27
2 XMSU 22 - Perl 56 38.01 +/- 6.16
2 Perl - Per2 100 15.34 W -  2.81
2 Per2 - V 99 30.46 +/- 4.11
2 v - xKSU 21 85 31.63 W -  4.53
2 xKSU 21 - xMSU 21 71 30.37 +/- 4.85
2 xMSU 21 - wst,,B 83 37.37 W -  5.02
2 xMSU 22 - Per2 56 41.22 +/- 6.39
2 Per2 - xKSU 21 86 38.49 +/- 5.00
2 Perl - V 99 38.37 W -  4.65
3 al - xKSU 32 81 36.85 +/- 5.04
3 xKSU 32 - xKSU 31 64 37.35 W -  7.23
3 xKSU 31 - Esf2 71 7.22 W -  3.17
3 Est2 - Estl 100 8.81 W -  2.11
3 al - xKSU 31 71 43.03 W -  7.14
3 xKSU 31 - Estl 71 13.97 +/- 4.39
4 i no significant linkage identified
5 Hor2 - Horl 92 13.91 W -  2.78
5 Horl - xMSU 51 78 38.81 W -  6.63
5 xMSU 51 - Pgd2 84 18.13 W -  4.58
5 Hor2 - xMSU 51 78 41.80 W -  6.77
5 Horl - Pgd 2 92 38.08 +/- 4.87
6 O no significant linkage identified
7 xMSU 74 - xMSU 73 68 31.61 W -  5.07
7 XMSU 73 - r 94 23.26 W -  3.63
7 r - xKSU 73 86 9.83 +/- 2.40
25
Table 4. (continued)
Chromosome
Number Linked loci
Mo. of 
progeny
Recombination frequency and 
S.D.
7 xKSU 73 - xMSU 72 83 8.85 +/- 2.32
7 XMSU 72 - s 91 24.36 +/- 3.78
7 s - xKSU 72 74 13.67 +/- 3.07
7 xKSU 72 - xKSU 71 72 43.50 +/- 5.75
7 xKSU 71 - Rrn2 74 7.54 +/- 3.17
7 Rrn2 - xMSU 71 73 9.64 +/- 11.57
7 xMSU 73 - xKSU 73 86 26.59 +/- 4.09
7 r - xMSU 72 91 14.93 +/- 2.90
7 xKSU 73 - s 86 27.29 +/- 4.15
7 xMSU 72 - xKSU 72 72 26.65 +/- 4.47
7 XKSU 71 - xMSU 71 82 11.58 +/- 3.72
Discussion
In this paper sixteen new informative genomic and c D M  clones for 
use in barley RFLP analysis were evaluated and released. These have 
been mapped using previously mapped marker loci and provide an initial 
analysis of the types of polymorphisms identified.
Four loci of the 34 loci tested, xKSU 11, xMSU 22, v, and xMSO 74, 
did not meet chi-square expectations for segregation of dominant 
alleles at a single locus. Linkage estimates with these loci are 
likely biased and will need confirmation with other crosses or progeny 
of these 100 Fz lines. Twenty-two percent of the loci evaluated by 
Landry et al. (1987), and 10 percent of the maize loci and 34 percent 
of the tomato loci evaluated by Helentjaris et al. (1986) segregated 
with abnormal frequencies. Without a careful estimates of gametic or 
zygotic selection, it is impossible to correct for distorted gene
26
CD
Tf
PO
'd-
Th
O
CO
ro
ro
CO
PO
CO
PO
N
CO
CXJ
IO
N
CXJ
- -WX
--xKSUII
n
IkZ
-XlVISU 12 
-xMSU Il
Th
CO
PO
Q
CO
PO
I r IO1 r
IO
CO
PO
IO
O
PO
Th
dro
Th
ic
PO
--xMSU 22
Per I 
Per 2
- - v
--XKSU2I
--xMSU 21
— Wst11B
Figure 9. Barley genetic linkage maps of chromosomes I and 2. 
Distances listed are in percent recombination, not centimorgans. All 
the data were analyzed by the maximum likelihood method using 
computerized program, Linkage-1. The detailed distances show in table 
4.
43
.0
27
- - a  I
0)
CD
ro
d^-
Sro
Mi*
03 
CO
-xKSU 32
xKSU 31 
Est 2
Est I
a O) n
*1*
CDro
CQ
<d- CO
COro
Hor 2 
Hor I
XlVISU 51 
Pgd 2
Figure 10. Barley genetic linkage maps of chromosomes 3 and 5. 
the data are listed in Table 4.
All
28
1Q a
0)v
IO
ro
ro
CO
CU
CO
(d
CVl
; 1 i
ro
’d-
OvJ
(T) 
CO 4r 
COa
- Y C D w __
ro
rd
CVJ
CO
R5
•xMSU 71 
Rrn 2 
xKSU 71
rxKSU 72 
s
■xMSU 72
xKSU 73  
r
--xMSU 73
--xMSU 74
Figure 11. Barley genetic linkage map of chromosome 7. All the data 
are listed in Table 4.
29
segregation (Heum, 1987) . He therefore recommend caution in utilizing 
data involving these loci and their probes.
The probes and markers utilized in this project span 680 
recombination units of the barley genome, approximately 50 percent of 
its estimated recombinational length. Kleinhofs et al. (1988) recently 
published an RFLP map of barley chromosome 6, increasing the amount of 
coverage of the barley genome to well over 50 percent.
Release of these clones along with release of the 100 mapping 
lines (detailed in Chapter 4) produced in this effort provides two sets 
of tools which will simplify the mapping of additional RFLP loci.
30
CHAPTER 4
,
PHYSICAL MAPS OF SEVENTEEN INFORMATIVE DNA MARKERS 
AND DESCRIPTION OF 100 MAPPING LINES
Introduction
The recently developed technique, restriction fragment length 
polymorphism (RFLP) analysis, provides the potential for an unlimited 
number of genetic markers which could be utilized not only to saturate 
genetic linkage maps but also to estimate intervarietal and 
interspecific genetic relationships among plant genomes. RFLPs are 
dependent upon mutational events, either point mutations, insertion and 
deletion events, or rearrangements and inversions. To evaluate 
polymorphic DNA clones and identify the genomic locations of their 
homologous alleles in barley lines, well characterized genetic stocks 
are required. Recently, recombinant inbred lines (RIL) were developed 
in maize for the rapid mapping of molecular probes to chromosomal 
locations (Burr et al., 1988).
