Fine mapping of a semi-dwarf gene brachytic1 in barley



Li, M.1 Kudrna, D.2 Kleinhofs, A.2



1The State Key Lab of Plant Cell and Chromosome Engineering, Institute of Genetics, Chinese Academy of Sciences, Beijing 100101, P. R. China

2Department of Crop and Soil Sciences, Washington State University, Pullman, WA 99164-6420, U. S. A.



Abstract

RFLP markers isolated from barley, wheat and rice were used to construct a fine structure map of brachytic1, a semi-dwarf gene located on chromosome 1(7H) short arm. The map covers 15.1 cM with the average distance 0.5cM between markers. A barley cDNA clone, MWG2074B co-segregated with brh1 gene in the test population. Another major band of this clone MWG2074A was 0.8cM away from brh1 toward centromere. CDO545 and BCD129 were two flanking markers mapped on both sides of brh1, toward distal and centromere, respectively. CDO545 fitted the sytenic region of rice genome, chromosome 6 short arm perfectly. But two major bands of MWG2074 could not be mapped to the target position of rice.



Introduction

Plant height mutants have been collected, identified and mapped for a long time (Franckowiak et al 1992; Szarejko 1985; U. M. Barua et al., 1993). Among all the semi-dwarf mutant classes in barley (Hordeum vulgare), brachytic phenotype is considered to be a distinctive group (Franckowiak 1995). Brachytic plants have short leaves, culms, spikes, awns, and seeds. The coleoptile and the foliage leaf are shorter than normal. Their seeds are small and yields are low (Franckowiak 1997). This mutant is easy to phenotype at all growth stages. Two alleles, the br1 (ari-i) locus on chromosome 1(7H) short arm and br2 on chromosome 4(4H) short arm are known for the controlling brachytic in barley (Tsuchiya 1974, Tsuchiya 1976).

The semi-dwarf mutants are insensitive to gibberellic acid (GA3) treatment (Boulgeret al 1982). One of the explanations is mutation has changed the balance between growth-promoting and growth-inhibiting hormones either with respect to the amounts of these hormones or to their biological activities (Stoy et al 1967). This mutant was first found in a spontaneous mutant in Himalaya and controlled by a single recessive gene (Power 1936).

As a very good morphological marker, br1 (brh1) locus was associated with RFLP markers using bulked segregant analysis (Michelmore 1991). A Steptoe brh1 mutant - FN53 was developed and crossed with Morex and F1 seeds were produced. The brh1 locus was mapped between RFLP markers BCD129 and ABG320 with approximate distance of 3 cM (Kudrna 1996). More over, it also provides an excellent marker for the distal region of 1(7H) with very close linkage to Rpg1, a stem rust resistant gene (Yu Jin et al., 1993; Kleinhofs 1996). Based upon these, fine structure map of brh1 will not only facilitate the map-based cloning of the gene itself, which may disclose the expression of a growth and development related gene, but also provide a larger population for screening Rpg1 recombinants.

Using rice as an intergenomic mapping vehicle, success has been made toward the map-based cloning of the barley Rpg1 and rpg4 genes (Killian et al 1997). Rice YAC, BAC clones can be used as probe sources to saturate the syntenic region in rice and establish BAC contigs in barley.

In this report, we present the progress in fine RFLP mapping of brh1 in barley and syntenic mapping in rice using segregating populations.



Material and Methods

Mutant development and selection--Barley (Hordeum vulgare cv Steptoe) seeds were exposed to fast neutrons to induce mutations. The M1 and M2 generations were grown in field and brachytic1 mutant - FN53 was selected. FN53 was crossed with Morex and F2 seeds were produced in greenhouse. We grew two F2 populations in growth chamber and greenhouse respectively. Sample F2 plants (63 individuals) were given the conditions of 16-18hr photoperoid, 18°C day and 13°C night and fertilized with Nitrate Nutrient Solution during tillering time. Homozygous brachytic individuals were identified at seedling stage. Young inflorescences/tillers were harvested from each plant and lyophilized for extracting genomic DNA. Polymorphisms were determined for four restriction enzymes (EcoR I, Hind III, Dra I and Xba I) with two parents (FN53 and Morex), using Steptoe as a reference to detect mutations other than deletion or insertion.

Mapping--Mapping techniques are described in Kleinhofs et al (1993). Rice probes were hybridized to barley genomic DNA at 58-60°C and washing stringency were adjusted to a lower percentage of mismatch.

Linkage analysis--Maximum likehood method was used to estimate recombination frequency of all pairs of polymorphic loci defined by single restriction fragments. The calculation is described in Ritter et al (1991). In this mapping study, only homozygous FN53 mutants were analyzed, the recombination frequencies were calculated within the recessive class of 1:2:1 segregating F2 population according to the following formula: P=(h+2b)/2n, where P= recombination frequency, n=number of homozygous recessive individuals in F2, h=number of heterozygous recombinant individuals in F2, b=number of homozygous recombinant individuals in F2. (Allard 1956) as described in Hinze et al (1991).



