The gene responsible for serrate leaflets in P. sativum ssp. abyssinicum is on linkage group III tightly linked to an STS marker

Lorenzi, V.                                                                                         E.S.I.T.P.A., Rouen, France

Murphy, R.L. and Weeden, N.F.                                      Montana State Univ., Bozeman, MT, USA

      The strongly serrate leaflet margins constitute the most obvious morphological character defining Pisum sativum ssp. abyssinicum (1, 2, 13).  The serration is not observed on leaflets of the first two true leaves but is strongly expressed for approximately the next five nodes (see cover).  Sutton (11) reported on this phenotype in W808, a line from Palestine that has been confirmed to possess the Abyssinicum genotype (13).  He determined that the phenotype was controlled by a dominant allele at a locus he designated Ser.  Wellensiek (14) suggested that the symbol Td be used.  Rosen (9) used td, believing the gene was recessive or only semi dominant, and Lamprecht (5) used both Ser and Td to describe serrate phenotypes.  Finally, Smirnova (10) suggested that a different locus, Td’, might exist in material he examined.  At present, W808 is used as the type line for Td and is recessive for ser, whereas W1414 is the type line for Ser, as defined by Lamprecht (5).  Thus the literature is very confusing, because the gene responsible for the strongly serrate leaflet margins in line W808 has been referred to as both Ser and Td.  In addition, Lamprecht’s (5) drawing of leaflet morphology for the genes Td, Ser and Inci places the phenotype exhibited by W808 squarely in the Ser category.

      The location of Td (as defined by the type lines) on the pea linkage map also has been a subject of discussion in the last 20 years.  Marx (7) pointed out inconsistencies between his data and those of Lamprecht.  Lamprecht’s data suggested linkage between Td and Fa at a distance of about 30 cM (4), whereas Marx (7) found linkage between B and Td at an intensity of 14 and 17 cM.  Marx reasoned that because Fa was on chromosome 4 (Lamprecht’s definition) and B was on chromosome 3, one of the two linkages must be incorrect.  Within the following decade several investigators (3, 8, 12) explored linkage relationships of genes generating serrate leaflet margins. Święcicki (12) observed linkage between Inci and B, but not between Td and B or Ser and B.  His experiments did not involve the markers Le or Np but did test linkage with many standard morphological markers, including St.  Two papers reported linkage between Td and markers on linkage group III.  Grajal-Martin and Muehlbauer (3) reported weak linkage (20 to 30 cM) between Lap1 and Td, and concluded that their results indicated that either Td was on linkage group III or that there were more than one gene producing the serrate phenotype.  In two crosses Polans (8) reported linkage between Td and St and/or B.  The two lines he used as sources of the serrate phenotype (82-14n and A778-26-6) were a P. s. ssp. elatius line selected from PI268480 by N.F. Weeden and a P. s. ssp sativum line with an introgressed Td allele derived from P.s. ssp. abyssinicum (see source in ref. 3), respectively.  The data obtained by Polans placed Td between St and B, a surprising location because Święcicki (12) should have observed linkage between Td and St in his experiments.  As indicated above, most of the reported linkage intensities between the gene generating the serrate leaflet phenotype in P.s. ssp. abyssinicum and standard marker loci have been greater than 20 cM and therefore are open to question.  In this study we attempted to generate more conclusive data for the position of this gene through the use of DNA markers. 

      A cross was made between P. s. ssp. sativum cv ‘Sparkle’ and P. s. ssp abyssinicum line PI358617.  The F₁ plants were tall and displayed a serrate leaflet phenotype matching that in PI358617 (most prominent on leaflets from nodes 5 through 10).  The F₁ plants were also partly sterile, indicative of chromosomal rearrangements known to exist between Abyssinicum pea and most P. sativum ssp. sativum lines (Lamprecht, 6).  Thirty-six F₂ plants were scored for Le, Td and the STS marker, CipPor.  Both Le and CipPor are known to be on linkage group III of the consensus map, with CipPor mapping approximately 7 cM from Np on the opposite side from Le (S. Brauner, R. Murphy. J. Przyborowska, and N. Weeden, unpublished).  The primer sequences used to amplify the CipPor fragment were 5’-ACTGCTAAGGCTTTGGCTGA and 5’-AGATTTTGTTAGGCTTGGATCACT with standard amplification conditions and an annealing temperature of 60 C.  The resulting 1100 bp fragment was cut with HaeIII to reveal a polymorphic restriction site (the CipPor fragment from ‘Sparkle’ possessed a HaeIII site near the middle of the fragment, whereas the CipPor fragment from PI358617 did not).

      Segregation of the individual loci gave monogenic ratios [Le:le = 27:7, c² =0.37, p<0.01; Td:td = 20:16 c² =4.6, p<0.05; CipPor (het + PI358617):CipPor (‘Sparkle’) = 20:16].  Joint segregation analysis is shown in Table 1.

Table 1.  Joint segregation analysis of Le, Td and CipPor in the ‘Sparkle x PI358617 F₂

¹Phenotype designations: D=dominant; R= recessive

The Td and CipPor segregation ratios deviate significantly from expectation (3:1), but the deviation may be attributed to linkage with a lethal or some other factor causing the partial sterility.  Although neither CipPor nor Td display significant linkage with Le, if we arrange the loci so that the number of recombinant and double recombinants are both minimized, we obtain a locus order with Td in the middle.  From the position of CipPor on the consensus map we know the order of the loci is Lap1—Pepc—CipPor—Np—Le.  Thus our current results would place Td near Np, although the data do not permit a determination of the distance between Np and Td or which locus in closer to CipPor.  It should be emphasized that we mapped the gene in P. s. ssp. abyssinicum, and that the gene in the P. s. ssp elatius accession used by Polans (8) may be different.

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