It is fortunate for barley geneticists to have available a well- 
marked master recessive stock (Wolfe, 1984) that provides a relatively 
large number of 'benchmarks'. Genotypes of one hundred F2 lines using 
10 morphological characters, 5 isozyme loci, 2 hordeins, and 17 RFLP 
loci were characterized (detailed in Chapter 3). In this chapter along 
with the genotypic matrix of 100 mapping F2 lines and thirty-four loci, 
seventeen polymorphic DNA fragments that have been mapped in barley 
linkage groups were characterized in detail by restriction mapping
31
analysis. At least 8 different restriction endonucleases were 
utilized to digest them either singly or in combination. DMA sequence 
analysis was perfomed according to Sanger et al. (1977) to provide a 
detailed physical map of pxMSU 21.
Materials and Methods
Genotype Characterization
Plant materials, isozyme loci, hordein loci, experimental 
techniques for DMA work and linkage analyses were described in Chapters 
2 and 3. Experimental materials were derived from the hybrids of MMS x 
Apex. The methods of Milan (1964) and the USDA handbook 'Barley'
(1979) were adapted to identify the genotypes for morphological 
characters. The pedicellate lateral selection method of Gilbertson and 
Hockett (1986) was used to identify genotypes for the v and i alleles.
Restriction Mapping
Plant materials, cloning, transformation, and clone selection 
procedures were identical to the previous chapters (Chapter 2 and 3). 
About 500 ng of plasmid DMA was digested with appropriate restriction 
endonucleases, either Bam HI, Hind III or Pst I, followed by at least 
7 different restriction endonucleases either singly or in combination. 
DMA fragments were separated in 0.8 % agarose gel, stained by ethidium 
bromide, and photographed. The size of each fragment was determined 
using the Hind III digested fragments of the phage Lambda as standards. 
The exact clockwise direction of each fragment in plasmid pBR322 was
also determined.
32
A fully detailed restriction map of pxMSU 21 was compared to 
double digestions of total barley genomic DNA from the cultivars 
'Betzes1 and "Robust" to identify the mechanism by which allelic 
variation was generated at the locus xMSU 21.
Nucleotide Sequencing
The Bam HI and Sst I digested fragment of pxMSU 21 containing 
approximately 500 base pairs was cloned into M13mpl8 and nucleotide 
sequenced using the dideoxynucleotide chain termination method of 
Sanger et al. (1977). This M13mpl8 RF and the bacterial strain NM522 
were utilized in this purpose as per manufacture's instruction.
Results and Discussion
Genotypes of 34 Alleles
The genotype matrix of 100 Fg mapping lines and 34 loci is listed 
in Table 5. Nomenclature for RFLP loci was explained in a previous 
chapter (Chapter 3). Digit labeled "11" are homozygous for the allele 
from "MMS", "12" is heterozyous, '22' indicates homozygosity for the 
allele from "Apex", and '99' means no data identified.
All the morphological characters were relatively simply 
determined. For isozyme loci wheat-barley addition lines and 
ditelosomic series were utilized to evaluate whether the alleles at the 
polymorphic loci in 'MMS' and 'Apex' were identical to previously 
located alleles. Twelve of seventeen RFLP loci and the isozyme Pgd2 
showed codominant segregation; otherwise, dominant patterns were 
observed in the Fg population.
Table 5. Genotypes of 34 loci in 100 mapping lines
L ocu i Him#
Mo t i t  t i t  P e r  P e r  Pgd HSU HSU H orHor HSU HSU wet HSU HSU HSU HSU HSU KSU KSU Kro KSU KSU KSU KSU KSU
wx e  lk 2  r  i l  o I  r  I I  2 I  2 2 13 21 2 I 12 22 , .B  71 11 12 7« SI 32 12 2 73 11 11 21 31
I 12 11 11 22 22 11 11 11 22 12 12 12 12 22 12 22
2 12 22 22 22 22 22 22 22 12 12 22 22 22 22 22 22
3 22 12 12 11 12 12 22 12 12 11 11 11 11 22 12 12
4 12 22 22 12 22 12 12 12 12 22 22 11 12 12 12 12
S 22 12 12 22 12 11 11 11 11 11 11 12 12 12 12 11
6 12 12 12 22 11 12 11 12 11 12 12 11 11 11 11 11
I 12 22 22 22 11 12 12 12 22 12 12 22 22 12 12 12
S 12 12 22 12 12 22 11 22 12 12 12 11 12 11 22 11
9 12 11 11 22 11 12 22 22 22 22 22 22 22 12 22 12
10 22 22 22 22 22 22 12 12 12 12 12 22 22 11 11 12
11 12 22 22 22 12 12 22 22 11 22 22 12 12 12 12 12
12 22 12 12 12 22 22 12 22 12 22 22 11 11 22 22 11
13 12 12 12 12 22 11 11 12 12 12 12 12 12 11 12 22
14 12 12 12 11 12 22 12 12 12 22 12 22 11 12 12 11
IS 12 12 12 12 12 12 12 12 12 22 12 12 12 22 22 12
16 22 11 12 22 12 11 22 12 22 11 12 12 12 12 12 12
11 11 12 12 12 12 11 11 11 11 22 12 11 12 11 11 22
IB 11 12 12 11 22 22 11 11 12 22 22 12 22 12 99 99
19 22 12 22 22 12 22 22 11 11 22 22 22 22 12 12 12
20 12 12 12 12 12 12 11 11 11 11 11 12 12 12 12 12
21 11 12 12 22 11 12 12 11 12 12 12 22 22 12 11 12
22 12 12 11 11 11 22 12 22 22 11 11 12 12 11 12 12
23 11 12 12 22 22 22 11 11 11 11 12 12 12 12 99 99
24 12 11 11 22 22 11 12 12 12 11 11 12 22 12 12 22
