Translocation derived duplications involving short arm segments of barley
chromosome 5 as defined genetically by physical RFLP mapping.
G. Künzel, A. Sorokin and F. Marthe
Institute of Plant Genetics and Crop Plant Research, D-06466 Gatersleben, Germany

Applying the principles described by Hagberg and Hagberg (1991) two duplication lines, DU1 and DU2, were produced by crossing suitable translocation lines (T-lines) involving the same two chromosomes each (Table 1). Both pairs of parental T-lines, T5-7ab/T5-7aj and T5-7ac/T5-7ah, have interchanged chromosomes which are distinguishable from each other and the remaining chromosomes by their Giemsa N-banding patterns and/or arm ratios (Marthe and Künzel 1994). This allowed us to identify homozygous duplications by studying Giemsa N-banded mitotic metaphase chromosomes from F/2/ seedlings.

As compared to their parental T-lines, the plants of both the DU1 and DU2 line show somewhat reduced tillering capacity and have approximately 10% and 25% reduced kernel yields, respectively.

In principle, the translocation technique used allows precise predictions of the cytological size and location of duplicated segments if the breakpoint positions of the crossed T-lines are precisely known as in the case of T5-7ab. However, in T5-7ac, T5-7ah, and T5-7aj the breakpoints could be assigned so far only to defined segments of different size (Table 1). This is restricting the predictability of position and extension of the duplicated segments. Thus, in DU1 resulting from the combined translocation chromosomes T57aj and T75ab, duplicated segments of uncertain size can be presumed to be located within the distal half of the short arm of chromosome 5 (5S) in addition to the proximal part of the Nucleolus Organizing Region (NOR) together with an adjacent segment of 7S. Similarly, in DU2 derived from T57ac and T75ah, duplicated segments can be expected within the distal half of 5S and/or the middle region of 7L.

To confirm the involvement of 5S segments in duplications and to determine the genetic "make-up" of these segments, DNA markers localized in linkage group 5 of the German Igri/Franka RFLP map (Graner et al. 1994) were partly sequenced for constructing sequence-specific primers to be used in polymerase chain reactions (PCR). Then, the DNA of each of the eight different translocation chromosomes of the four parental T-lines was used as a template for PCR as described by Sorokin et al. (1994). The chromosome 5-specific RFLP loci were assigned to each of the two pairs of translocation chromosomes, T57aj/T75ab and T57ac/T75ah, constituting DU1 and DU2, respectively. These studies revealed the locus MWG758 to be duplicated in DU1 and the loci MWG758 and MWG2056 to be duplicated in DU2 (Table 2).

Using additional RFLP probes and translocations involving chromosome 5, MWG758 and MWG2056 were physically assigned to neighbouring segments within the distal half of 5S at the positions 25-30 mGN and 30-46 mGN, respectively (Künzel at al. 1995 and Sorokin unpublished). Thus, as predicted, DU1 proved to contain a duplication spanning about 5 mGN at the position 25-30 mGN, and DU2 proved to contain a duplication spanning about 21 mGN at the position 25-46 mGN.

Tab. 1. Characterization of parental T-lines, F/1/ hybrids, and duplications derived in F/2/ from the crossed T-lines

________________________________________________________________

DU1
  Parental T-lines
     Line¹          Arms involved and break positions²
      T5-7ab     T57ab: 5S (25 mGN)³     T75ab: 7NOR (43 mGN)
      T5-7aj     T57aj: 5S (25-40 mGN)   T75aj: 7S (28-43 mGN)

  F1 hybrid T5-7ab x T5-7aj
    Meiosis:                   7 bivalents
    Pollen fertility^4^:       70%
    Seed set:                  73%
        
  Karyotype of the derived duplication DU1 
    Translocation chromosomes T57aj and T75ab in addition to
    normal chromosomes 1, 2, 3, 4 and 6
________________________________________________________________

