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Contents
The Concept     1. Brief summary of Mendelian Inheritance
Phenotype Descriptions    2. Head and kernel characters
Educational Resources    3. NEPs segregating in Wolfe Barley
Maps and Map Data    4. How to score some morphological traits
Quantitative Trait LOCI (QTLs)  
Getting Seed of OWBs
Protocols
Who is Involved and How?

Brief Summary of Mendelian Inheritance

Segregation, independent assortment, and linkage: examples from the OWB

  1. Monohybrid Model(Segregation)
  2. Dihybrid Model


1.Monohybrid Model (Segregation)

Example: Number of kernel rows (Vrs1/vrs1) in barley (Hordeum vulgare)

Generation:             Parent 1        X       Parent 2 
Genotype:               Vrs1Vrs1                vrs1vrs1
Phenotype:              Two-row                 Six-row

Generation:                             F1 
Genotype:                               Vrs1vrs1
Phenotype:                              Two-row

If the F1 is selfed to give an F2 generation,  the  genotypic ratio  will be    
1Vrs1Vrs1; 2Vrs1vrs1; 1vrs1vrs1
and the phenotypic  ratio will be 
3 two-row: 1 six-row

Since the OWB is a doubled haploid population there are no heterozygotes.
Thus, the expected phenotypic and the genotypic ratios are the same 
1 Vrs1Vrs1: 1 vrs1vrs1
1 two-row: 1 six-row

The actual numbers in the OWB are 40 Vrs1Vrs1 and 54 vrs1vrs1.

A chi-square test can be used to test the goodness of fit of the expected and observed 
ratios.

Phenotype       #Observed       #Expected       O - E           (O - E)2/E
Vrs1Vrs1        40              47             -7               1.04
vrs1vrs1        54              47              7               1.04
Totals          94              94              0               2.08 (chi square; X2)
at 1 df  0.10<(chi-square greater than or equal to 2.08)<0.25

This chi-square is well within the realm of acceptance, so we conclude that there is indeed a 1:1 
ratio of Vrs1Vrs1:vrs1vrs1 genotypes in the OWB.

2. Dihybrid Model

a. Segregation and independent assortment

Example: Number of kernel rows (Vrs1/vrs1) and seedling white stripe (Wst/wst) in barley

Generation:             Parent 1        X       Parent 2 
Genotype:               Vrs1Vrs1/WstWst         vrs1vrs1/wstwst
Phenotype:              Two-row                 Six-row


                       Normal seedling White stripe seedling

Generation:                             F1 
Genotype:                        Vrs1vrs1/Wstwst
Phenotype:                      Two-row; Normal seedling

In the OWB DH population, the expected frequencies of  female gametes used to produce 
haploid plants are:     
  0.25 Vrs1/Wst     0.25 Vrs1/wst             0.25 vrs1/Wst             0.25 vrs1/wst

After chromosome doubling, this would give the genotypic ratio: 
1 Vrs1Vrs1/WstWst; 1 Vrs1Vrs1/wstwst; 1 vrs1vrs1/WstWst; 1 vrs1vrs1/wstwst
and the phenotypic ratio: 
1 two-row/normal:1 two-row/white stripe: 1 six-row/normal: 1 six-row/white stripe.

Data from the OWB can be used to test the goodness of fit to a two-locus model with 
independent assortment: 

Phenotype       #Observed       #Expected       O - E           (O - E)2/E
WstWst/Vrs1Vrs1  24             23.5            0.5             0.011
WstWst/vrs1vrs1  24             23.5            0.5             0.011
wstwst/Vrs1Vrs1  16             23.5           -7.5             2.394
wstwst/vrs1vrs1  30             23.5            6.5             1.837
Totals           94             94              0               4.253 (chi square; X2)

at 3 df  0.10<(chi-square greater than or equal to 4.253)<0.25

Accept hypothesis.  

