Genetics of Barley Hooded Suppression

Corresponding author: Francesco Salamini, Carl-von-Linnè-Weg 10, D-50829 Cologne, Germany., salamini{at}mpiz-koeln.mpg.de (E-mail)

Communicating editor: V. SUNDARESAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The molecular basis of the barley dominant Hooded (K) mutant is a duplication of 305 bp in intron IV of the homeobox gene Bkn3. A chemical mutagenesis screen was carried out to identify genetical factors that participate in Bkn3 intron-mediated gene regulation. Plants from recurrently mutagenized KK seeds were examined for the suppression of the hooded awn phenotype induced by the K allele and, in total, 41 suK (suppressor of K) recessive mutants were identified. Complementation tests established the existence of five suK loci, and alleles suKB-4, suKC-33, suKD-25, suKE-74, and suKF-76 were studied in detail. All K-suppressed mutants showed a short-awn phenotype. The suK loci have been mapped by bulked segregant analysis nested in a standard mapping procedure based on AFLP markers. K suppressor loci suKB, B, E, and F all map in a short interval of chromosome 7H, while the locus suKD is assigned to chromosome 5H. A complementation test between the four suK mutants mapping on chromosome 7H and the short-awn mutant lks2, located nearby, excluded the allelism between suK loci and lks2. The last experiment made clear that the short-awn phenotype of suK mutants is due to a specific dominant function of the K allele, a function that is independent from the control on hood formation. The suK loci are discussed as candidate participants in the regulation of Bkn3 expression.

THE floret of grasses is protected by two leafy organs, the lemma and the palea, both representing reduced vegetative leaves (ARBER 1934 ; DAHLGREN et al. 1985 ; CLIFFORD 1988 ; POZZI et al. 2000 ). In several species of the family, the upper part of the lemma develops into the awn, a long distal appendage. In barley, several mutants are known in which the development of the lemma is affected (discussed in POZZI et al. 1999 ). Among these are the recessives calcaroides and leafy lemma (POZZI et al. 2000 ) and the dominant mutant Hooded (STEBBINS and YAGIL 1966 ). In Hooded plants, an extra flower develops at the site of transition between lemma and awn. In this region, the mutant differs from the wild type (WT) in the size of the cells of the adaxial epidermis and in the direction of cell division (STEBBINS and YAGIL 1966 ). In the subepidermal layer of the K awn primordium, periclinal divisions generate a dome, from which ectopic floral organs differentiate in an inverted orientation with respect to the lemma proper (MULLER et al. 1995 ). The molecular basis of this phenotype is a mutation in the homeobox gene Bkn3, which is a member of the knox plant homeodomain family (MULLER et al. 1995 ).

In plants, knox genes play an important role in the establishment and development of leaf primordia (BHARATHAN and SINHA 2001 ). Some of them, referred to as class I (KERSTETTER et al. 1994 ), drastically alter the meristematic activity and the shape and compoundness of leaves when expressed in transgenic plants (MULLER et al. 1995 ; CHUCK et al. 1996 ; PARNIS et al. 1997 ; LIN and MULLER 2002 ). A particular feature of the barley Bkn3 gene (MULLER et al. 1995 ) and of its maize ortholog Kn1 (VOLLBRECHT et al. 1991 ) is that dominant, homozygous viable mutations are associated with DNA insertions in the large intron (IV in barley, III in maize). The importance of this noncoding region as the putative regulatory region was recently put forward (INADA et al. 2003 ; SANTI et al. 2003 ). This noncoding region makes it possible to screen for second-site mutations to identify loci coding for factors that putatively participate in intron-mediated regulation of Bkn3. This article describes such a suppressor screen, together with the mapping to linkage groups of the concerned loci.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plant material and generation of K-suppressed lines:
The barley line KBGS152 [Barley Genetics Stock Center (BGSC), Fort Collins, CO) is homozygous for the dominant allele K of the Hooded locus and for the recessive allele vrs1 of the Two/six row spike locus. The line was propagated at the Max-Planck-Institut für Züchtungsforschung (MPIZ, Köln, Germany) and was used for the suppressor screen.