DU2
  Parental T-lines
    Line¹          Arms involved and break positions²
     T5-7ac     T57ac: 5S (30-46 mGN)³  T75ac: 7L (32-48 mGN)
     T5-7ah     T57ah: 5S (11-32 mGN)   T75ah: 7L (36-57 mGN)
        
  F1 hybrid T5-7ac x T5-7ah 
    Meiosis:                  7 bivalents
    Pollen fertility^4^:       67%
    Seed set:                 72%
        
  Karyotype of the derived duplication DU2 
    Translocation chromosomes T57ac and T75ah in addition to
    normal chromosomes 1, 2, 3, 4 and 6
________________________________________________________________
¹) according to Künzel 1993
²) according to Sorokin et al., this volume
³) breakpoint positions in milliGeNomes according to Jensen and Linde-Laursen 1992
^4^) based on aceto carmine staining

The programme for producing "directed" duplications of short arm segments of chromosome 5 via translocations was initiated to duplicate agronomically important loci such as Mla conferring powdery mildew resistance. However, in the meantime it has been demonstrated that nearly all of the studied translocation breakpoints of chromosome 5 occur within regions of suppressed recombination while the overwhelming majority of the hitherto mapped RFLPs (and genes ?) belong to very distal chromosome segments expressing high recombination rates (Sorokin et al., this volume). Therefore, translocation techniques, as used in this study, have to be considered less promising in duplicating genes positioned within chromosome regions of high recombination.

Tab. 2. Chromosome 5-specific RFLP markers assigned to translocation chromosomes involved in DU1 and DU2

___________________________________________________________

                    DU1                      DU2     
              _____________           _________________
              T57aj     T75ab         T57ac      T75ah
              _______________         ________________
   MWG913¹      -         +             -           +
   MWG2056      -         +             +           +
   MWG758       +         +             +           +
   MWG800       +         -             +           -
   Centromere
____________________________________________________________
+/-: RFLP probe-specific PCR fragment present/absent

¹ RFLP markers genetically localized in linkage group 5 of the German Igri/Franka RFLP map (Graner et al. 1994, Sorokin et al. 1994) in order from the distal region of 5S to the centromere

Acknowledgements

We are grateful to Andreas Graner at the Institute of Resistance Genetics, Grünbach, Germany for supplying the RFLP probes. This research was supported by the Federal Ministry for Research and Technology (Grant No. 0319960B).

References

Graner, A., E. Bauer, A. Kellermann, S. Kirchner, J.K. Muraya, A. Jahoor, and G. Wenzel. 1994. Progress of RFLP-map construction in winter barley. BGN 23: 53-59.

Hagberg, A., and P. Hagberg. 1991. Production and analysis of chromosome duplications in barley. In: T. Tsuchiya and P.K. Gupta (Eds.), Chromosome Engineering in Plants: Part A 'Genetics-Breeding-Evolution', Elsevier Sci. Publ., Amsterdam, pp. 401-410.

Jensen, J., and I. Linde-Laursen. 1992. Statistical evaluation of length measurements on barley chromosomes with a proposal for a new nomenclature for symbols and positions of cytological markers. Hereditas 117: 51-59.

Künzel, G. 1993. Coordinators report: translocations and balanced tertiary trisomics. BGN 22: 80-102.

Künzel, G., A. Sorokin, and F. Marthe. 1995. Progress in relating genetic to physical distances of marker sequences on microisolated barley chromosomes. In: "Kew Chromosome Conference IV", Proc. 4th Kew Chromosome Conference, submitted.

Marthe, F., and G. Künzel. 1994. Localization of translocation breakpoints in somatic metaphase chromosomes of barley. Theor. Appl. Genet. 89: 240-248.

Sorokin, A., F. Marthe, A. Houben, U. Pich, A. Graner, and G. Künzel. 1994. Polymerase chain reaction mediated localization of RFLP clones to microisolated translocation chromosomes of barley. Genome 37: 500-555.

Sorokin, A., F. Marthe, and G. Künzel. Integration of 37 translocation breakpoints of barley chromosome 5 into the Igri/Franka derived RFLP map. BGN 24: