Mutants with roots showing an abnormal response to gravity have been isolated several times in pea [2, 3]. In these mutants, the root generally fails to display geotropism, and if seeds are germinated in soil, the main root will often break through the soil surface and grow horizontally above it. Blixt [1] reviewed the history of three such mutants and demonstrated that all three were alleles at the same locus, Age. Attempts to map the locus were unsuccessful, although many regions of the genome were tested [1]. With the present availability of many mapped DNA markers, we attempted to map this locus using molecular means.

Seeds of JI819 (age) were obtained from Thomas Bjorkman at Cornell University. A cross between this line and line ‘Slow’ was made in order to confirm the segregation of the phenotype and to test for linkage with many standard markers. A convenient method for identifying mutant plants was developed. Seeds were planted in 10 cm clay pots containing Cornell mix, a light artificial soil. One week after planting, the pots were checked for emergence of epicotyl. In those cases where no epicotyl could be observed, or where the radicle had emerged instead, the seed was gently dug from the soil and replanted so that the radicle was directed downward. Although this screening identified many of the mutant plants in a segregating population, the most reliable screen was performed two to three weeks later, when the appearance of many secondary roots at the soil surface clearly identified the mutant homozygote.

^a-Phenotype designations: 1 = dominant or allele in wildtype parent, 2 = recessive or allele in mutant parent.
^b-Due to seedling death and experimental design not all plants in the populations were scored for the DNA markers see text for details).
^* P < 0.05, **P < 0.01

The JI 819 x Slow F2 population did not reveal linkage between age and d, i, a, wb, st, le, gp, oh, or several isozyme markers (data not presented). Therefore, three segregating F2 populations were analyzed for segregation of age and DNA markers. C97-8 was derived from VIR1987 (Age) ? A97-5-5 (a homozygous age derivative of the JI 819 x Slow F2). A98-41-44 was derived from a homozygous age derivative of C97-8 (C97-8-17) crossed with 87-19 I-d, an inbred line from the JI1794 ? Slow mapping population mentioned in Weeden et al. [5]. C98-1 was derived from C97-8-17 ? A1078-239, the latter being a multiple marker line obtained from Dr. G.A. Marx. In the first two F2 populations mentioned, the segregation of age deviated significantly from the expected 3:1 ratio (Table 1), with the mutant phenotype being in surplus. However, in the C98-1 population, age segregated normally. In population C 97-8, DNA was extracted from only 20 plants (8 wild type and 12 mutant). These 20 DNA samples were screened with a set of primers that in the JI 1794 ? slow mapping population generated RAPD fragments that gave excellent coverage of the linkage map. Joint segregation analysis indicated that markers on linkage group IV consistently displayed linkage with the mutant (Table 2). The marker P393 is a sequence tagged site (STS) mapped to linkage group IV by Gilpin et al. [4] and has proven to be a particularly good marker for this linkage group in several crosses analyzed at Geneva, NY. P603B600, Sympgm and VM16A are amplified fragments generated by single long primers (16 to 21 bp in length) as was described in Ye et al. [6].

Table 2. Joint segregation analysis of age with markers on linkage group IV

Cross	Markers	No. with designated phenotypea						Total	c2	% Recomb. + S.E.
		1,1	1,H	1,2	2,1	2,H	2,2
C97-8	Age/P393	4	4	0	0	4	8	20	11.0	14 + 8
A98-41-44	Age/P603B	31		3	9		18	61	22.3	17 + 12
C98-1	Age/P603B	5	8	1	0	1	3	18	8.5	12 + 8

^a Phenotype designation: 1 = dominant or homozygous for allele in wildtype parent, 2 = recessive or homozygous for allele in mutant parent, H = heterozygous.

The results from populations C97-8 and A98-41-44 would generally be regarded as questionable because of the distorted segregation of age. However, in both populations we had access to many of the over 800 markers generated for the JI 1794 ? Slow RILS . The use of a selection of these markers to cover the genome revealed independent assortment between age and other regions of the map. Furthermore, the only region to display distorted segregation was the one end of linkage group IV. Hence, the correlation between the segregation pattern for age and that for DNA markers on linkage group IV is almost certainly due to direct linkage. Results from the third small population (C98-1), in which age does segregate normally, confirm the linkage between this gene and the marker P603B600.

The position of age appears to be about 14 cM from the P393 STS. Because Sympgm maps very close to P393 on the consensus map, whereas P603B600 and VM16A map closer to the end of that linkage group, age also must be toward the lower end of linkage group IV as it is commonly depicted. This region does not currently contain a convenient morphological mutation that can be used to ‘tag’ this region of the genome in general mapping studies. We feel that the age mutation is sufficiently easy to score that it could serve such a purpose.

1. Blixt, S. 1970. Pisum Newsl. 2:11-12.
2. Blixt, S., Ehrenberg, L. and Gelin, O. 1958. Agri Hort. Genet. 16:238-250.
3. Blixt, S., Ehrenberg, L. and Gelin, O. 1958. Agri Hort. Genet. 22:178-216.
4. Gilpin, B.J., McCallum, J.A., Frew, T.J. and Timmerman-Vaughan, G.M. 1997. Theor. Appl. Genet. 95:1289-1299.
5. 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.
6. Ye, G.-N., Hemmat, M., Lodhi, M.A., Weeden, N.F. and Reisch, B.I. 1996. BioTechniques 20:368-371.