Three consecutive mutagenic treatments were carried out. In the first one, 500 g of seeds were soaked for 18 hr at 5° in H₂O and treated for 2 hr with 0.001 M sodium azide (NaN₃) in 0.1 M phosphate buffer (pH 3.0) at room temperature. Seeds were washed in running tap water for 30 min and sown in the field. Single ears were harvested from each M₀ plant, and 2697 M₁ families of 10 plants each were grown at the Istituto Sperimentale per la Cerealicoltura, Fiorenzuola, Italy. Families segregating plants in which the K phenotype was suppressed were identified and single non-Hooded plants were harvested, while remaining lines were harvested in bulk. A seed sample of this bulk harvest was again mutagenized. The second treatment, involving 7 x 10⁴ seeds, was carried out at the MPIZ using NaN₃ under the conditions described. The M₂ families that did not segregate for the WT-reverted phenotypes were mutagenized a third time, using approximately the same number of seeds. About 4.5 x 10⁴ plants were harvested in bulk after the second and third mutagenic treatments and phenotypes other than vrs1 and K were discarded during field selection. Field mutagenesis was carried out without ear bagging.

Two lots of 8 x 10⁴ seeds, obtained from the third mutagenic treatment, were germinated in micropots (96-well plates of 50 x 33 cm with wells 2 cm in diameter and 2 cm deep) under greenhouse conditions. Each plant produced from one to three fertile seeds and ear morphology was sufficiently clear to allow selection of K-suppressed lines.

The KAtlas strain was made available by G. L. Stebbins (Department of Genetics, University of California, Davis, CA). The wild type (WT) lines Nudinka (ears with two rows), Vogelsanger Gold (six rows), and Atlas (six rows), used in genetic studies and phenotypic comparisons, were from the MPIZ collection. The lks2 mutant was from the BGSC.

Identification of second-site K suppressors:
The crossing program was conducted by manual cross-pollination: two ears were emasculated for each cross and fertilized using anthers of the appropriate genotype. A total of 6–18 F₁ seeds were harvested from each cross and F₁ plants were grown for phenotypic assessment. When necessary, F₂ seeds were harvested from single F₁ plants for further genetic analysis. Molecular fingerprinting and testcrosses were carried out to eliminate contaminants due to accidental outcrossing and to distinguish between intragenic and second-site mutations, respectively.

Amplified fragment length polymorphism (AFLP) molecular profiles diverging even marginally from those of the mutagenized KBGS152 line were assumed to originate from contaminants. Contaminating lines were found to have awn length comparable to that of WT lines. Moreover, contaminants derived from Vrs1 (two-rowed ear) pollen were also easily detected, because the phenotype conferred by this allele is dominant.

Testcrosses were conducted by crossing K-suppressed lines to the WT line Nudinka (genotype kk). The symbol suK was assigned to extragenic suppressor, the lowercase initials indicating the recessive nature of the suppressing alleles. The genotype of these suppressed lines is indicated as suK suK, K K, the WT condition being SuK SuK, k k. Dominance was assessed through crosses of K-suppressed lines with KBGS152 and with KAtlas.

Complementation tests and phenotypic comparisons of suK lines:
For suK lines, complementation tests were carried out using various crossing schemes. Each cross was repeated up to six times, and each repetition generated from 4 to 28 F₁ seeds. All F₁ plants were grown to maturity and their lemma phenotypes were recorded. A further validation of the complementation test was carried out on the basis of the analysis of F₂ populations. This concerned a specific set of crosses among suK mutants mapped to chromosome 7H, sublinkage groups 5–7. Their F₂ generations were grown and the presence of K and suK phenotypes controlled, as well as their segregation ratio.

F₂ plants or F₃ segregating families from the cross suK x KAtlas were used to compare plant and ear traits of suK and K phenotypes. Plants were grown in 20-well plastic plates of 50 x 33 cm in the greenhouse or in the field in rows of plants spaced 10 cm apart with 20 cm between rows. In the same F₂ populations, grown in either the greenhouse or the field, segregation ratios were also recorded.