2S 12 11 11 22 12 12 11 12 11 22 22 12 12 11 22 99
26 12 22 22 12 12 12 12 12 12 22 22 12 12 11 12 99
27 22 12 22 11 11 12 12 22 22 11 11 12 12 12 22 11
28 12 12 11 12 12 12 22 12 12 12 12 12 12 11 22 11
29 22 12 12 22 22 22 11 11 22 12 12 12 12 22 11 12
30 12 11 11 12 22 12 11 11 11 12 22 12 12 12 12 12
31 22 12 12 22 12 11 12 12 12 22 22 22 12 11 12 12
32 12 11 11 22 12 22 12 12 12 11 11 12 22 11 12 11
33 12 11 12 22 12 12 12 12 12 12 12 12 12 12 22 12
34 12 22 22 12 12 12 12 12 12 22 22 11 11 12 12 12
35 12 11 11 22 12 11 22 22 12 12 12 11 12 12 12 12
36 22 12 12 22 12 22 22 12 12 22 22 22 22 12 12 12
37 12 11 11 12 11 12 12 11 12 22 22 12 12 11 11 11
38 22 12 22 22 12 12 12 12 22 12 12 11 12 12 12 11
39 12 22 22 12 11 12 11 11 12 12 12 22 22 11 12 12
40 22 12 11 11 12 12 11 11 12 11 11 12 22 12 11 12
12 12 11 12 22 11 11 11 22 22 12 11 99 11 12 11 12 11
12 12 22 12 22 22 22 22 12 22 99 22 11 22 11 12 22 11
12 12 12 11 12 11 11 11 22 22 12 22 11 12 22 12 11 11
12 12 12 12 12 11 11 11 12 22 11 12 11 12 22 11 22 11
22 22 11 12 12 22 12 11 12 22 12 11 22 11 11 22 11 11
12 12 11 99 12 11 12 11 22 11 99 11 11 11 22 12 11 11
11 12 12 12 22 11 22 22 12 11 99 12 11 12 99 12 12 11
22 22 22 11 12 11 11 11 11 11 99 11 99 22 99 12 11 11
11 11 22 12 12 11 11 11 11 22 99 22 11 22 11 n  J J  J J
12 11 12 12 11 11 11 11 U  11 99 i j  U  12 j j  22 J J  U
12 12 22 12 12 11 11 22 22 99 12 22 11 22 12 12 22 22
12 12 22 12 11 11 11 22 22 22 22 12 11 22 22 11 12 22
12 22 12 11 11 22 11 22 12 11 12 11 99 12 22 12 12 11
22 22 11 11 22 11 12 11 22 22 12 12 99 11 22 11 11 H
12 22 12 99 12 11 11 11 22 22 12 12 11 12 22 11 11 22
12 11 12 12 12 22 12 11 11 11 12 22 11 12 22 22 12 11
22 22 11 12 11 11 12 11 12 11 99 99 99 99 99 99 99 99
99 99 22 99 11 99 99 99 99 99 99 99 99 99 99 99 99 99
99 99 12 12 22 22 22 22 12 99 12 12 99 12 22 12 12 22
12 22 11 12 22 11 11 11 12 99 12 11 99 11 12 11 H  H
99 99 11 22 12 11 12 99 11 22 12 99 99 11 12 22 12 11
22 22 12 12 12 99 22 99 11 22 12 99 11 12 22 12 11 11
12 12 99 99 12 99 99 99 99 99 99 99 99 99 99 99 99 99
99 99 12 12 12 11 22 99 22 22 22 99 11 12 99 12 22 11
12 22 11 12 12 11 12 11 12 22 12 99 99 12 22 11 22 22
22 22 12 12 12 11 12 22 11 11 99 99 99 12 12 I J  i j  J 2
12 22 22 22 12 11 12 11 22 11 12 99 11 22 12 12 11 11
12 12 12 22 11 22 11 11 12 11 12 99 11 11 12 12 12 11
12 12 11 22 11 11 12 11 11 22 12 99 99 99 22 99 12 11
99 99 11 12 12 11 22 11 11 11 22 99 11 11 11 H  J J  n
12 12 12 12 22 22 12 11 99 11 99 99 22 99 99 99 99 99
11 12 12 12 22 11 12 11 99 22 12 12 11 12 22 11 12 11
12 12 12 12 22 11 12 22 99 22 22 11 11 12 12 11 22 11
99 99 12 11 11 22 12 22 99 22 22 12 22 12 22 22 11 22
12 12 22 12 22 11 22 22 99 22 12 22 11 99 11 99 11 11
99 99 12 22 11 22 12 11 99 22 11 12 J J  12 I J  J j  i j  j 2
22 22 11 99 12 11 22 99 99 11 99 99 99 99 99 99 99 99
12 12 99 99 11 11 11 99 99 99 99 99 99 99 99 99 99 99
12 12 11 12 22 99 11 99 99 11 99 99 11 11 99 12 99 99
11 11 11 12 22 11 12 99 99 22 99 99 11 11 99 11 99 99
Table 5. (continued)
Plant
■x n lk2 v si o s r
41
«2
43
44 
4 S
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60 
61 
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
12 22 22 
22 12 12 
11 22 22 
12 12 12 
22 12 22 
12 22 12 
22 12 12 
12 11 12 
11 12 22 
12 11 11 
12 12 12 
22 22 22 
12 11 12 
11 12 22 
12 22 22 
12 11 11 
12 12 11 
22 12 12 
22 11 11 
12 22 12 
12 12 22 
12 22 22 
12 12 11 
12 22 12 
22 22 12 
11 22 22 
12 22 12 
22 22 22 
12 22 22 
11 12 22 
11 22 22 
12 12 12 
12 11 11 
11 22 22 
12 12 12 
11 12 12 
12 12 22 
22 11 12 
12 12 12 
12 22 12
11 12 12 11 12 
11 22 12 22 12 
12 12 11 11 12 
12 12 11 22 22 
12 12 12 12 12 
12 22 22 22 22 
11 12 11 11 22 
12 22 11 11 11 
22 12 12 12 12 
22 11 12 22 22 
12 22 11 22 12 
22 22 12 22 12 
12 22 11 22 22 
22 11 22 12 22 
12 22 11 12 12 
12 22 11 12 11 
12 12 22 11 12 
11 12 11 12 22 
12 12 11 11 11 
12 12 12 22 11 
12 22 22 11 12 
12 12 22 12 12 
22 12 12 12 12 
12 11 12 12 11 
12 11 11 22 12 
22 22 22 11 12 
12 12 12 22 12 
12 22 12 22 22 
12 11 12 12 12 
12 12 12 11 11 
22 22 12 12 22 
12 11 11 22 12 
11 22 22 12 22 
22 11 22 22 22 
22 22 12 12 12 
12 12 12 12 12 
22 11 11 22 12 
12 12 11 12 12 
12 12 22 12 12 
22 12 12 11 12
I
22
22
12
11
12
12
12
12
12
12
12
12
11
22
12
11
12
12
12
12
12
12
22
12
12
11
22
12
22
12
22
12
22
22
11
12
12
12
11
12
Locus Uses
Est Kst Per Par Pgd MSU MSU Mor Mor MSU MSU wet MSU MSU MSU MSU MSU ESU ESU
I 2 I 2 2 73 21 2 I 72 22 Tl n 12 74 51 32 72
11 11 11 12 12 99 11 99 99 12 11 12 11 12 99 11 22 12 11
11 11 12 12 12 99 12 22 22 12 12 12 11 12 99 99 22 22 12
22 22 12 12 12 99 22 12 12 12 12 11 11 12 99 99 22 11 12
11 11 22 12 22 11 22 11 11 22 12 11 11 12 99 11 22 11 12
11 11 12 22 12 12 11 22 22 12 11 11 11 22 99 12 11 22 12
12 12 22 22 11 22 11 12 12 12 99 11 11 22 99 22 11 22 12
22 22 11 11 11 12 11 22 22 12 99 11 11 12 99 11 11 99 11
