Aconitase isozymes -- new gene markers for tomato.

Tanksley, S.D.

Aconitase (E.C. 4.2.1.3) catalyzes the reversible interconversion of citrate to isocitrate via the intermediate cis-aconitate:

citrate  -->    (cis-aconitate)   -->    isocitrate

This reaction is part of the TCA cycle and thus one isozymic form is expected to be found in the mitochondria. In most species, an additional isozyme is found in the cytosol. Using the histochemical staining procedure given below, it has been possible to visualize the sites of tomato aconitase isozyme activity in starch gels. In extracts from leaf or root tissue of inbred tomato lines, two well resolved bands are visible in the anodal slice of histidine gels. In L. esculentum cv. VF36 and most other esculentum cultivars the bands are 30 and 40 mm from the origin. A survey of various populations of tomato species has revealed that the position of the two bands can vary independently suggesting that they are controlled by alleles at two loci. The proposed loci have been given the symbols Aco-1, coding for the faster migrating isozymes, and Aco-2 coding for the slower migrating form. To study the joint segregation of the two loci, a single plant of L. pimpinellifolium LA 1661, found to be heterozygous for both loci, was testcrossed to another individual homozygous for both loci. The results given in Table 1 support the two-locus hypothesis and further indicate that the two loci are not tightly linked.

Using the interspecific cross L. esculentum cv. VF36 x L. pennellii LA716 BC pennellii, it has been possible to test the segregation of Aco-1 with a number of previously mapped enzyme-coding genes (see Table 2). Independent segregation was observed with all but two loci, Pgm-2 (chromosome 4) and Pgi-1 (chromosome 12) (deviation significant at 0.05 probability level). To decide which, if either, of these deviations was due to true linkage, a plant carrying a variant Aco-1 allele was crossed to primary trisomics for chromosome 4 and chromosome 12. Only in the triplo 12 hybrid trisomic was allele dosage observed indicating that the locus is located on that chromosome. As a further manner of proof, a triplo 12 plant was synthesized which carried three distinct alleles for Aco-1. These combined data unequivocally prove that Aco-1 is on chromosome 12. The exact placement of Aco-1 relative to other markers on the same chromosome, Est-4 and Pgi-1, can be determined by the joint three locus segregation data (Table 3). These data suggest the following gene order: Est-4 -- 13 cM -- Pgi-1 -- 38 cM -- Aco-1.

Although it was known that Aco-1 and Aco-2 are not tightly linked (Table 1) it is possible that the two loci still reside on the same chromosome. To test this possibility, a variant allele for Aco-2 was also crossed to triplo 12. In this case, the trisomic hybrids showed no allele dosage indicating that Aco-2 is on a chromosome other than chromosome 12. At present the chromosomal location of Aco-2 is unknown.

STAINING  RECIPE  FOR  ACONITASE

1M Na citrate pH 7.0       2 ml
NADP+                     10 mg
MTT                       10 mg
PMS                        2 mg
IDH (isocitrate           30 units
     dehydrogenase)
0.1 M Tris buffer pH 7.5  50 ml

 - incubate in dark for  1  hr  -

Table 1. Joint segregation of Aco-1 and Aco-2 in testcross.

                   Aco-1
              +/+   +/1  Total

Aco-2    +/+    6    10     16
         +/1    6     5     11  X^2  (independence)  0.75
       Total   12    15     27



Table 2.  Backcross segregation of Aco-1 against other 
mapped enzyme markers. + = L. esculentum cv VF36 allele; 
p = L. pennellii allele.

                             Aco-1
Locus Chromsome      p/p  p/+       p/p p/+   Probability
_________________________________________________________
Prx-1      1    p/p  27   20  p/+   15   21     0.15
Est-3      1    p/p  20   18  p/+   19   18     0.91
Sikdh-1    1    p/p  20   18  p/+   22   22     0.81
Prx-2      2    p/p  17   19  p/+   24   19     0.44
Est-7      2    p/p  15   13  p/+   18   20     0.61
Tpi-2      4    p/p  26   14  p/+   15   26     0.01
Pgm-2      4    p/p  27   18  p/+   14   23     0.05
Aps-1      6    p/p  19   20  p/+   23   20     0.66
Got-2      7    p/p  21   24  p/+   21   16     0.36
Aps-2      8    p/p  20   15  p/+   22   25     0.35
Prx-4     10    p/p  25   17  p/+   17   24     0.10
Est-4     12    p/p  21   13  p/+   20   28     0.07
Pgi-1     12    p/p  25   14  p/+   17   27     0.02
_________________________________________________________

Table 3.  Joint segregation data for Est=4, Pgi-1 and Aco-1.

Est-4   Pgi-1   Aco-1      #
_____________________________
p/p    p/p    p/p          21
+/p    +/p    +/p          28
p/p    +/p    +/p           3
+/p    p/p    p/p           4
p/p    p/p    +/p          11
+/p    +/p    p/p          19
+/p    p/p    +/p           4
p/p    +/p    p/p           1

                  Total =  91

Est-4 -- Pgi-1  12/91  = 0.13 = 13 cM
Est-4 -- Aco-1  37/91  = 0.41 = 41 cM
Pgi-1 -- Aco-1  34/91  = 0.37 = 37 cM