The allelism test between suK mutants and the lks2 line was carried out by analyzing the F₁ and F₂ generations, as described for the suppressor lines. In this case, the phenotypic description included the measurements of the length of the awn considering those of the more distal spikelets of the ear. For awn length, three classes were considered (Table 4): A, long awn; B, short awn (awn length 2–8 cm; control genotype was lks2, with an average awn length of 7.5 cm, evaluated in 19 plants; suKB-4 was 5.1 cm in 12 plants; suKC-33 was 5.9 cm in 15 plants; suKD-25 was 5.1 cm in 15 plants; suKE-74 was 4.3 cm in 13 plants; suKF-76 was 6.5 cm in 15 plants); C, WT or almost awnless (9–15 cm; control genotype was the variety Proctor, awn of 12.9 cm evaluated in 15 plants, and the variety Nudinka, 13.7 cm in 15 plants).

Amplified fragment length polymorphism analysis:
Populations used in mapping experiments were generated from crosses between suK mutants and KAtlas and between KAtlas or KBGS152 and the WT line Nudinka. F₂ plants were harvested, DNAs were extracted (DNeasy plant mini kit, QIAGEN, Hilden, Germany) and pooled if necessary or used as such for molecular fingerprinting. The procedure and primer combinations used for AFLPs were as described in CASTIGLIONI et al. 1998 .

Mapping of suK loci:
The mapping procedure described by CASTIGLIONI et al. 1998 was used and integrated as follows. Mutants representative of each of the different complementation groups were crossed with KAtlas. Homozygous suK suK, K K (awned lemma) or SuK SuK, K K (Hooded lemma) F₃ lines were harvested. AFLP analysis of 15 bulked DNAs for both types of lines allowed the identification of amplified fragments in linkage with the suK loci. In the next step, the suK-linked AFLP fragments were amplified from single F₃ lines. Four to seven AFLP loci were sufficient to define a map around each suK locus; loci were not considered with genetic distances among each other if exceeding 10 cM.

To position the suK-linked AFLP loci into the map of CASTIGLIONI et al. 1998 , crosses were performed between the K genotypes KBGS152 and KAtlas and the WT line Nudinka. In these crosses, linkage of at least two suK-linked AFLP loci with AFLP bands already mapped by CASTIGLIONI et al. 1998 was established for each SuK locus.

The segregation data were analyzed with the MAPMAKER program (UNIX version/EXP3; LANDER et al. 1987 ) with a LOD score value of 3 and a maximum distance of 50 cM. A virtual marker, showing 100% linkage to the mutant phenotype in F₂ plants, represented the locus of interest.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation and complementation analysis of extragenic suK mutants:
The large dimension of the barley genome made it necessary to design a recurrent mutagenesis experiment during which only mutants in the M₂ generation were selected. A total of three recurrent mutagenic treatments, conducted on 1.6 x 10⁵ KK seeds, yielded 41 second-site suppressed mutants, exhibiting an awn in place of the extra floret present on the lemma of Hooded plants. AFLP fingerprinting of all the isolated WT revertants confirmed that they represent suK mutations as opposed to pollen contaminations. Testcrosses were conducted to distinguish among intragenic and extragenic suppressors of the K phenotype. Contaminants and lines bearing Bkn3 intragenic mutations were expected to generate kk F₁ plants with the non-Hooded phenotype, while extragenic suppressor lines were expected to generate F₁'s with the K phenotype (Fig 1A). Testcross analysis allowed the identification of the mutants suK4, suK9 to suK35; 74 to 78; and 81 to 86, 88, and 89 as representing extragenic suppressors of the K phenotype.

View larger version (46K): In this window In a new window Download PPT slide   Figure 1. (A) Phenotypic analysis supporting the nonintragenic nature of the suK mutant with respect to the K locus. The appearance of the Hooded phenotype in F1 supports the recessivity for the suK locus. Thus, the genotype assigned to the mutant line is suKD-25 suKD-25, K K and to the WT variety, Nudinka SuKD-25 SuKD-25, k k. N, WT variety Nudinka. (B) Short-awn phenotype of suK suK, K K mutant compared to Hooded KBGS152 (K). Arrows, instability of the suK phenotype in mutants suKB-4 and suKC-33.