12 22 22 12 11 11 12 12 12 11 11 22 11 11 99 11 11 11 11
22 22 12 11 12 12 12 11 11 22 12 12 22 99 99 12 22 11 12
11 12 22 22 12 22 11 22 22 22 99 22 11 12 99 22 11 12 22
11 11 11 12 11 12 11 12 12 12 99 11 99 11 ii 22 11 12 22
12 12 22 22 22 12 12 12 12 12 99 22 11 12 11 12 22 11 12
12 12 12 12 12 22 12 11 11 22 99 12 99 99 11 99 22 22 22
11 11 11 12 12 11 22 12 12 12 99 12 11 22 22 11 22 11 12
22 22 11 11 12 12 22 22 22 12 99 22 11 12 22 99 22 12 11
12 22 12 12 12 12 22 12 22 12 99 12 11 12 11 99 11 22 99
11 11 12 12 12 12 12 11 11 11 99 11 11 22 11 99 11 11 11
11 11 12 12 11 22 12 12 12 12 99 12 11 22 99 99 11 12 12
12 12 22 12 11 11 12 12 12 11 99 12 22 12 99 n 11 11 11
12 12 12 22 11 11 11 11 11 12 99 11 11 12 11 12 11 12 12
22 22 22 22 12 11 11 12 22 12 12 11 11 22 99 11 22 12 11
22 22 22 22 12 12 99 22 12 12 11 12 11 11 11 11 22 22 11
11 11 12 12 12 12 12 22 22 12 12 22 11 12 11 11 22 12 12
22 22 ii 12 22 11 12 11 11 12 11 22 11 12 11 11 22 12 12
12 22 12 12 11 12 99 22 22 12 12 12 11 11 11 12 22 11 22
22 22 12 12 22 22 11 22 22 12 12 12 11 12 11 11 22 12 11
11 12 11 11 12 11 99 12 12 12 99 11 11 11 11 11 22 12 12
12 12 12 12 22 22 99 22 12 12 99 22 11 22 22 12 22 12 22
12 12 22 22 11 11 22 22 22 22 99 12 22 12 11 99 11 11 12
11 11 12 12 12 12 11 11 11 12 12 22 11 22 11 99 22 12 11
12 12 22 22 12 22 12 22 22 22 11 12 11 22 22 11 22 22 12
22 22 12 12 11 11 11 11 11 22 11 22 11 12 11 11 22 11 12
11 11 12 12 22 12 12 12 12 22 11 12 11 11 11 12 22 11 12
12 12 12 22 22 22 12 11 11 22 12 12 22 11 99 22 22 12 22
12 12 22 22 11 12 12 22 22 99 12 12 11 12 11 12 11 22 12
11 11 12 12 12 11 22 22 22 99 99 22 11 12 11 11 11 12 99
12 12 12 12 22 22 22 11 11 12 12 22 11 12 99 22 22 22 12
12 12 12 12 22 12 99 12 12 22 12 12 11 12 ii 12 22 12 22
12 12 12 12 11 22 99 12 12 99 11 12 22 12 99 11 22 12 12
12 22 22 22 12 12 12 11 12 12 99 12 11 11 «9 12 22 12 99
Krn KSU KSU KSU KSU KSU 
2 73 11 71 21 31
99 99 12 99 99
12 99 12 11 99
99 99 12 12 99
22 99 12 12 99
99 99 12 11 99
12 99 12 12 99
99 99 99 99 99
11 99 12 12 99
12 99 22 22 99
22 99 11 12 99
12 22 22 12 99
12 22 12 22 99
22 11 12 22 99
12 99 12 12 99
12 12 12 22 99
11 11 12 12 22
11 99 11 12 11
22 12 11 12 11
11 12 22 12 11
11 11 11 12 11
12 22 11 11 22
12 12 11 11 22
12 12 12 12 11
11 22 12 12 22
12 22 12 11 11
12 99 11 12 22
12 12 11 12 11
22 12 12 12 11
12 22 22 12 11
11 12 12 11 11
22 99 12 12 99
22 99 12 11 11
22 11 99 12 11
22 99 22 12 11
12 22 11 22 11
12 99 12 12 11
22 12 12 22 11
22 12 11 11 11
12 22 22 12 11
12 99 11 22 99
Table 5. (continued)
Plant
Ho.
wx n 112 V al O • r I
Kat
I
Bat
2
Iar
I
Par
2
Pgd
2
81 22 12 12 22 11 12 12 ii 12 12 12 12 12 12
82 12 11 11 22 12 22 22 12 22 12 12 12 12 11
83 12 22 22 12 22 12 11 12 12 11 11 22 12 12
84 12 12 12 11 12 12 12 12 22 ii 11 11 11 22
85 12 12 11 12 22 11 11 12 12 22 22 12 22 12
86 12 12 12 12 22 12 11 11 12 22 22 12 12 12
«7 22 11 11 12 12 22 12 22 12 12 12 12 12 11
88 11 12 12 12 22 12 22 11 12 22 12 11 11 12
89 22 11 11 12 22 12 12 12 12 22 22 12 12 22
90 12 22 22 22 22 12 22 12 11 11 22 12 12 12
91 22 11 11 22 22 22 22 12 12 12 12 11 11 22
92 U 22 12 12 22 22 11 11 12 22 22 22 12 12
93 12 22 22 12 12 12 12 12 12 12 12 22 22 12
94 12 12 12 11 22 22 11 11 12 11 11 22 12 12
95 11 99 99 99 11 99 99 99 99 11 11 22 22 22
96 22 12 12 11 12 22 12 22 22 12 22 11 11 12
97 22 12 12 11 11 11 22 12 12 22 22 12 n 22
98 22 22 22 22 22 11 12 12 22 11 11 22 22 22
99 11 12 22 22 ii 22 12 12 22 12 12 12 12 11
100 12 22 22 12 11 22 22 12 12 12 12 12 12 22
Locua Warn# ~
O lor Bor HSU HSU .at HSU HSU HSU HSU HSU ESU ESU Irn ESU ESU ESU ESU E 
" J I 72 22 ,, B 71 11 12 74 SI 12 72 2 71 11 Tl Ji
11
12
11
11
12
11
12
12
*»
12
22
11
22
11
12
12
12
22
12
22
99
99
99
99
99
99
99
99
99
99
99
99
99
99
99
99
99
99
99
99
22
11
22
12
11
22
12
22
11
22
22
12
12
11
12
12
12
11
22
22
11
99
11
22
99
22
11
11
11
11
11
11
11
99
22
11
11
11
11
11
99
99
99
99
99
99
99
99
99
99
99
99
99
99
99
99
99
99
99
99
11
11
22
99
11
99
22
22
11
11
99
99
99
99
99
99
99
99
99
99
11
11
11
12
22
22
12
22
22
12
99
99
99
99
99
99
99
99
99
99
22
22
22
22
22
11
11
22
22
22
99
99
99
99
99
99
99
99
99
99
12
11
12
22
12
12
11
12
12
12
99
99
99
12
12
12
12
22
12
11
12
22
11
12
11
11
12
22
12
12
99
99
99
99
22
12
11
12
99
12
11
11
11
22
11
22
22
11
11
11
11
11
99
22
99
22
22
11
11
11
11
12
11
11
12
11
12
11
12
12
99
11
22
11
12
12
12
12
12
12
99
99
12
12
11
99
99
11
12
12
99
99
99
99
12
22
22
22
99
22
12
12
11
22
12
22
12
12
12
12
99
11
11
22
11
12
12
12
12
12
U»
in
36
Release of these 100 mapping lines along with 17 informative DNA 
clones will provide a 'master template' for locating additional RFLP 
loci in the barley linkage mapL Development of saturated genetic 
linkage maps with the relatively unlimited DNA markers will likely 
allow the identification of agronomically important quantitative trait 
loci.
