# **Barley Genetics Newsletter (2005) 35:36-38**

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# **Genetic analysis of several root hair mutants of barley**

I. Szarejko, A. Janiak, B. Chmielewska, M. Nawrot

Department of Genetics, University of Silesia,

Jagiellonska 28, 40-032 Katowice, Poland e-mail: szarejko@us.edu.pl

### Introduction

Root hairs are specialized epidermal cells that play an important role
in uptake of water and nutrients from rhizosphere. Each hair is a long,
tubular, non-dividing cell characterized by tip growth, i.e. by
deposition of new membrane and cell wall precursors at a cell-growing
tip. Several root hair mutants have been isolated and genetically
characterized in *Arabidopsis thaliana* (Parker, 2000, Shiefelbein et
al., 2000) however, besides a few reports in maize (Wen and Schnable,
1994), there is a lack of information about genetic basis of root hair
development in monocotyledons. Recently, Engvild and Rasmussen (2004)
described some barley mutants with changes in root hair phenotype, but
for now without information on their genetic basis. Nevertheless, the
growing resources of such barley germplasm can help to reveal the
mechanisms underlying the process of root hair morphogenesis in
monocotyledon species.

### Material and Methods

The mutagenic treatment of barley varieties 'Rudzik', 'Dema', 'Krona',
'Diva', 'Karat', and 'Delisa' was performed with the use of
N-methylo-N-nitroso-urea (MNU) and sodium azide. To generate high
frequency of gene mutations, the combined treatment with both mutagenes
(Szarejko and Maluszynski, 1999) or double treatment of MNU was applied.
The doses for treatment of Rudzik and Dema varieties were as follows:
1.5 mM NaN~3~/3h -- inter-incubation germination/6h -- 0.75 mM MNU/3h
and for variety Krona: 1.75 mM NaN~3~/3h -- inter-incubation
germination/6h -- 0.9 mM MNU/3h. For the varieties Diva, Karat and
Delisa double treatment of MNU was used: 0.7 mM MNH/3h. -
inter-incubation germination/6h - 0.7 mM MNH/3h. The selection of root
hair mutants was performed in the M~2~ or M~3~ generations. Subsequent
generations (M~4~-M~6~) were used to perform homozygosity test and
multiply seeds of homozygous lines with mutated root hair phenotype. The
observations of root hair zone were done for plants growing in aeroponic
conditions (Fig.1). The inheritance of the root hair phenotypes was
analyzed in the F~1~ and F~2~ generations from the crosses between
mutants and their parent varieties. Allelism test between mutants was
performed.

# **Results**

#  Seventeen mutants with changes in the root hair development and morphology have been identified after mutangenic treatments of barley varieties Rudzik, Dema, Krona, Diva, Karat, and Delisa. The mutations affected different stages of root hair development and were classified as belonging to one of four main root hair phenotypes (Table 1): 

-   root hairless mutants, designated rhl,

-   mutants with development of root hairs blocked at the initial stage,
    forming only small root hair primordia, seen as 'bulges', designed
    rhp,

-   mutants with very short root hairs, designed rhs,

-   mutants with irregular pattern of root hairs, designed rhi.

The genetic analysis revealed that each of the characters in the
analyzed mutants was controlled by single, recessive gene. According to
the results of allelism tests, the locus number and letter symbol of
allele was added to the three-letter code of the mutant name. The three
root hairless mutants derived from variety Karat proved to be allelic.
Similarly, one gene controlled the phenotype of four mutants developing
root hairs blocked at the primordium stage. The rhp1.a -- rhp1.d mutants
showed slightly elongated root hair primordia, indicating that root hair
development has been stopped at the stage of transition to tip growth.
All three mutants with very short root hairs represented three different
loci. The root hair length in these mutants has not reached 10% of the
parent variety. Additionally, the mutant rhs1.a was characterized by the
short seminal roots and short spike (reaching about 50% of the length of
parent variety). These characters were controlled by separated genes
linked with the gene controlling the root hair length. The other mutant
with short root hairs, 522DK, not yet characterized according to the
allelism with other forms, showed about 40% reduction of root length and
30% reduction of the shoot length in comparison to the parental form
(Nawrot, 2005).

The remaining group of four mutants with irregular root hairs was
representing two loci. In mutants rhi2.a and rhi2.c, the length and
density of root hairs varied between roots and different parts of the
same root, but never reached the length and density of root hairs
characteristic for the parent variety. The other two mutants: rhi1.a and
rhi2.b develop only a few, very sparsely located root hairs of
differentially reduced length. The root hair density was lower than in
two mutants described above. Another mutant, 800Q with irregular root
hairs develops very short root hairs, similar in length to the rhs1.a
mutant, but spread on the root in a very irregular pattern.

The analysis of genetic relationships between genes of study was
performed for four loci: *rhl1* (mutants 834Q and 931Q), *rhp1* (mutants
DM 209.1.3.1 and DM 204.1.8.5), *rhs1* (mutant 225DV) and *rhi1* (mutant
DM 204.1.8.12). Gene *rhl1*, responsible for the lack of root hairs was
epistatic to three other genes involved in further steps of root hair
formation, i.e., formation of root hair primordia (gene *rhp1*),
location of root hair bearing cells (gene *rhi1*) and root hair
elongation (gene *rhs1*). Similarly, genes: *rhp1* and *rhi1* were
epistaic to the *rhs1* locus. The genes: *rhp1* and *rhi1*, however,
give hairless phenotype in a double recombinant. Further analysis will
be performed to describe relationships between the remaining loci.