Diallelic F₁ populations:
Sets of diallelic crosses were allowed to conclude allelism for 37 mutants (suK9 to 32, 34, 35, 77 to 79, 81 to 86, 88, and 89). The result was achieved in two steps: at first, six mutants were found allelic upon phenotypic inspection of the F₁ generations of all their possible pairwise crosses. A second diallelic cross, involving one of the previous six mutants and an additional four, again revealed complete allelism, suggesting the predominance among the isolated mutants of alleles belonging to one locus. Eventually, suK 25 and 28, representative of this locus, were crossed to all remaining mutants: 37 of 41 suK resulted in being allelic and the locus was designated suKD. The four mutants that complemented suKD were tentatively assigned the symbols suKB-4, suKC-33, suKE-74, and suKF-76. The assumption that they represented alleles of four different complementation groups was verified by diallelic crosses among the mutant lines suKB-4, suKC-33, suKD-25, suKE-74, and suKF-76. All the resulting F₁ plants were Hooded, supporting the conclusion that the 41 suK mutants represented five different complementation groups. For the loci suKB, C, E, and F, only one allele was recovered.

Allelism tests in F₂ populations:
Allelism of those suK mutants mapping to a short region of chromosome 7H was controlled also in the F₂ generation. Short-awn, non-Hooded F₂ populations were expected in the case of allelism among suK mutants. Because of the tight linkage existing between the genetic loci involved, a shift in the segregation ratio from 9 K and 7 suK to 1 K and 1 suK was expected. F₂ data were available for the crosses suKB-4 x suKC-33 [the result of the segregation was 34 K and 45 suK; , statistically not significant (NS)], suKB-4 x suKE-74 (66 K and 56 suK; , NS), suKB-4 x suKF-76 (88 K and 85 suK; , NS), and suKC-33 x suKF-76 (94 K and 84 suK; , NS). It was concluded also that F₂ generation analysis indicated that the suK mutants mapping to chromosome 7H represented four different genetic loci.

Phenotypic and genetic analyses of suK mutants and of their F₁ hybrids:
Crossing of representative mutants belonging to the different complementation groups with the line KAtlas (Table 1) supported the conclusion that each locus behaved according to Mendelian rules, with suppression occurring only when the recessive alleles were homozygous. In the F₂'s, the homozygous recessive class was slightly underrepresented, particularly for suKB-4 and E-74. In all mutants, the length of the awn was reduced by 50% compared to WT. The suppressed phenotype was unstable in that few lemmas, from otherwise fully suppressed ears, developed a rudimentary extra floret (arrows in Fig 1B). This phenotype was analyzed in detail for mutants suKB-4, C-33, D-25, E-74, and F-76. Only suKB-4 and suKC-33 showed an intraplant phenotypic penetrance <100%, at least when plants were grown under field conditions. The incomplete penetrance of suKC-33 was more obvious in ears produced from lateral tillers (data not shown). When suK mutants were crossed to the WT two-rowed Nudinka line, the resulting suK SuK, k K F₁ plants had lemmas carrying extra florets in a more elevated position as compared to KBGS152 (Fig 1A). The Hooded phenotype recorded in complementing F₁'s involving suKB-4 showed a small percentage of lemmas with a rudimentary awn, suggesting that this allele is not completely recessive. All other complementing F₁'s were indistinguishable from KBGS152. Homozygous suK plants from suK x KAtlas crosses were smaller when compared to K plants (Table 2) and characterized by reduced spike length and number of fertile nodes. However, only suKE-74 was significantly different from K in plant and ear characters.

Mapping suK loci to barley linkage groups:
Data derived from suK x KAtlas F₂ populations allowed the construction of an AFLP map around each suK locus. The segregation of the same AFLP suK-linked markers in the crosses between K and WT (Nudinka) genotypes made it possible to precisely position these AFLP loci ("bridge markers") in the Nudinka x Proctor map of CASTIGLIONI et al. 1998 .

The suppressor loci suKB, C, E, and F all mapped to barley chromosome 7H in linkage subgroups 5, 6, and 7 (Fig 2A). The locus suKD was assigned to linkage group 5H between linkage subgroups 66 and 67 (Fig 2B). A summary of the data on bridge AFLP markers, providing their distances in centimorgans from suK loci, is in Table 3.

View larger version (21K): In this window In a new window Download PPT slide   Figure 2. Assignment of suK loci to linkage groups and subgroups. (A) Mapping of suKB, C, E, and F loci by AFLP markers to a short region of chromosome 7H. Bridge markers are joined by a transversal dotted line and are mapped in the crosses suK x KAtlas (left) and KAtlas x Nudinka or KBGS152 x Nudinka (right). AFLP markers not in boldface type and included in the "subgroups" boxes are also mapped in the Proctor x Nudinka population (CASTIGLIONI et al. 1998 ) and can be used as links to the restriction fragment length polymorphism integrated map of CASTIGLIONI et al. 1998 . (B) Mapping of the suKD locus. Bridge AFLP markers have been mapped via KAtlas x Nudinka and KBGS152 x Nudinka crosses.