Physical Maps of DNA Markers
Fifteen clones out of seventeen polymorphic DNA markers were 
physically mapped using several restriction endonucleases as shown in 
Figure 12. Two clones out of seventeen were not utilized in this 
project, since pTA71 was characterized by Gerlach and Bedbrook (1979) 
and Saghai-Maroof (1984), and pxMSU 22 is available only in the virus 
vector EMBL4.
Twelve clones were well characterized by one or more restriction 
endonucleases (Figure 12). Three cDNA clones pxKSU 32, pxKSU 71 and 
pxKSU 73 had no site using 13 or 14 different restriction 
endonucleases, but had distinctive fragment sizes ranging from 500 to 
650 base pairs.
The clone pxMSU 21 was utilized to determine the mechanism by 
which variation was generated between the cultivars 'Betzes' and 
'Robust'. Total genomic DNAs of two barley cultivars, 'Robust' and 
'Betzes1, were doubly digested with combinations of Bam HI and either 
Sst I or Hind III, electrophoresed and transferred to a nylon membrane.
4
2.4 Kbp with Bam HI
37
pxMSU 11
Sst I 
Xho I 
Pst I
* *
1.4 0.7 0.3
* *
TTt o.4 O
* *
T H  ' 0.8 . oTI
2
2
2
(No site identified)
Bgl II, Xba I, Hind III, Eco RI, Eco RV, Dra I, Kpn I, and Bel I
pxMSU 12 2.5 Kbp with Bam HI
Xba I * * 2
Sst I
1.0 0.7 0.8
* I
Xho I
0.7 1.8
* I
Hind III
0.45 2.15
* I
Eco RI
2.1 0.4
* I
Eco RV
2.0 0.5
ft I
(No site
1.1 1.4
identified)
Bgl II and Dra I
pxKSU 11 0.73 Kbp with Pst I
Bgl II _____*_______________ I
0.17 0.56
Xho I ________*____________ I
0.28 0.45
Eco RV _________*___________ I
0.3 0.43
(No site identified)
Xba I, Sst I, Hind III, Eco RI, Dra I, Kpn I, and Bel I
Figure 12. Restriction maps of 15 informative DNA clones which have 
been mapped in barley chromosomes. All the sizes of fragments are in 
kilobase pairs. Total size of each clone is described with the cloned 
site restriction endonuclease
V38
Figure 12. (continued) 
pxMSU 21 2.7 Kbp with Bam HI
Sst I * ft 2
Hind III
0.5 0.8
*
1.4
I
BstE II
1.0
*
1.7
I
Pst I
0.4 2.3 
' * I
Taq I
1.6 
ft ft
t—I 
1—1
2
Rsa I
0.3 0.2 
* *
2.2
* 3
0.1 0.7 0.8 1.1
(Mo site identified)
Bgl II, Xho I, Eco RI, Eco RV, Dra I, Kpn I, Bel I, and Sal I
pxKSlf 21 0.56 Kbp with Pst I
Xho I _______*__________ I
0.23 0.33
(No site identified)
Bgl II, Xba I, Sst I, Hind III, Eco RI, Eco RV, Dra I, Kpn I, and 
Bel I
pxKSU 31 0.73 Kbp with Pst I
Kpn I __________________*____ I
0.6 0.13
(No site identified)
Bgl II, Xba I, Sst I, Xho I, Hind III, Eco RI, Eco RV, Dra I, and 
Bel I
pxKSU 32 0.65 Kbp with Pst I
None of the following enzymes identified restriction site.
Bgl II, Xba I, Sst I, Xho I, Hind III, Eco RI, Eco RV, Dra I, 
Kpn I, Bel I, and Bam HI
39
Figure 12. (continued)
pxMSU 51 2.7 Kbp with Bam HI
Xba I * I
1.9
Hind III *
0.8
I
Eco
1.7
RI
1.0
* I
Dra
2.3
I *
0.4
I
0.5 2.2
(No
Bgl
site identified)
II, Sst I, Xho I, and Eco RV
nxMSU 71 3.9 Kbp with Bam HI
Xho I ft I
2.6
Hind III *
1.3
ft 2
Eco
0.4 2.4 .
RT ft
i.i
I
2.2 1.7
(No
Bgl
site identified)
II, Xba I, Sst I, Eco RV, and Dra I
DXMSU 72 2.1 Kbp with Hind III
Pst I * I
1.2 0.9
(No
Bgl
Kpn
site identified)
II, Xba I, Sst I, Xho I, 
I, and Bel I
Eco RI, Eco RV, Dra I, Bam HI,
DXMSU 73 5.B Kbp with Bam HI
Bgl II ft I
Xba
4.6
I * *
. I-2
2
Sst
2.5 0.6
I * *
2.7
2
0.3 2.0 3.5
40
Figure 12. (continued)
Hind III A ft 2
Eco RI
2.3 2.0 1.5
ft I
Eco RV
4.0
* a 
» OO
Dra I
3.6
*
1.4 0.8
* 2
1.0 4.1 0.7
(No site identified)
Xho I
pxMSU 74 1.5 Kbp with Hind III
Bgl II __________*_____ I
1.0 . 0.5
Eco RV ____ -*__________ I
0.5 1.0
Dra I ____*___________ I
0.4 1.1
(No site identified)
Xba I, Sst I, Xho I, and Eco Rl
pxKSU 71 0.65 Kbp with Pst I
None of the following enzymes identified restriction site.
Bgl II, Xba I, Sst I, Xho I, Hind III, Eco.RI, Eco RV, Dra I, 
Kpn I, and Bel I
pxKSU 72 0.6 Kbp with Pst I
Bgl Ii _________*___ I
0.45 0.15
Sst I ___*_________ I
0.15 0.45
(No site identified)
Xba I, Xho I, Hind III, Eco RI, Eco RV, Dra I, Kpn I, and Bel I
41
pxKSU 73 0.56 Kbp with Pst I
None of the following enzymes identified restriction site.