These two loci show independent assortment. Alleles segregate independently; members 
of different allelic pairs assort independently into gametes. Independent assortment 
applies to allelic pairs on different chromosomes, or to pairs sufficiently distant on the 
same chromosome.  Based on previous reports (Shin et al. 1990) we know that these loci 
are both on chromosome 2.  However, they are sufficiently far apart that they show 
independent assortment.

b. Segregation and linkage:

Example: Short awn (Lks2/lks2) and naked kernel (Nud/nud) are
two easily scored phenotypes in the OWB. 
When we test for independent assortment (1:1:1:1), however,  we find a very poor fit. 

 Lks2Lks2/NudNud  Lks2Lks2/nudnud  lks2lks2/NudNud  lks2lks2/nudnud
O     42              12              5              35
E     23.5            23.5            23.5           23.5

Overall chi square for a 1:1:1:1 ratio is 40.38 (3 df).  This is not a good fit.



We then compute separate chi squares for each of the two traits to determine if there is 
aberrant segregation:

        Lks2Lks2               lks2lks2               Total
O       42 + 12 = 54           5 + 35 = 40                94
E          47                      47                     94

Chi square (1 df) = 2.09. Reasonable fit.


 Likewise:       NudNud                 nudnud               Total
O    42 + 5 = 47            12 + 35 = 47             94 
E       47                        47                 94

Chi square (1 df) = 0. Excellent fit.



Alleles at each of the two loci are showing  expected segregation, but there is not 
independent assortment.  This indicates that lks2 and nud may be linked.  That is, they are 
located on the same chromosome and sufficiently close together that independent 
assortment does not occur. 

The next step is to calculate a chi square for linkage:

If parental combinations are Lks2/Nud and  lks2/nud, each of the four classes of progeny 
can be classified as parental if Lks2/Nud or lks2/nud;  or recombinant if Lks2/nud or 
lks2/Nud.  With independent assortment,  the frequency of recombinant types = frequency 
if parental types. 


       (Parental phenotypes)                       (Recombinant phenotypes)
      Lks2Lks2/NudNud + lks2lks2/nudnud            Lks2Lks2/nudnud + lks2lks2/NudNud
O               77                                              17
E               47                                              47

Chi square (1 df) = 38.30. Reject null hypothesis that frequency of parental = frequency of 
recombinant.  With confidence conclude there is linkage.

If loci are linked, they are located on the same chromosome and they are close enough 
that crossovers do not occur often enough  to give independent assortment.  In the above 
example, 17 out of 94 progeny represent recombinant types. The recombination 
frequency between Lks2 and nud is 18%. 

We can use this recombination value to make a map: 


lks2            18               nud

___________________________________


This was the first step in building the OWB linkage map!

Building a linkage map with multiple loci is not something you do by hand! Linkage map construction is facilitated by mapping software. In our lab we have experience with MAPMAKER and G-Mendel, and can offer assistance with these packages.

Shin, J.S., S. Chao, L. Corpuz, and T. Blake. 1990. A partial map of the barley genome incorporating restriction fragment length polymophism, polymerase chain reaction, isozyme, and morphological marker loci. Genome. 33:803-810.



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Description for Barley Cultivars in North America


Head and kernel characters of barley

From American Malting Barley Association Barley Variety Dictionary


Head— Parts of the barley head are identified in Figure 1. Barley heads may have either six rows or two rows of kernels. In 6-row barley there are three kernels at each joint (node) on alternate sides of the head, resulting in six rows of kernels. In 2-row barley only one kernel develops at each joint on alternate sides of the head, resulting in two rows of kernels, Figure 2.

The number of rows of kernels is a stable character and is very useful for identifying barley varieties. The difference in the appearance of threshed samples of 6-row and 2-row barley varieties is distinct and useful in variety identification. In threshed samples of 6-row varieties, two-thirds of the kernels are twisted in appearance. These are the lateral or side kernels which twist as they grow alongside and overlap the central kernels. In threshed samples of 2-row varieties, all of the kernels are straight and symmetrical because there are no lateral kernels. These differences can be observed in the individual variety descriptions. In order to identify 6-row and 2-row barley in mixed samples, it is necessary to distinguish between central kernels of 6-row varieties, which are straight and symmetrical, and kernels of 2-row varieties. Kernels of 2-row varieties are generally broader and do not taper as sharply towards the attachment end of the kernel as is the case in central kernels of 6-row varieties, Figure 3.