The unexpected localization of all four nonallelic suK mutants, B-4, C-33, E-74, and F-76, to a short linkage region of chromosome 7H was confirmed by AFLP analysis in the KAtlas x Nudinka cross (Fig 2A). Loci suKF-76 and suKE-74 were separable by crossing over as indicated by their distal and proximal, respectively, recombination with the AFLP marker E41M46-5. A similar observation was made for suKE-74 and suKC-33 and the AFLP marker E42M43-2. On the basis of recombination data, the possibility cannot be ruled out that suKC-33 and suKB-4, although complementing, are alleles of the same genetic locus.

The short awns of suK mutants suggest a test of allelism to the short-awn mutant lks2:
On the basis of mapping information, the short-awn mutant lks2 was a candidate to correspond to one of the four suK loci mapping on chromosome 7H, sublinkage groups 5–7. In fact, the marker loci E35M40-1 and E34M40-4, which are tightly linked to the lks2 locus (POZZI et al. 2003 ), also mapped very near to suKF-76 and suKC-33 (Fig 2A).

A complementation assay based on F₁ and F₂ generations of lk2 x suK crosses was designed to test the allelic state of the two types of mutants (Table 4 and Fig 3). The phenotypes recorded in the F₁ (genotype Lks2 lks2, SuK suK, K k) were unexpected in that the lks2 allele demonstrated that it could suppress, to a different degree, the formation of the hood in K k plants in the presence of a WT SuK allele. For the same F₁, a short-awn phenotype, even in the presence of the long awn dominant allele Lks2, was noted and interpreted as due to a dominant effect of the K mutation (see below and DISCUSSION). Under this assumption, in addition to the control of hood formation, K should provide a second specific function related to the reduction of awn length.

View larger version (31K): In this window In a new window Download PPT slide   Figure 3. Phenotypes recorded in the F1 and F2 generations of the crosses lks2 x suK. (A) lks2 x suKD-25; (B) lks2 x suKC-33; (C) lks2 x suKE-74. Note in C that the short-awn phenotype of the F1 is also characterized by a very elevated hood.

In detail, F₁ plants of the crosses between lks2 and suKB-4 and lks2 and suKC-33 resulted in nonhooded with short-awn lemmas (Fig 3B). F₁ plants from lks2 crosses with suKE-74 and suKF-76 were less sensitive to lks2 repression of hood formation, showing a very elevated hood phenotype (Fig 3C). The presence of short-awn characters in at least two different F₁'s did not allow the establishment of the complementation state between lks2 and the mutants suKB-4, suKC-33, suKE-74, and suKF-76.

F₂ populations were generated. The goal was to search, in a cross of a specific suk mutant, for a discriminatory large fraction of kk (WT, non-Hooded) plants with a long awn phenotype, concomitantly with Hooded plants. This would have been considered an indication of the nonallelic state of lks2 with the suK allele. Putative interactions between suK mutants and lks2 are well illustrated by the results of the cross lks2 x suKD-25, which, involving loci mapping to different chromosomes, represents a control case for the absence of allelism, because in F₂ the cross allows the combination of all alleles of considered loci (Fig 3A). In F₂, awn length was under a complex control: 11 of 65 non-Hooded plants were awnless while 17 had normal awns (the latter are kk genotypes in which the K function on awn length is absent; Fig 3A). A fraction of Hooded plants was also observed. The hypothesis that in the F₂ population the Lks2-, suKD-25 suKD-25, kk genotypes occur within the long awn phenotypic class is accepted in Table 4 at the 5% confidence interval (). The segregation data of this cross (Table 4) fit, statistically, the phenotypic ratio expected under the following assumptions: (i) lks2 dominantly suppresses the hood formation in Kk plants (allowing the appearance of short-awn phenotypes); (ii) lks2 recessively conditions the short-awn phenotype in kk (WT) plants; and (iii) in K plants, homozygous recessive for the suKD-25 allele, the hood is suppressed.