Bgl II, Xba I, Sst I, Xho I, Hind III, Eco RI, Eco RVr Dra I,
Kpn I, and Bcl I
Labeled pxMSU 21 was hybridized to the filter (Figure 13). In the Bam 
Hl/Sst I digestion, fragments of 800 bp and 1.4 Kbp of Robust were the 
same size as the Betzes fragments. However, the Betzes allele 
contained about 200 base pairs of inserted sequence within the 500 bp 
fragment, as is shown in Figure 13.
Sequence of Polymorphic Region
When sequence information for allelic variants is available, 
allele-specific oligonucleotides (ASOs) (Erlich et al., 1986) can be 
synthesized that detect single base substitutions and identify allelic 
variants using simple dot blot procedures. In human genetics, 
synthetic oligonucleotides were applied to detect genetic diseases, 
sickle cell disease and thalassemia traits in the prenatal stage 
(Conner et al., 1983; Kazazian, 1985). The rationale of using short 
DNA oligomers as probes for the detection of oligonucleotide 
polymorphisms in agricultural species was reviewed and suggested by 
Beckman (1988).
An approximately 500 bp size fragment digested by Bam HI and Sst 
I, which identifies polymorphisms, was sequenced using the
Figure 12. (continued)
42
Figure 13. Autoradiograph of double digested genomic blot hybridized 
with probe pxMSU 21. (Right); All digits indicate kilobase pairs. 
Arrows indicate polymorphic bands. Letters indicate as following; BHl; 
Bam HI, SI: Sst I, H3: Hind III, R: Robust, B: Betzes. (Left); Betzes 
allele contains about 200 base pairs (bp) of inserted sequence within 
the 500 bp fragment. Letters indicate as following; B: Bam HI, S: Sst 
I, H: Hind III.
43
dideoxynucIeotide chain termination reaction (Sanger et al. , 1977) 
(Figure 13). A total of 474 base pairs including the cloning site 
were sequenced (Table 6). The sequence was AT rich (70 %) and 
contained inverted repeat sequences which were eight base pairs and 
six base pairs long. The locus xMSU 21 probably contains a deletion 
in the allele in 'Robust' relative to the 'Betzes' allele. To prove 
this hypothesis, further sequencing in the allelic polymorphic 
fragment in the Betzes type cultivars is required.
Table 6. Sequence data of 474 bp from sequence gel of Figure 14. 
Bases underlined exhibit inverted repeat sequences.
GRATCCCATA TTTATGATAT TTTGGTCTTT CATRTACCTA CCTAVTATGC GTATCCATAA 60
Bamril
TARATACATA CCAATTTATC CAAGTACATA ATCAGAAAAC GCAAGACTAA AGATGCCCAT 120
Gi TGTTGGAT CAGCCATTTC TAGT TTCA I C CTTGTGCCTT GTAACICAAA ACAGTGTAAA 1 Go
CAGCTTGGAC AACCGCACAA ACT AGCCCGG TGATACCTGC CAGGARCCCA AI GAGCAGCC 240
AGGGATTGCT AAAGTATT TC TGCCCTAGCC ACACCATCGA TC TCCGAAAA TAAACT TGGC TOi)
GGATATGGCT CCRRRACCGG GACCGCATGT CTAGCTTCAA GCA TATCTI C AiCABGTAiGTT T60
GC-TRGCC T I G TTGTTAGGGT TC, AACAAGA T CCCTTGCAAG AT CACCGAAG CACTCGGCCA 420
CCTCRTCATT ATTGCGGTGG TTRTGCTTGA T GACiiCA T TC C ICGACAGRA RCTCGAATTC 480
Sstl
GTAATCAtRG TCAT 494
44
T G C A
Figure 14. Sequence of Bam HI and Sst I double digested pxMSU 21 
fragment that identified a polymorphism. The double digested fragment 
was cloned into M13mpl8 and sequenced using a single-stranded DNA 
template. Part of sequence gel is shown. Letters indicate as 
following; A: Adenine, C: Cytosine, G: Guanine, T: Thymine, Bam HI: Bam 
HI cut sequences which are located at the cloning site.
45
CHAPTER 5
BARLEY VARIETAL DISCRIMINATION 
USING RFLP AND HORDEIN MARKERS
Introduction
Estimates of genetic relationships among cultivars provide useful 
information to solve the related problems of varietal identification, 
purity and origin. Several approaches have been utilized to provide 
these estimates. Common methodologies are based on pedigree analysis 
(Cox et al., 1985; Delannay et al., 1983; Smith and Smith, 1988), 
quantitative characters (Jain et al, 1975; Martinez et al., 1983; Price 
et al., 1984), isozyme variation (Linde-Laursen et al., 1987; Nielsen 
and Johansen, 1986), and polymorphisms in storage proteins (Gebre et 
al., 1986; Linde-Laursen and Doll, 1982; Shewry et al., 1978). The . 
advantage of biochemical markers over most morphological markers is 
that molecular markers are generally selectively neutral, often large 
in number, and may be evaluated at many plant growth stages.
Levels of genetic diversity, geographic structure and 
environmental correlation with allozyme variation have been studied in 
natural populations of wild barley, Hordeum spontaneum (Brown et al., 
1978; Brown et al., 1980; Nevo et al., 1986) . Two concepts of genetic 
diversity were utilized. First, 1 allelic richness1, which is the 
average number of alleles per locus, indicates the number of distinct 
kinds of alleles encountered in a sample of a particular size. The 
second component of genetic diversity, 'evenness of allele
46
frequencies', is related to the distribution of allelic frequencies. 
'Gene diversity' (Nei, 1973) is the most common measure of evenness 
within and between populations. This method is applicable to any 
population without regard to the number of alleles per locus, the 
pattern of evolutionary forces such as mutation, selection, and 
migration, and the reproductive method of the organism used.
In previous chapters, seventeen barley RFLP DNA markers were 
selected, characterized, and their genomic locations mapped. The 
probes selected identified different alleles in 'MMS' and 'Apex'. In 
this chapter, the number of different banding patterns identified using 
each probe in Southern blot hybridizations to DNA from 21 barley 
cultivars was estimated. The amount of genetic diversity identified 
with each probe was then compared to that identified by the seed 
storage protein loci. The objective of this chapter was to determine 
the relative utility of RFLP markers in. cultivar identification.
Materials and Methods
Plant Materials
I
Twenty-one barley cultivars grown in North America and Europe for 
malting and feed were selected. Their parentage, origin, spike row 
number, and common usage is given in Table 7.
Data Collection
SDS-polyacrylamide gel electrophoresis for hordeins was performed 
as previously described (Blake et al., 1982). The DNA procedures for 
Southern blot analysis were the same as described in Chapter 2.
47
Table 7. Barley cultivars utilized to identify genetic relationships 
using polymorphisms of hordeins and RFLP DNA markers.