The density of a head of barley depends on the length of the rachis internodes. If these internodes are short, the kernels are crowded close together and the head is dense. If the internodes are long, the head is lax as the kernels are spaced farther apart. Among varieties there are all gradations between dense and lax. This character is influenced to a moderate extent by environment and is therefore of limited value for identification purposes.

In 6-row barley the rows of lateral kernels may overlap each other in varying degrees. The extent of overlapping ranges from none to almost the entire length of the head, Figure 4. Overlapping of the lateral kernels is expressed first at the tip of the head and progresses towards the base of the head as the amount of overlapping increases. In most cases overlapping is found only in the upper third of the head. In varieties having no overlapping or high degree of overlapping, this character is useful in identification.

Position of head on the maturing barley plant can be described in three main groups: erect, inclined, nodding. There is considerable variation in this character within varieties but it is nevertheless a useful descriptive character.

The stem of the barley head (rachis) is flattened. The edges of this flattened stem have varying degrees of hairiness. Most varieties have numerous hairs, some varieties are intermediate and only a few have non-hairy edges, Figure 5.

This is a useful identification character in varieties differing from the majority and can generally be used in threshed samples because part of the rachis remains attached to some kernels.


Awns — Heads of barley may be awned, awnless or hooded. awns may be long or short with various combinations of awn length on the same head, Figure 6. Awnless and hooded types are shown in Figure 7.

Awns can be classified into three groups based on the degree of barbing; rough, semismooth and smooth, Figure 8. Environment has some effect on the development of barbs on the awns, resulting at times in some overlapping in the smooth and semi-smooth awn groups. Occasionally in the semi-smooth group, awns may be almost fully barbed. However, this character is generally stable and is useful in variety identification.


Glumes — Location and appearance of glumes are shown in Figure 1.

Hairiness of Glumes — Variations in the length and location of hairs on the glumes can be seen in Figure 9.
In some varieties this character is constant while in other varieties it is variable.

Length of Glume Awn— Glume awns are measured in relation to the length of the glume. In most barleys the glume awn is either equal to the length of the glume or twice the length of the glume, Figure 1.


Hull — Barley varieties have either covered or naked kernels, generally referred to as hulled or hull-less, Figure 10.

This character is stable and therefore very useful. In hulled varieties there are differences in the tightness of adherence of the hull. The degree of adherence of the hull is subject to variation due to environment but is a useful character in some varieties.

Wrinkling of the hull can be described in three classes as shown in Figure 11.

The degree of wrinkling is highly dependent on environment and is more fully expressed in mature barley than in immature barley. Certain varieties have distinctly characteristic wrinkling and this character is useful in these cases.


Aleurone — There are two major classes of aleurone color: colorless and colored or blue. There are several degrees of blue color, the expression of which is highly influenced by climatic conditions. Color in blue aleurone varieties is expressed more fully under arid and semi-arid conditions than under humid conditions. In some cases under humid conditions, this color may not develop enough to be readily seen, particularly in varieties that have inherently light blue aleurone color. Examination for aleurone color in hulled varieties can best be made by using a barley pearling machine to remove the hull.


Rachilla — Length of the hairs on the rachilla is one of the most stable characters for identifying barley varieties, Figure 13. The abundance of hairs in varieties with long rachilla hairs is useful in a few cases. The length of the rachilla itself is of minor taxonomic value. Abortive rachillas, Figure 13, may vary from none to as high as 50% in some varieties. This is a useful identifying character only for those varieties which normally contain a high per cent of abortive rachillas. Expression of this character is dependent to some extent on environment.


Veins — Prominence, continuity and shape of the central and lateral veins (Figure 12) are of some value in variety identification. Varieties differ in the number of barbs on the lateral veins, Figure 14. The development of barbs is affected by environment. Some varieties are not pure for this character and may show a wide range in number of barbs.


