Mapping of two thiamin deficiency genes of pea

Hagh Nazari, A., Sharma, B. and                                         Div. of Genetics, Indian Agric. Res. Inst.

Mishra, S.K.                                                                                                         New Delhi, India

      Kumar and Sharma (1) isolated 20 stable auxotrophic mutations for thiamin biosynthesis in pea.  These mutations were induced by 0.05% ethylmethane sulphonate (EMS) and 0.0057% nitrosomethyl urea (NMU).  Based on complementation tests, the mutations were recessive alleles (thiA, thiB, and thiC) at three loci.  The mutants are characterized by yellowing of the leaves about two weeks after germination, followed by death of the plants.  The mutants were viable and indistinguishable from normal plants when thiamin was applied exogenously at the concentration of about 0.2 mg/ml.

      ThiA has been mapped very close to K on linkage group II (10).  ThiB was mapped near Pl in linkage group VI (11.3 crossover units), and ThiC was located 20 crossover units away from St in linkage group III (1).  However, the precise positions of each locus in relation to other loci on the respective linkage groups have not been studied.  The thiamin auxotrophic mutants can serve as good seedling markers in genetic studies.

      Two of these three mutants, i.e. thiB (SK 25) and thiC (L 102), are maintained at the Division of Genetics, I.A.R.I., New Delhi.  In order to determine the precise position of these two genes, the mutants were crossed with lines having several known markers of linkage groups III and VI.  The data for thiB were obtained from the cross of SK 25 (thiB) with P 1746-2 (Pl, Arg) while the data for ThiC were obtained from crosses of line L 102 (thiC) with P 1297-1 (apu, uni), P 1759-2 (wel), and P 1563 (st). A cross between the lines P 841 (st) and P 1297-1 was also made to determine the distance between the loci St and Apu. These crosses also segregated for the genes A, D, Af, Le, Gp, Tl, Con, and Wlo, which belong to other linkage groups. The distance between Apu and Wel was estimated from a cross between P 1297-1 and P 1759-2. In all the crosses, the F₁ plants were fully fertile. The F₁ plants in the crosses involving the thiB and thiC mutants and normal green parents were normal, confirming the recessive nature of these two mutations. Details of the crosses, single locus and joint segregation with c² values and recombination fractions (R.F) obtained by using a computer program, CROS, provided by Dr. S.M. Rozov (http://pisum.bionet.nsc.ru) are presented in Table 1. No mapping function was applied, and the map distances are expressed as recombination frequencies.

Table 1.  Joint F₂ segregation of thiamin-deficient mutations (thiB, thiC) with other morphological markers.

^a,  ^b, ^c  Mean P < 0.01, P < 0.001, P = 0.0001, respectively.

¹ D = dominant allele and r = recessive allele

² C = coupling and R = repulsion phase linkage

      The F₂ segregation analysis showed that ThiB and ThiC as well as all other marker loci segregated in the 3:1 ratio (Table 1).  In the cross SK 25 x P 1746-2, thiB was estimated to be located at about 18.3 map units away from Arg, and 16.5 map units from Pl. The linkages ThiB–Arg (P < 0.0001) and Pl–ThiB (P < 0.0001) were highly significant. The Pl–ThiB map distance of 16.5 is reasonably close to 11.3 reported by Kumar and Sharma (1). The map distance between Pl and Arg on the same chromosome was 4.3 (Table 1).  This distance was reported as 3 map units by Marx (2), 2 map units by Weller and Murget (10) and 2.3 map units by Święcicki and Kruszka (9).

      Based on the segregation analysis of ThiB, Arg and Pl, these loci can be arranged in the following order:

      From the cross L 102 x P 1759-2, ThiC has been located at the distance of 16.6 map units from Apu, 27.5 map units from St, and 33.3 map units from Wel.  Marx (3) located St about 8.8 map units away from Wel.  Subsequently this distance was reported as 12.5 map units (5).  Sharma and Kumar (8) did not find any linkage between St and Uni, but this linkage was estimated to be 36.4 map unitsd by Marx (5).  However, Rozov et al. (6) reported 57 Kosambi units as the distance between St and Uni.

      The results of the cross L 102 x P 1297-1 showed that ThiC is located about 18.2 map units away from Uni.  The distance between Uni and Apu being 24.6 shows that ThiC is placed between these two genes.  The linkages ThiC-Apu and ThiC-Uni were highly significant (P < 0.0001).

      Marx (4) reported 23 map units distance between Apu and Uni, 10 map units between St and Apu, and 30 map units between St and Uni.  Marx (5) located Apu at the distance of 33.9 map units from Uni, while Sarala and Sharma (7) reported 20 map units distance between these two loci.

      From the cross P1297-1 x P841, St has been mapped 13 map units away from Apu.  This distance is reasonably close to 15 map units reported by Sarala and Sharma (7).  Finally, from the cross P1297-1 x P1259-2, the distance between Wel and Apu was estimated about 17.9 map units which is very close to the distance (19.8) reported by Marx (5).

      On the basis of joint segregation of thiC, wel, apu, uni and st, the following arrangement is proposed:

      Thus, our results confirm that the two thiamin auxotrophic mutations, thiB and thiC, are recessive.  The locus ThiB is located in the lower part of linkage group VI and ThiC belongs to linkage group III between Apu and Uni .  The exact position of ThiC with respect to the isozyme markers (Adh-1 and Acp-3) is under investigation.  The two thiaminless mutations can be used for further studies on the biochemical pathway of thiamin biosynthesis as well as in mapping programs.

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02. Marx, G.A.  1982.  Pisum Newslett. 14: 50-52.

03. Marx, G.A.  1983.  Pisum Newslett. 15: 47.

04. Marx, G.A.  1984.  Pisum Newslett. 16: 46-48.

05. Marx, G.A.  1986.  Pisum Newslett. 18: 49-52.

06. Rozov, S.M., Borisov, A.Y. and Tsyganov, V.E.  1994.  Pisum Genetics 26: 24-25.

07  Sarala, K. and Sharma, B.  1994.  Pisum Genetcs 26: 28.

08. Sharma, B. and Kumar, S.  1981.  Pulse Crops Newslett. 1(3): 21-22/

09. Święcicki, W.K. and Kruszka, K.  1995.  Pisum Genetics 27: 22.

10. Weller, J.L. and Murfet, I.C.  1994.  Pisum Genetics 26: 41-43.