Preliminary cytological studies on a maize strain carrying the latent1 supergene
--Pierozzi, NI, Miranda, LEC de, Miranda, LT de*
*deceased

The latent1 gene complex (ltp - lte-1 - Lsc) has been studied and mapped in the short arm of chromosome 2 of maize by Miranda and Miranda (MNL 62:39, 1988) and Miranda et al. (MNL 58:48-50, 1984; MNL 60:29, 1986; MNL 61:27-29, 1987; MNL 64:35-36, 1990). This complex has been referred as a supergene and is involved in resistance against drought, heat, and frost. The cellular basis is protoplasmatic resistance by hydrophobic proteins and by sulfhydryl-disulfide bonds. The latent1 supergene was first discovered in a Michoacan race, and it is formed mainly by Fas - Flt - Lsc - ltp - lte and B genes. All these genes are brought together and are involved in an efficient stomatal and heat control mechanism associated with anthocyanin pigment presence. According to Miranda et al.'s hypothesis (MNL 67: 21-24, 1993) the latent complex is mantained together because: (1) the distance between these genes is so small; and (2) the complex is near a knob in the short arm of chromosome 2 (2S) in such a way that crossing over is reduced or not formed inside the complex, favoring the supergene's existence. In a breeding program developed in IAC, some directed crossing involving races such as TuxpeÒos and Catetos with the latent1 donor Michoacan race has been done. Cateto was an autochthonous Brasilian race similar to the Coastal Tropical Flints (McClintock et al., Colegio de Postgraduados, Chapingo, 1981). It has some resistance fators such as aluminium tolerance mapped on chromosome 10, but does not have latent1 supergene (Miranda et al. MNL 58:38-46, 1984).

Preliminary cytological analyses were done in two stocks of Cateto Assis Brasil sub-race, native to the Brasilian southern region, as part of the breeding program. One of these stocks, named Ip 48-5-3, was obtained by 3 cycles of selfing. It was considered as the ìcontrolî, because it does not have the latent1 complex in chromosome 2. The other stock was the Ip 48-5-3 lte-1 ìisolineî which was obtained through crossing Ip 48-5-3 with a latent1 stock derived from Michoacan race 21 compuesto 1-104 germplasm (IAC Maya latente). Both inbred lines were field tested in different experiments to detect the presence of the latent1 character.

Seeds of both Cateto Ip 48-5-3 ìcontrolî and Ip 48-5-3 lte-1 were germinated in moist filter paper at 26 C. Roots were collected, treated in para-dichlorobenzene saturated solution (p-DB) for 3 hours at 16 C, fixed in Carnoy 3:1 solution (ethyl alcohol p.a. and acetic acid p.a., respectively) and stored at -20 C until the cytological preparations were done. Softening of tissues was done by treating the roots with an equal enzymatic solution mixture of pectinase 20% and cellulase 2%. We employed the C-band procedure as described by Pierozzi and Jung-MendaÁolli (Cytologia 62:80-91, 1997) for metaphase chromosome characterization. C-banding technique was chosen because there is a good correspondence between pachytene knobs and mitotic metaphase C-bands, as demonstrated by Aguiar-Perecin and Vosa (Heredity, 54:37-42, 1985).

Preliminary data have shown that this variant of C-bands was suitable for visualizing knob heterochromatin. Ip 48-5-3 Cateto control has a C-band only in chromosomes 3, 6 and 7 and none in the short arm of chromosome 2 (Figure 1A). Isoline Ip 48-5-3 lte-1, which carries the latent1 supergene, has C-bands in chromosomes 3, 6, 7 and 8 and also in the 2S position (Figure 1B). Knob heterochromatin number revealed by C-bands in the control stock differs from that obtained by McClintock et al. (Colegio de Postgraduados, Chapingo, 1981) for some Cateto samples. These authors found a higher knob number than that observed here and almost all chromosomes have knobs. It is possible that this Cateto sub-race was not plotted in that work.

The involvement of knob heterochromatin in the establishment of supergenes was first proposed by Kato (Colegio de Postgraduados, PhD thesis, 1976). Heterochromatin DNA has some properties of recombination inhibition near it, as cytologically demonstrated by authors such as Torrezan and Pagliarini (Caryologia 48:247-253 1995). In maize evolutionary history, knobs were set in some chromosome sites. Recombination events were probably reduced in their neighbourhood and in some cases this event may have favored supergene development which just conferred different adaptive values to the races (Kato, 1976). So, in this way the 2S knob, as visualized by C-banding technique in the isoline Ip 48-5-3- lte-1, must have helped the development of the latent1 supergene, as hypothesed by Miranda et al. (MNL67:21-24, 1993).

Figure 1. C-band karyotype of two stocks of Cateto Assis Brasi. A) Ip 48-5-3 considered as control with no C-band in chromosome 2 short arm. B) Isoline Ip 48-5-3-lte-1, carrying latent-1 supergene, with C-band in chromosome 2 short arm. In this isoline, one chromosome of pair number 1 was stretched. Bar = µm. 

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