Results and discussion

Saturated and high-resolution map of the brachytic1 region--Brachytic1 region was saturated with 29 RFLP markers where four markers mapped in this region of an integrated map Kudrna et al (1996). Seventeen barley genomic/cDNA clones, nine rice probes, two wheat probes and one oat clones were hybridized to the mapping filters. This fine map in the target region spans 15.1 cM (Fig.1) with an average distance of 0.8 cM between markers. Previous map shows brh1 is between BCD129 and ABG320. This map indicates that brh1 is far from ABG320 and one crossover from BCD129 toward the distal region. CDO545 is another flanking marker given the interval of 0.8cM on the distal side. A barley cDNA clone-MWG2074, which has two loci, has been mapped in the target region. B locus co-segregates with brh1 in the sample population. MWG2074 played a role of a flanking marker. Six rice clones in the rice syntenic region (Chromosome 6 short arm) hybridized to brh1genomic DNA. R3139 shows 0.8 cM away from brh1 toward centromere.

In the confirmation population with 89 homozygous recessive individuals, closely linked marker MWG2074 shows no segregation between its two loci. However, there is only one crossover from brh1 to BCD129. To map brh1 precisely, a relatively larger population that contains more brh1 lines is needed to produce and study. Meanwhile, the lager population can be used for Rpg1 recombinants screen. Furthermore, more makers may be generated for positional cloning of both genes.

Rice syntenic map of brh1 region--In order to prove the assumption that rice has brh1 gene in the syntenic position, we hybridized two flanking markers, CDO545 and MWG2074 to rice F3 population (186 individuals). The data shows that CDO545 fits the region fairly well. But two major bands of MWG2074 could not map to this region. One possibility could be that other band(s) of MWG2074 would map to this region. Thus the rice homology of brh1 gene will be confirmed to be present in rice syntenic region. Rice YAC or BAC between CDO545 and MWG2074 will be isolated, subcloned, sequenced and examined for barley brh1 candidate genes.

Map-based cloning requires the identification of RFLP markers that are tightly linked to the interest gene. Fine structure mapping of target gene provides closest flanking markers that allow to establish BAC contig. Some other GA3 insensitive genes that have been cloned and characterized or open reading frames can be probe sources to map brh1 gene as to determine if they co-segregate with it or not.




References



J. D. Franckowiak (1995) The brachytic class of semidwarf mutants in barley. BGN 24: 56-59.

T. Tsuchiya (1974) Allelic relationships of genes for short-awned mutants in barley. BGN 4: 80-81.

T. Tsuchiya (1976) Allelism testing of genes between brachytic and eractoides mutants. BGN 6: 79-81.

M. C. Boulger, R. G. Sears, and W. E. Kronstad (1982) An investigation of the association between dwarfing sources and gebberellic acid response in barley. Barley Genet. IV: 550-553.

R. W. Michelmore, I. Paran, and R. V. Kesseli (1991) Identification of markers linked to disease-resistance genes by bulked segregant analysis: A rapid method to detect markers in specific genomic regions by using segregating populations. Proc. Natl. acad. Sci. USA 88: 9828-9832.

Kudrna, D., A. Kleinhofs, A. Killian, and J. Soule (1996) Integrating visual markers with the steptoe x Morex RFLP map. In: A. slinkard, and B. Rossnafel (eds.) V International Oat Conference & VII Intrnational Barley Genetics Symposium., pp. 343. University Extension Press, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.

A. Kleinhofs(1996) Integrating Barley RFLP and Classical Marker Maps. BGN 26:105-112.

A. Kleinhofs, et al. (1993) A molecular, isozyme and morphological map of the barley (Hordeum vulgare) genome. Theor Appl Genet 86: 705-712.

A. Killian, J. Chen, F. Han, B. Steffenson and A. Kleinhofs (1997) Towards map-based cloning of the barley stem rust resistance genes Rpg1 and rpg4 using rice as an intergenomic cloning vehicle. Plant molecular Biology 35: 187-195.

E. Ritter, C. Gebhardt, and F. Salamini (1990) Estimation of Recombination Frequencies and Construction of RFLP Linkage Maps in Plants from Crosses between Heterozygous Parents. Genetics 125: 645-654.

R. W. Allard (1956) Formulas and tables to facilitate the calculation of recombination values in heredity. Hilgardia 24: 235-278.

Karin Hinze, Richard D. Thompson, Enrique Ritter, Francesco Salamini, and Paul Schulze-Lefert (1991) Restriction fragments length polymorphism-mediated targeting of the ml-o resistance locus in barley (Hordeum vulgare). Proc. Natl. Acad. Sci. USA 88: 3691-3695.