MAPPING OF A LEGUMIN SUBUNIT GENE ON PEA CHROMOSOME 7

The major pea seed storage protein legumin contains six subunit pairs. They may be divided into three types: conventional (M_r 53000-54000), big (M_r 55000-58000) and small (M_r 35000), referred to as Lg-1, Lg-2 and Lg-3, respectively (6). Each subunit pair consists of a - and b-subunits, joined by disulphide bonds. The majority of legumin is synthesized as precursor molecules, containing covalently linked a- and b-subunits (2,5). So the genetics of b-subunits should follow those of a-subunits.

It has been reported that Lg-1 a-subunits are encoded in a set of closely linked genes at a single locus on chromosome 7, located approximately 7 map units (3) or 17 map units (6) from r in the direction of tl. The genetics of other legumin subunits has not been studied in detail. Lg-2 a-subunits segregated independently from Lg-1 a-subunits in one cross and were linked with them in the other. Linkage was found between Lg-2 a- and Lg-3 b-subunits, but independent segregation for Lg-3 a- and Lg-3 b-subunits was suggested (6).

Three classes of legumin cDNA were identified (4). One class of legumin genes showed linkage to gene r and corresponds to the Lg-1 a-subunits. A second class has been mapped to a locus near a on chromosome 1. A third class of legumin genes also appears to be linked with gene a (4). The relationship between the two last classes of legumin genes and particular a- and b-subunits is unknown.

Analysis of the seed proteins of pea line VIR319 (ssp. sativum) by sodium dodecylsulphate gel electrophoresis revealed a legumin subunit pair which could not be identified with any known subunit pair. This polypeptide of uncertain identity is symbolized temporarily as Lg-u. The M_r of Lg-u is approximately 3000 less than the M_r of Lg-1 (Fig. 1, non-reducing conditions). The difference between Lg-u and Lg-1 subunit pairs is due to their a-subunits, while the b-subunits of these pairs seemed to be identical (Fig. 1, reducing conditions).

Line VIR319 (Lg-u⁺, present) was crossed with line WL1238 (Lg-u^-, absent). In F₁ hybrid seed Lg-u was expressed in a lesser amount (Lg-u⁺/^-) than in parent VIR319. An analysis of F₂ progeny (n=196) revealed segregation of Lg-u a-subunit classes + : +/- : - as 47 : 102 : 47, respectively. These results indicate Lg-u⁺ and Lg-u^- to be a single allele pair (c²₁ = 0.33). Segregation of morphological markers and Lg-u a-subunits in F₂ plants showed linkage of Lg-u with genes r and tl (Table 1A). 13 plants showed recombination between genes r and tl, 32 between tl and Lg-u, and 45 between r and Lg-u. Corresponding distances were calculated as 3.3 ± 0.9, 8.2 ± 1.4 and 11.5 ± 1.6 map units, respectively, which generates the following map:

r--3.3--tl------------- 8.2---------------- Lg-u

Mapping of gene Lg-u showed that it is located in the same region on chromosome 7 as gene His-1, coding for histone H1 the slowest fraction. The distance between His-1 and tl was found to be 7.6 crossover units (7) while the data above indicate a distance of 8.2 units for Lg-u and tl. These results were obtained in different crosses and the question arises as to which gene, Lg-u or His-1, is situated closer to gene tl. To answer this question I crossed lines 319.1 and Sprint, which differ not only in molecular variants of legumin and histone H1 the slowest fraction, but also in genes r and tl at one side of Lg-u and His-1, and gene Sa-K9 at the other side. Line 319.1 has genotype r, tl^w, His-1 F, Sa-K9 S, Lg-u⁺ and line Sprint has genotype R, Tl, His-1 S, Sa-K9 F, Lg-u^-. Only two F₂ plants among 132 had a recombinant phenotype in respect of genes Lg-u and His-1 (Table 1B). Thus, the map distance between Lg-u and His-1 is about 0.8 ± 0.5 map units. Recombinant plants had genotypes Rr Tltl^w Lg-u+/- His-1 S, Sa-K9 F and Rr Tltl^w Lg-u+/- His-1 F, Sa-K9 S.

These results indicate the following gene order:

r — tl — Lg-u — His-1 — Sa-K9.

The location of gene Lg-u on chromosome 7 coincides with that of Lg-1. Possibly, Lg-u is a member of the Lg-1 gene family and the Lg-u polypeptide may be the result of a deletion in one of the genes of this family. Several variant forms of the Lg-1 polypeptide have been described (1,6,8). Alternatively, Lg-u may be structurally different from the other major legumin polypeptides. Further study, e.g. sequencing or peptide mapping, is necessary to resolve these alternatives.

Table 1. Distribution of F₂ plants into phenotypic classes.

Fig. 1. Sodium dodecylsulphate polyacrylaramide gel electrophoresis of pea seed proteins in lines VIR319 (1) and WL1238 (2).

A - total protein extracts stained by Coomassie R-250,

B - legumin subunits (reducing conditions) and subunit pairs (non-reducing conditions) identified by "Western" blot analysis.

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