Crease — Shape of the crease is influenced by environment and the extent of development of the kernel. While the expression of this character is variable, it is useful for variety identification in a specific area. Many varieties have creases of similar shape. Distinct differences for this character can be found in certain varieties making it useful for identification purposes. The shape of the crease may vary from wide, open and V-shaped for the full length of the kernel to tightly closed for almost the entire length of the kernel, with many variations between these two conditions, Figure 15.


Point of attachment— The width of the point of attachment and shape and length of the narrowed part of the base of the kernel are also useful. Some varieties have a short stem-like projection from the point of attachment on some of the lateral kernels.Figure 16.

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Description for Barley Cultivars in North America

Dominant
Recessive
Chromosome location*
hulled barley

Nud: Hulled

naked barley

nud: hulless

7H
non waxy

Wax: Non Waxy (Blue stain)

waxy

wax: Waxy (purple stain)

7H
Image not available

Gth: barbs on lateral lemma veins

Image not available

gth: no barbs

2H
purple lemma and pericarp

Pre2: purple lemma and pericarp

non purple lemma and pericarp

pre2: non-purple lemma and pericarp

2H
2 row barley

Vrs1: 2-row (deficiens)

6 row barley

vrs1: 6-row

2H
no white stripe on seedlings

Wst: no stripes

white stripe on seedlings

wst: white stripes on seedlings

2H
zeo dwarf with dense head

Zeo: dwarf with dense head

normal non dense head

zeo: normal height

2H
normal green color of lemma and nodes

Alm: normal green color of lemma and nodes

albino lemma and nodes

alm: albino lemma and nodes

3H
brittle rachis

Btr1: brittle rachis

tough rachis

btr1: tough rachis

3H
pubsecence on upper leaf blades

Pub: pubsecence on upper leaf blades

lack of pubsecence on  leaf blades

pub: lack of pubescence

3H
hairs on lower leaf sheaths

Hsh: hairs on lower leaf sheaths

lack of hairs on lower leaf sheaths

hsh: lack of hairs on lower leaf sheaths

4H
hooded

Kap: hooded

awned

kap: awn

4H

Shiny head

Cer-yy: Shiny head(deficiens)

non-shiny head

cer-yy: non-shiny head

1H
black head

Blp: black head

non - black head

blp: non-black

1H
normal green lemma and nodes

Rob: normal green lemma and nodes

orange lemma and nodes

rob: orange lemma and nodes

6H
long hairs on rachilla

Srh: long hairs on rachilla

short hairs on rachilla

srh: short hairs

5H
rough awn

Raw1: rough awn

smooth awn

raw1: smooth awn

5H
 



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Instructions for Scoring contrasting Alleles in the Oregon Wolfe Barleys

Chromosome  Gene Designation  Brief description with instructions 

1

Wax wax Wax Blue stain of starch of seed with dilute KI solution;
wax purple stain of starch.
Adjust KI concentration with water to control speed of color change. Dog's toe nail clippers work well in cutting off small amounts of endosperm from the seed. Tincture of iodine works well, and can be bought in drug stores.

1

Nud nud Nud Hulled; 
nud hulless. 
The hulless phenotype lacks glue between the seed and the lemma and palea (the hulls). 

1

Lks2 lks2 Lks2 Normal or wild awn length, about 9-15 cm 
lks2 Short awn length, about 1/2 the length of the dominant long awn.
lks2 lks2 masks the expression of the hood, Kap Kap. Glume awn length may also be influenced by this gene.

2

Pre2 pre2 Pre2 Purple lemma and pericarp; 
pre2 non-purple lemma and pericarp
In black lines Pre2 is visible only just prior to development of black pigment. Check for it regularly where you only have one or two heads per plant. 

2

Vrs1 vrs1 Vrs1 Two-row (deficiens);
vrs1 six-row.
The two-row allele here conditions smaller vestigual lateral floral parts than found in most commercial two-row barley. The lateral six-row seeds tend to be somewhat smaller than those found in six-row barley, and seed set is lower. Under poor growing conditions, as is often found in greenhouses, there may be few or no seeds set in the lateral florets of some of these lines.

2

Zeo zeo * Zeo Semi-dominant dwarf with dense head;
zeo normal height.
The Zeo Zeo head is very short, as compared to the normal. The distance between nodes in the head is reduced. There is a tendency for some of the glumes to be enlarged.