#### References

Engvild, K.C. and S.K. Rasmussen. 2004. Root hair mutants of barley.
Bar. Genet. News. 34:13--15.

Franckowiak, J. and U. Lundqvist. 2004. Rules for nomenclature and gene
symbolization in barley. Barley Genet. Newslet. 34:132-136

Nawrot, M., Szarejko, I.and M. Maluszynski. 2005. Barley mutants with
short roots. Bar. Genet. News. 35:3-8.

Parker, J.S., Cavell, A.C., Dolan, L., Roberts, K. and C.S Grierson.
2000. Genetic interactions during root hair morphogenesis in
*Arabidopsis*. The Plant Cell 12:1961-1974.

Szarejko, I. and M. Maluszynski. 1999. High frequency of mutantions
after mutagenic treatment of barley seeds with NaN~3~ and MNH with
application of inter-incubation germination period. Mutation Breed.
Newslet. 44:28-30.

Schiefelbein, J.W. 2000. Constructing a plant cell. The genetic control
of root hair development. Plant Physiology 124:1525-1531.

Wen, T-J. and P.S Schnable. (1994. Analysis of mutants of three genes
that influence root hair development in *Zea mays* (*Gramineae*) suggest
that root hairs are dispensable. American Journal of Botany 81:833-842.

![](media/image2.jpeg){width="2.311111111111111in"
height="3.477777777777778in"}

Fig. 1. Aeroponic culture of barley seedlings

.

+--------------+-----------------+-----------------+------------------+
| Table. 1.    |                 |                 |                  |
| List of      |                 |                 |                  |
| barley       |                 |                 |                  |
| mutants with |                 |                 |                  |
| changes in   |                 |                 |                  |
| root hair    |                 |                 |                  |
| morphology   |                 |                 |                  |
+--------------+-----------------+-----------------+------------------+
| **No. of     | **Parent        | **Phenotypic    | **Symbol of      |
| mutant       | variety**       | class**         | locus and        |
| line**       |                 |                 | allele\***       |
+--------------+-----------------+-----------------+------------------+
| 834Q         | Karat           | root hairless   | *rhl1.a*         |
+--------------+-----------------+-----------------+------------------+
| 931Q         | Karat           | root hairless   | *rhl1.b*         |
+--------------+-----------------+-----------------+------------------+
| 934Q         | Karat           | root hairless   | *rhl1.c*         |
+--------------+-----------------+-----------------+------------------+
| DM 209.1.3.1 | Dema            | root hair       | *rhp1.a*         |
|              |                 | primordia       |                  |
+--------------+-----------------+-----------------+------------------+
| DM 204.1.8.5 | Dema            | root hair       | *rhp1.b*         |
|              |                 | primordia       |                  |
+--------------+-----------------+-----------------+------------------+
| DM 201.11.3  | Dema            | root hair       | *rhp1.c*         |
|              |                 | primordia       |                  |
+--------------+-----------------+-----------------+------------------+
| RD 103.1.1   | Rudzik          | root hair       | *rhp1.d*         |
|              |                 | primordia       |                  |
+--------------+-----------------+-----------------+------------------+
| 225 DV       | Diva            | root hair short | *rhs1.a*         |
+--------------+-----------------+-----------------+------------------+
| DM 55.1      | Dema            | root hair short | *rhs2.a*         |
+--------------+-----------------+-----------------+------------------+
| 740Q         | Karat           | root hair short | *rhs3.a*         |
+--------------+-----------------+-----------------+------------------+
| DM           | Dema            | root hair       | *rhi1.a*         |
| 204.1.8.12   |                 | irregular       |                  |
+--------------+-----------------+-----------------+------------------+
| # RD 100.1   | Rudzik          | root hair       | *rhi2.a*         |
|              |                 | irregular       |                  |
+--------------+-----------------+-----------------+------------------+
| RD 101.2.7   | Rudzik          | root hair       | *rhi2.b*         |
|              |                 | irregular       |                  |
+--------------+-----------------+-----------------+------------------+
| DM 208.1.6   | Dema            | root hair       | *rhi2.c*         |
|              |                 | irregular       |                  |
+--------------+-----------------+-----------------+------------------+
| 522DK        | Delisa          | root hair short | n/a              |
+--------------+-----------------+-----------------+------------------+
| 800Q         | Karat           | root hair       | n/a              |
|              |                 | irregular       |                  |
+--------------+-----------------+-----------------+------------------+
| KR 36.6      | Krona           | root hair       | n/a              |
|              |                 | irregular       |                  |
+--------------+-----------------+-----------------+------------------+

\* - symbols designated according to the genes symbolization in barley
(Franckowiak and Lundquist, 2004)

n/a -- not available, mutants not yet characterized according to the
locus and allele number