No. Cutivar Pedigree Origin
Row
type Common use
I Klages Betzes/Domen USA 2 Malting
Malting2 Andre Klages/Zephyr USA 2
3 barker Traill/UM 570 USA 6 Malting
4 Ingrid Balder/(Binder/Opal) SWE 2 Feed
5 Robust Morex/Manker USA 6 Malting
6 Bellona Aramir 2*/Bomi NET 2 Feed
7 Clark Hector/Klages USA 2 Malting
8 Azure Bonanza/Nordic/NBD130 USA 6 Malting/Feed
9 Piroline W.M.C.P./Morgenrot GER 2 Malting/Feed
10 Menuet L92/Minerva//Emir/3/Zephyr NET 2 Feed
11 Hazen Glenn/4/Nordic//Dickson
/Trophy/3/Azure
USA 6 Feed
12 Harrington Klages/S72114 CAN 2 Malting
13 Morex Cree/Bonanza USA 6 Malting
14 Compana from Composite Cross I USA 2 Feed
15 Columbia USA 6 Feed
16 Apex Aramir/4/CB6721/3/Julia 3* 
/Volla/LlOO
NET 2 Feed
17 Moravian III Moravian/Firlbecks III// 
Moravian/Saxonia
USA 2 Malting
18 Dicktoo Winter barley field selection USA 6 Feed
19 Hector Betzes/Palliser CAN 2 Feed
20 Summit HPl203/Zephyr/Tern ENG 2 Feed
21 Traill Kindred/Titan USA 6 Malting
Abbreviation used in origin; USA: United States of America, SUE: 
Sweden, NET: Netherlands, GER: Germany, CAN: Canada, ENG: England
The products of the Hor-I and Hor-2 have been well characterized 
by several authors (Shewry et al., 1978; Marchylo and Laberge, 1981; 
Gebre et al., 1986; McCausland and Wrigley, 1977). Each B or C hordein 
variant identified was given a number, the banding patterns identified 
among the Southern blots of the 21 cultivars evaluated were also each 
given a number and different banding patterns assumed equivalent to 
different alleles.
48
Statistic Analysis
Allelic frequency for each locus and estimates of gene diversity 
were calculated (Brown and Weir, 1983; Nei, 1973) using following 
equations;
(1) Allele frequency at a locus:
frequency of an allele at a locus
x = _________________________________
total number of tested samples
(2) Gene diversity:
H = I -  Gene Identity = I - £ x 2
A data matrix of scored 'alleles' was utilized to construct a 
dendrograph using the phylogenetic analysis program, PAUP (Swofford, 
1985) . This dendrograph provided a graphic representation of the 
estimated intervarietal genetic distances among these 21 barley 
cultivars.
Results and Discussion
Polymorphic banding patterns for each of the listed RFLP loci are 
shown in Figures 15 - 21. Using the data matrix described in Table 8, 
allele number for each locus (ranging from 2 to 10) and allele 
frequency at each locus (ranging from 0.05 to 0.95) are presented in 
Table 9. The average number of alleles per locus which was calculated 
has the obvious merit of emphasizing one component of diversity, namely 
allelic richness.
Hordeins showed much more richness of mean alleles {= 9.00) than 
of polymorphic DNA markers (=3.571). The high allelic richness of
x KSU 21
Figure 15. Polymorphic allelic patterns of pxKSU 21. Fifteen 
micrograms of total barley DNA of 11 cultivars were digested with Eco 
RV restriction endonuclease, transferred to nylon membrane and 
hybridized by probe pxKSU 31. Digits indicate cultivars identical to 
numbers of Table 8. Digits indicate polymorphic alleles identified. 
Letters indicate as following; Ha: Hazen, Hr: Harrington, Mo: Morex,
Co: Compana, Cl: Columbia, Ap: Apex, M3: Moravian III, Di: Dicktoo, He: 
Hector, Sn: Summit, Tr: Traill.
50
Figure 16. Polymorphic allelic patterns of pxKSU 32. DNA from 11 
barley cultivars were digested with Eco RV restriction endonuclease. 
Digits indicate polymorphic alleles identified. Letters indicate as 
following; Kl: Klages, An: Andre, La: Larker, St: Steptoe, In: Ingrid, 
Ro: Robust, Be: Bellona, Cr: Clark, Az: Azure, Pr: Piroline,
Me: Menuet.
51
Figure 17. Polymorphic allelic patterns of pxMSU 21. DNA from 9 
barley cultivars were digested with Hind III restriction endonuclease
Figure 18. Polymorphic allelic patterns of pxMSU 11. DNA from 11 
barley cultivars were digested with Hind III restriction endonuclease
52
. %
Ha Hr Mo Co Cl Ap M3 Di HeFsu Tr
Figure 19. Polymorphic allelic patterns of pxKSU 11. DNA from 11 
barley cultivars were digested with Hind III restriction endonuclease.
Ha Hr Mo Co Cl Ap M3 Di He Su Tr
IMi
1 1 1 3 2 1 1 1 3 3 1
x KSU 71
Figure 20. Polymorphic allelic patterns of pxKSU 71. DNA from 11 
barley cultivars were digested with Bam HI restriction endonuclease.
53
Ha Hr Mo Co Cl Ap M3 Di He Su Tr
-«*#**» ***## #
—
I 4 3
• #
3 3 3 5 1 3 1
x KSU 31 ^
Figure 21. Polymorphic allelic patterns of pxKSU 31. DNA from 11 
barley cultivars were digested with Hind III restriction endonuclease.
54
hordeins is the reason for their common use in varietal identification. 
The mean allele number of the tested DNA markers was about one-third of 
the hordeins. As the polymorphic DNA markers are unlimited in number 
and as the total allele number of the seven DNA markers (=25) was more 
than that of 2 hordeins (=18), this technology can be utilized to 
supplement problems in varietal identification insoluble through the 
sole use of the hordeins.
Table 8. Data matrix of polymorphic alleles among 21 barley cultivars.
Cultivar xKSU
21
xKSU
32
Polymorphic loci 
xMSU xMSU xKSU xKSU 
21 11 11 71
xKSU
31
Hor
2
Hor
I
Klages I I I I I I I I I
Andre I I I I 2 I . I 4 I
Darker I I 2 2 I I I 5 6
Ingrid I .1 I 2 2 3 2 3 2
Robust I I 2 2 I I 3 5 6
Bellona I 2 3 2 2 3 I 7 3
Clark I I I I I 3 I I I
Azure I I 2 I I I I 8 6
Piroline I I I 2 3 3 3 9 4
Menuet I I I 2 3 3 I 4 2
Hazen I I . 2 2 I I I 5 6
Harrington I I I I 2 I 4 3 I
Morex I I 2 2 I I 3 5 6
Compana I I I 2 3 3 I 9 . 4
Columbia I I 2 I I . 2 3 I 8
Apex I 2 4 2 3 I 3 4 2
Moravian III I I 5 2 2 I 3 4 2
Dicktoo 2 I 2 2 4 I 5 10 7
Hector I I I I I 3 I I 4
Summit I I 6 I 5 3 3 I 5
Traill I I 2 2 I I I 6 6
The probabilities of nonidentity of 9 genetic loci were given by 
the Equation 2 and described in Table 9. The probability of 
nonidentity, H, is a measure of genetic variation of a population and
55
usually called heterozygosity. The H value in this varietal 
identification population implies sufficient gene diversity to 
discriminate cultivars. The overall mean of gene diversity was 0.554. 