2

Wst wst Wst Normal green leaves;
wst white stripes on some of the seedling leaves.
Penetrance fair, but most if not all plants homozygous for the recessive can be determined. The stripe is most easily visible under cool growing conditions in the field on spaced plants at about the 6 tiller stage. The stripes cannot be seen in later stages of the plant's life cycle, when the leaves have lost their green color.

3

Pub pub Pub Pubescence on upper leaf blades;
pub lack of same.
The hairs are very short, and give a slightly rough feel to the leaf. Misting the leaves and use of a good light will aid in determination of this trait.

3

Alm alm Alm Normal green color of lemma and nodes;
alm albino lemma and nodes.
The recessive allele conditions a loss of green color in the central part of the lemma. The nodes also appear white. Score before masked by Blp, and before ripening and loss of green color occurs.

3

Btr1 btr1 * Btr1 Wild or brittle rachis;
btr1 tough domesticated rachis.
The top part of the normal-length brittle rachis head breaks up or disarticulates at maturity. One internode of the rachis remains with each seed in two- row barley, and with each group of three seeds in six- row. This is because the abscission layers in the rachis are just above the points of attatchment of the seed. In domesticated barley the development of this abscission layer is suppressed, and upon threshing the break generally occurs at the base of the seed. When one forces a separation at the abscission layer on the rachis of a normal-length ripe head, the break-point is smooth when the Btr1 allele is present, and somewhat ragged when one of the two known `mutations of domestication' for tough rachis, btr1 or btr2, are present.

4

Hsh hsh Hsh Hairs on lower leaf sheaths;
hsh lack of hairs.

4

Kap kap Kap Hood;
kap awn
The hood is a misplaced, usually vestigual, floret. It does not express in the presence of the short awned, lks2 lks2, phenotype.

5

Blp blp Blp Black head;
blp non-black.
The black allele here is strong. However some difficulty may be experienced in determining its presence in combination with Pre2 and the orange lemma, rob.

5

Cer-yy cer-yy Dr. Jerry Franckowiak identified Cer-yy, a dominant shiny head, in the multiple dominant stock. It may also be in the recessive stock.

6

Rob rob Rob Normal green lemma and nodes;
rob orange lemma and nodes.
The orange lemma gives an orange cast to the lower part of the lemma, both when it is green and on non-black ripe seed, although its presence can generally also detected on ripe heads carrying the black allele. The orange coloration also tends to show up on stem nodes. The orange lemma in combination with the albino lemma, alm, can be quite beautiful in immature heads.

7

Srh srh Srh Long hairs on rachilla;
srh short hairs on rachilla.
The two alleles here may be the only two in the species, although there seem to be modifiers for hair length. The short haired allele has hairs up to about a length equal to the width of the rachilla. The long haired allele has hairs considerably longer than this.

7

Raw1 raw1 Raw1 Rough awn;
raw1 awn smooth at base on edges.
In the smooth awned plant the tip of the awn is usually rough. In some plants carrying the smooth awned allele the roughness can extend most of the way down the awn. This smooth awned allele is also pleiotropic for reduced branching of stigma. The barbing on the mid-rib of the awn seems to be controlled by one or more other genes. Barbing or lack of it may also show up at the base of the hood. The barbing effect is produced by small almost microscopic thorns.

-

Lga lga Lga  Long glume awn;
lga  short glume awn.
Several dominant genes may be involved. Both parents have short glume awns.

*Bt is masked by the homozygous dominant Zeo Zeo, and is probably present in about 50% of the Zeo Zeo lines. Bt is expressed in about 50% of the zeo zeo segregants. The Bt rachis, and consequently this doubled haploid material, should be treated with caution. The presence of the brittle rachis allele, Bt, in the master dominant stock was a surprise to us. Although I understand the wild counterpart of domestic barley, Hordeum vulgare var. spontaneum, is not considered weedy where it is found, it does self-seed, as will some of this material. One might consider not planting the Master Dominant or its progeny in the field. Also, the seed should be killed before it is discarded.



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