The following data, summarized in Table 4, consider the crosses between lsk2 and the four suK mutants mapping on chromosome 7H. The presence in all F₂ populations of a substantial fraction of long-awn plants supported the conclusion that lks2 was not allelic to any of the four suK mutants tested. The numerical analysis of the F₂ phenotypic segregation ratios of the four F₂ populations was not attempted because of the existence of tight linkages among the concerned loci and of the different degree of dominance of lks2 on hood suppression, resulting in F₁'s either with short-awn phenotypes (as in suKB-4 and suKC-33) or in lemmas with a very elevated hood (as in suKE-74 and suKF-76).

F₂ data, nevertheless, supported the concept, already evident from the consideration of F₁ results, that the short-awn phenotype of all suK mutants is a character under control of the K allele and not of the suK recessive alleles.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plant homeobox genes have a role in the formation of leaf primordia (POETHIG 1997 ; reviewed in BRUTNELL and LANGDALE 1998 ; POZZI et al. 1999 ; SINHA 1999 ), as evident from the effect of mutations in class I Knox genes (VOLLBRECHT et al. 1991 ; KERSTETTER et al. 1994 ), which result in dominant mutant phenotypes (CHEN et al. 1997 ; PARNIS et al. 1997 ; CHAN et al. 1998 ). Overexpression of the same genes has morphogenetic effects on leaf shape (KANO-MURAKAMI et al. 1993 ; MATSUOKA et al. 1993 ; SINHA et al. 1993 ; LINCOLN et al. 1994 ; MULLER et al. 1995 ; CHUCK et al. 1996 ; SATO et al. 1996 ; TAMAOKI et al. 1997 ; WILLIAMS-CARRIER et al. 1997 ), while loss-of-function mutants (with some exceptions; LONG et al. 1996 ) have no obvious phenotypes.

Models considering homeobox gene regulation should take into account the finding that knox genes show partial functional redundancy (CHEN et al. 1997 ; MARTIENSSEN and DOLAN 1998 ; BHARATHAN et al. 1999 ; POZZI et al. 1999 ). Furthermore, classes I and II of knox genes interact with each other and with members of the Bell homeobox family (BELLAOUI et al. 2001 ; MULLER et al. 2001 ), supporting the possibility that the loss of single components of the system may have relatively minor effects on phenotype. In this context, the Hooded mutation represents a special case. In the Hooded mutant, the barley knox gene Bkn3 is overexpressed due to a 305-bp duplication in intron IV (MULLER et al. 1995 ). The regulatory role of this intron is consistent with the existence of similar dominant insertional mutants in the corresponding intron of the maize ortholog Knotted1 (reviewed in SANTI et al. 2003 ). To shed light on the molecular network involving Bkn3, a number of experimental strategies have been adopted. The 305-bp duplicated element in the intron of Bkn3 has been used as a bait in a yeast one-hybrid screen. This approach led to the isolation of four barley cDNA clones encoding for putative binding proteins, one of which one is extensively characterized (SANTI et al. 2003 ). The screen for second-site suppressors of Hooded, described in the present article, represents a complementary genetic support to the molecular investigation of intron-mediated homeobox gene regulation (SANTI et al. 2003 ). Our results indicate that five genetic loci exist that, when mutated, suppress in trans the Hooded syndrome, producing the replacement of the ectopic K flower with awns much shorter than those in the WT. Genetic tests designed to clarify whether suK mutations mapped to loci controlling awn length (SOGAARD and VON WETTSTEIN-KNOWLES 1987 ; FRANCKOWIAK 1997 ; FRANCKOWIAK et al. 1997 ; LUNDQVIST et al. 1997 ; POZZI et al. 2003 ) allowed us to understand that the K allele present in suK lines—and not the suK alleles per se—induced the appearance of the short awn. The finding that the gene Lks2 (whose recessive allele causes the formation of short awns) mapped in the vicinity of the suK cluster on chromosome 7H (POZZI et al. 2003 ) prompted us to test its allelism with suKB-4, suKC-33, suKE-74, and suKF-76. The allelic state was not assessed, but, interestingly, plants carrying the lks2 allele together with K were characterized by the absence of hood formation and reduction in awn length. This led to the assignment of two distinct functions of the dominant K allele: the reduction of awn length and the formation of the hood. Only the latter can be suppressed by both suK and lks2 alleles. In Fig 4, phenotypic interactions and genotypes supporting this conclusion are presented. The reported findings imply that short-awn and awnless loci should be considered as candidates in participating in the regulatory network controlling homeobox gene expression during barley lemma formation.