Average gene diversities from two different methodologies showed that 
of hordeins is greater than that of DNA markers (0.829 > 0.476). This 
estimate is well associated with the allele richness. However, the 
degree of gene differentiation of this population using DNA markers 
(0.476) was higher than the previously reported average gene diversity 
across 30 allozyme loci in Hordeum spontaneum populations (0.096) (Nevo 
et al., 1986).
Table 9. Analysis of allele frequency in a locus and gene diversity 
among 21 barley cultivars.
Locus
No. of 
alleles
No. of 
sample
Allele frequency 
at a locus
Gene
diversity
pxKSU 21 2 21 0.95, 0.05 0.095
pxKSU 32 2 21 0.90, 0.10 0.180
pxMSU 21 6 21 0.43, 0.38, 0.05 x 4 0.660
pxMSU 11 2 21 0.38, 0.62 0.471
pxKSU 11 5 21 0.48, 0.24, 0.19, 0.05 x 2 0.785
pxKSU 71 3 21 0.57, 0.05, 0.38 0.528
pxKSU 31 5 21 0.52, 0.33, 0.05 x 3 0.613
Mean 3.571 0.476
Hor-I 8 21 0.19 x 2, 0.29, 0.14, 0.05 x 4 0.814
Hor-2 10 21 0.24, 0.19 x 2, 0.09 x 2, 0.843
0.05 x 4
Mean 9.000 0.829
Some cultivars undifferentiated by hordeins were well separated 
using subset of the DNA markers. In the first case, while 'Klages' and
56
'Clark' both had the same allele types of hordeins, they were 
differentiated ('Klages' = I and 'Clark' = 3) by pxKSU 71. Second, 
'barker', 'Robust', 'Hazen', and 'Morex1, all had allele type 5 in Hor- 
2 and 6 in Hor-I. 'barker1 and 'Hazen1 were different from 'Robust' 
and 'Morex1 in pxKSU 31, but 'barker' and 'Hazen', and 'Robust' and 
'Morex1 were not further separated from each other. To discriminate 
between those two, more polymorphic DMA markers should be tested.
Third, 'Piroline' and 'Compana' were further discriminated by pxKSU 31. 
Finally, The polymorphic DMA markers pxKSU 32, pxMSU 21, pxKSU 11, and 
pxKSU 71, readily discriminated among three cultivars 'Menuet', 'Apex' 
and 'Moravian III1 that showed the identical allelic patterns in 
hordeins.
The tree of genetic relationships (dendrograph) among these was 
constructed by genetic similarity and genetic distances using the 
computer program, PAUP (Figure 22). As in previous papers (Shewry et 
al., 1978; Gebre et al., 1986), cultivars related by common ancestry 
tended to cluster together. Varieties were divided into 6 major 
groups; (I) Western 2-rowed malting and feed barley cultivars derived 
from Betzes (Klages, Andre, Harrington, Hector, and Clark), (2)
European 2-rowed malting and feed cultivars (Ingrid, Bellona, Menuet, 
Moravian III, Apex, Piroline, and Compaha), (3) Western 6-rowed feed 
barley (Columbia), (4) blue aleurone colored 6-rowed midwestern malting 
barley (Azure), (5) white aleurone 6-rowed malting cultivars (Robust, 
Morex, Hazen, Traill, and barker), (6) old 6-rowed winter barley 
cultivar (Dicktoo).
57
: ' /./V.'.T.
*. . - ‘
A
A
A
ft
ft
ft
ft
32
* I KLAGES
40
A * 2 ANDRE
***6*22
A Aftftftftftft 12 HARRINGTON
ft ft*** 4 INGRID
ft *6*6*24
A ***30 **************
ft ft ft
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Figure 22. Dendrograph of 21 barley cultivars using the genetic 
distances given by computer program, PAUP.
58
Although barley varieties can be divided into groups on the basis 
of relatively discrete morphology, polymorphic DMA markers and 
biochemical markers identify variation within groups with similar 
morphologies and growth habit. DMA markers as well as biochemical 
markers could be utilized to differentiate barley cultivars in the case 
of seed mixture or varietal mislabeling.
y
59
CHAPTER 6 
SUMMARY
This study focussed on the linkage analysis of molecular genetic 
markers based on restriction fragment length polymorphisms in the 
barley genome. For this purpose, barley random genomic DNA libraries 
were constructed using the plasmid vector pBR322 and the phage vector 
EMBL4. Repeat-free sequences from these libraries were screened 
against total genomic DNA using Southern blot analysis. Nine genomic 
clones and seven cDNA clones were identified which produced clear, 
consistent results in Southern blot analyses of segregating progeny. A 
multiple recessive marker stock as one parent provided seventeen 
'benchmark' loci which had been previously mapped.
Seventeen RFLP markers, which utilized ten morphological markers, 
five isozyme loci and two hordeins as reference points in map 
construction, were located in barley linkage groups with the exceptions 
of chromosomes 4 and 6. The probes and markers utilized in this work 
span 680 recombination units of the barley genome, approximately 50 
percent of its estimated recombinational length.
Physical maps of fifteen out of seventeen polymorphic DNA markers 
were constructed in detail using several restriction endonucleases. 
Twelve DNA clones were well differentiated using one or several 
restriction endonucleases. Three cDNA clones had no restriction site 
in 13 to 14 different restriction endonucleases, but had characteristic 
fragment sizes ranging from 500 to 650 base pairs. A total of 474 base
60
pairs of the polymorphic region of pxMSU 21 were also characterized by 
sequence analysis.
Seven polymorphic DMA markers and hordein traits were 
characterized for estimation of genetic diversity and allele frequency 
among 21 barley cultivars. Some cultivars undifferentiated by hordeins 
were well separated using a subset of the DMA markers. This technology 
could be utilized to supplement problems in varietal identification 
insoluble through the sole use of the hordeins.
The release of new informative RFLP markers with detailed 
restriction maps and the description of genotypes at thirty-four loci 
in 100 mapping lines provides two sets of tools which will simplify the 
mapping of additional RFLP loci in barley. Me believe that this will 
provide the starting point for tracking agriculturally important 
quantitative trait loci in barley breeding programs.
IV‘-
61
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