The position of Age and Wsp on linkage group IV

Weeden, N.F., McBroom, M.,                                         Dept. of Plant Sciences and Plant Pathology

                                                                              Montana State University, Bozeman, MT  59717

Przyborowski, J. and                                                      Dept. of Plant Breeding and Seed Production

                                    University of Warmia & Mazury in Olsztyn, 10-724 Olsztyn-Kortowo, Poland

Burger, P.                             Research Inst. for Fruit Vine and Wine, Stellenbosch, 7599, South Africa

      The locus Age (Ageotropic) has been determined to be on linkage group (LG) IV approximately 14 cM distal to the STS marker P393a (3).  Last year the senior author reported that the locus Wsp is linked to Age (2); however, the distance between Age and Wsp was only roughly approximated, and the relative order of the loci on the linkage group was not determined.  Both age and wsp are easily scored in seedlings, and the region of LG IV distal to P393a has relatively few convenient markers.  Hence, both loci have great potential as standard anchor markers for general mapping studies, and it is important to clearly establish their relative positions.  We present here the order of the loci on LG IV and a more precise estimation of the distance between them.  In addition, we present evidence that the number of RAPDs in this region is relatively low, further emphasizing the importance of these morphological mutants for mapping studies.

      Three experiments were performed to more precisely determine the linkage relationships among markers around Age.  One involved a classical linkage analysis with the alleles age and wsp in coupling phase.  The second attempted to place Wsp directly on the consensus map, and the third was an attempt to use bulked segregant analysis (1) to identify additional DNA markers near Wsp.  Only the first experiment was successful, but the lack of success of the other two may reveal important information about the DNA sequences in this region of the genome.

      A plant homozygous for both age and wsp was selected from the F₂ mapping population described in (2).  This plant was crossed to two Age Wsp lines of unrelated parentage (B285-500-5 and cv. Sparkle).  The two F₂ populations derived from these crosses were grown in a 1:1 peat:silt soil mix in Conetainers in the greenhouse.  Seed were examined after five days to insure age plants were oriented so that the primary root was directed downward and the epicotyl had a clear route to the surface.  After two weeks, seedlings were scored for wax deposition and root orientation as follows:  plants lacking wax on stem and upper surface of stipule were scored wsp; plants with normal wax on stem and upper surface of stipule were scored Wsp; plants with secondary roots protruding above soil surface were scored age; plants without roots on surface as Age.  Most plants were then transferred to pots, enabling the root orientation to be further examined.  In all cases the appearance of roots at the soil surface had correctly revealed the homozygous age genotype. 

      Segregation ratios indicated that in both populations each gene segregated normally.  Joint segregation analysis gave distances of 16 and 23 cM for the single population recombination estimates (Table 1).  Heterogeneity tests indicated the two results could be combined, and the combined linkage value was 19 + 11 cM.  This distance is in reasonable agreement with the previous estimate of <15 cM (2), which was based on a small population with alleles in repulsion phase.  The distance is also similar to that between Age and P393a (3).  Hence, Wsp must either be very near P393a or at the distal end of the known map for LG IV.

Table 1.  Joint segregation analysis of the loci Age and Wsp in two F₂ populations

      In order to determine the relative order of P393a, Age and Wsp, a cross was made between a recombinant inbred line from a mapping population derived from the cross MN313 x JI1794 (J. Przyborowski and N.F. Weeden, unpublished) and a line homozygous for wsp.  An F₂ population was generated from this cross and joint segregation analysis performed on P393a, wsp and several RAPD markers known to be on LG IV.  Only very weak (non-significant) linkage was observed between P393a (or RAPD markers closely flanking this STS) and wsp (data not presented).  Although age was not segregating in this population, the combination of a lack of significant linkage between P393a and wsp and the 14 cM linkage between Age and P393a reported in (3), suggest that Wsp is distal to Age and that this region of LGIV can be represented as follows:

P393a––––14 cM–––––––Age–––––19 cM––––––––Wsp.

Although a number of RAPD markers were identified distal to P393a on the consensus map (4), none of these segregated in the F₂ used in the second experiment.  Hence, we could not locate Wsp very precisely on the consensus map.  The approximately 33 cM between P393a and Wsp is similar to the distance (34 cM) between P393a and the most distal marker on the consensus map.  Thus, it would appear that Wsp is an excellent marker for the most distal portion of this arm of LG IV (chromosome 7). 

      Our third experiment attempted to identify RAPD markers linked to Wsp by bulked segregant analysis.  We used F₃ plants from a cross made by Dr. G.A. Marx (A686-353 [wsp U] x A486-115 [det]).  Ten F₃ plants were grown from each of 30 F₂ plants. F₂ plants homozygous wsp or homozygous Wsp were identified by examination of the F₃.   DNA was extracted from five homozygous wsp F₃ plants (each derived from a different F₂) and from five homozygous Wsp F₃ plants (again derived from different F₂ plants).  The DNA was combined into a wsp bulk and a Wsp bulk and the bulks compared using 281 primers obtained from Operon Technologies, Inc. or the University of British Columbia biotechnology laboratory.  We failed to identify a RAPD marker linked to Wsp despite the low stringency of the screen (markers within 20 cM should have been easily recognized in this survey because we included only homozygous lines and only five individuals in each bulk).  The few differences that were observed between bulks proved to be ‘false positives’ unlinked to Wsp when tested on the whole population. 

      In other bulked segregant analysis studies in pea involving this number of primers, we have usually been able to identify several RAPD markers linked to the locus of interest.  Hence, we conclude that this region of the pea genome contains a relatively low number of RAPD-generating sequences.  Whether this low level of RAPDs is due to a lack of amplifiable sequences in this region or a low level of sequence polymorphism could not be determined from our data.

1.  Michelmore, R.W., Paran, I. and Kesseli, R.V.  1991.  Proc. Natl. Acad. Sci. USA 88: 9828-9832.

2.  Weeden, N.F.  1999.  Pisum Genetics 31: 35.

3.  Weeden, N.F., Morrell, K. and Boone, W.E.  1998.  Pisum Genetics 30: 25-26.

4.  Weeden, N.F., Ellis, T.H.N., Timmerman-Vaughan, G.M., Swiecicki, W.K., Rozov, S.M. and Berdnikov, V.A.  1998.  Pisum Genetics  30: 1-4.