View larger version (23K): In this window In a new window Download PPT slide   Figure 4. Schematic of phenotypes, genotypes, and genes affecting lemma organization, including the role played by the Bkn3 homeobox gene.

suK mutants, particularly suKE-74, are somewhat weaker than K (Table 1). This is also evident in the F₂ segregation as a deficiency in the homozygous recessive class (Table 2), findings consistent with some of the pleiotropic effects noted when plant knox genes are expressed in model species (CHAN et al. 1998 ; discussed in MULLER et al. 2001 ). Also, the phenotypic instability of some suK mutants is in agreement with the observations of YAGIL and STEBBINS 1968 . These authors reported that variations in light and temperature could cause metabolic changes in the distal end of the lemma, resulting in variable penetrance of the K phenotype. Recent data support the hypothesis that hormones can mediate homeobox gene expression and their possible interactions with the environment (TSIANTIS et al. 1999 ).

The experiments reported in this article were performed under the hypothesis that the products encoded by suK loci may be related to the intron-mediated regulation of Bkn3 (MULLER et al. 1995 ). Evidence has been provided that a 305-bp fragment of the Bkn3 intron IV controls Bkn3 gene expression in specific developmental domains in planta. This is mediated by a (GA/TC)₈ sequence binding site of the nuclear-targeted BBR protein (SANTI et al. 2003 ). The BBR gene is one of four cDNAs isolated on the basis of a yeast one-hybrid screen and comaps with the lks5 locus on chromosome 4H, sublinkage group 38 (SANTI et al. 2003 ). The expression of BBR in tobacco leads to a pronounced leaf shape modification, as expected if BBR interacts with leaf primordia-related homeobox gene expression. The isolation of the BBR gene supports the assumption that distinct genetic loci affect the Bkn3-mediated phenotype. A further cDNA isolated during the one-hybrid screen, indicated with the symbol Beil, maps to the barley sublinkage group 6 on chromosome 7H (WANG 2001 ), and it is a candidate to represent one of the suK loci mapping to the same chromosomal region.

The genetic control of awn length in barley is well characterized: lks loci affecting the trait have been described (TSUCHIYA 1973 ; FRANCKOWIAK 1997 ; FRANCKOWIAK and LUNDQVIST 2002 ), and one of these loci hosts the lks2 mutant (TAKAHASHI et al. 1953 ; the symbol has been modified in lks2: FRANCKOWIAK and LUNDQVIST 2002 ). The position of the locus has been recently integrated into a barley molecular linkage map (POZZI et al. 2003 ) and assigned to chromosome 7H, sublinkage group 6. Also, the loci suKB, C, E, and F map close together on chromosome 7H. This observation is striking in light of the fact that for each of the four tightly linked loci only one allele was recovered. The possibility was considered that the four linked mutants on chromosome 7H might represent mutually complementing alleles of the same genetic locus. However, recombination occurring between their loci and linked AFLP markers and the analysis of F₁ and F₂ generations of suK crosses exclude the one-locus hypothesis. Correspondence of the four suK loci to lks2 was evaluated in consideration of the hypotheses that the suK short-awn phenotype could have originated from (i) the recessive state at a short-awn locus of the original (unknown) WT variety, which generated the dominant mutation Hooded, or (ii) the hood-suppressive effect of suK recessive alleles, which also controls awn length. Both hypotheses turned out to be incorrect, also on the basis of lks2 x suK cross results, which showed that the short-awn phenotype of suK mutants is contributed by the K allele itself. Moreover, due to the dominance of the K allele in F₁ crosses, the short-awn phenotype was also found to be dominant over the awn length control carried out by the WT allele Lks2.

	FOOTNOTES

¹ These authors contributed equally to this work.
² Present address: Fondazione Parco Tecnologico Padano, via Haussmann 7, 26900 Lodi, Italy.

	ACKNOWLEDGMENTS

We thank Donata Pagani for careful technical assistance.

Manuscript received August 4, 2003; Accepted for publication January 9, 2004.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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