Maize Genetics Cooperation Newsletter vol 87 2013

 

Buenos Aires, Argentina.

IEGEBA-CONICET and  LACyE, Departamento de Ecología, Genética y Evolución, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires.  

 

Karyotypical studies of two maize races from Northeast Argentine (NEA): DAPI-banding and Fluorescent In Situ Hybridization (FISH)

 

Realini MF*; L Poggio and GE González

* [email protected]

 

            Knobs  heterochromatic blocks occur in all Zea species with 2n = 20, varying in size and number across maize races and their wild relatives (Kato, Mass. Agric. Exp. Stn. Bull. 635: 1-185, 1976; McClintock et al., Colegio de Postgraduados, Chapingo, México, 1981; González & Poggio, Genome 54: 26–32, 2011; Poggio et al., Cytogenet. Genome Res. 109: 259–267, 2005). Variation in DNA content has been proposed to be principally due to differences in the amount of heterochromatin, which is mainly located in distal knobs (Laurie & Bennett, Herediy 55:307-313, 1985; Poggio et al., Ann. Bot. 82:115-117, 1998). These structures can be observed as subtelomeric bands by DAPI-banding, and using Fluorescent In Situ Hybridization (FISH) (González et al., Chrom. Res. 14: 629-635, 2006). Knobs have been described as highly repeated tandem arrays of 180-bp and  TR-1 (350 bp) sequences, both repeated in different proportions constituting different knobs (Dennis & Peacock, J. Mol. Evol. 20: 341-350, 1984; Ananiev et al., Proc. Natl. Acad. Sci. 95: 10785-10790, 1998).

In this study we present the karyotypic formulae, asymmetry indexes and the position and composition of the knobs of two races of maize from Argentine Northeast (NEA), Tupi Amarillo (VAV 6563) and Rosado (VAV 6565). DAPI-banding and FISH techniques were applied. The plant material has been provided by the Vavilov Lab. of Universidad de Buenos Aires (UBA), and cultivated in the greenhouse of the Facultad de Agronomía, UBA.

DAPI-banding was performed according to Summer (Chromosomes banding. Unwin Hyman, London, 1990). DAPI fluorochrome (4’-6-diamidino-2-phenylindole) preferentially stains AT-rich heterochromatin in plants (Guerra, Genet. Mol. Biol. 23: 1029-1041, 2000).

The 180-bp and TR-1 knobs sequence of maize was obtained from GenBank (http://www.ncbi.nlm.nih.gov/). These sequences were isolated and amplified from total genomic DNA of maize by PCR methods. The sequences obtained were labeled with biotin and digoxigenin by PCR as well by enzymatic methods. For the latter we used enzymatic kits: BioNick Labeling System (Invitrogen) to label with biotin and Dig High Prime (Roche) to label with digoxigenin. FISH was performed according to González et al. (Chrom. Res. 14: 629-635, 2006). FISH slides were observed with a Zeiss AxioPhot epifluorescence microscope (Carl Zeiss, Germany), and microphotographs were taken with a Leica CCD digital camera.

Chromosomal parameters were measured using the freeware program MicroMeasure 3.3 (http://www.colostate.edu/depts/ biology/micromeasure). The relative chromosome length, arm ratio, and centromeric indexes were calculated to determine the karyotypes. The chromosomes were ordered from the largest to the smallest, as usual for maize, and chromosome morphology was described according to Levan et al. (Hereditas 52: 201-220, 1964). To estimate the karyotype asymmetry, two numerical parameters were used, according Romero Zarco (Taxon 35: 526-530, 1986): A1 (intrachromosomal asymmetry index) and A2 (interchromosomal asymmetry index).

            In each race a maximum of 25 individuals were studied (3-5 individuals per maize ear) and at least 10 cell per individual were analyzed.

The karyotype parameters analysis let us to elaborate the idiograms from the two maize races (Figure 1 A and 2A).

DAPI banding allowed identified and locates the knobs as DAPI-positive bands on mitotic metaphase chromosomes (Figure 1 B and 2 B). In FISH experiments, simultaneous hybridization with the 180-bp and TR-1 probes showed that these sequences co-localized with all the DAPI-positive bands in both races. Different intensities of hybridization signals with each probe suggest that the DAPI-positive knobs are composed by different proportions of 180-bp and TR-1 sequences.

In table 1 the karyotypic formulae, A1 and A2 indexes, percentage of heterochromatin and number of knobs are presented for Tupi Amarillo and Rosado maize races.

We observed that the percentage of heterochromatin of Tupi Amarillo is about 8% higher than Rosado (Table 1), this difference between both maize races is due to the higher number of knobs in Tupi Amarillo. This race showed higher intrachromosomal asymmetry (A1) but lower interchromosomal asymmetry (A2) compared with Rosado. This is due to differences in the size and the distribution of the knobs on the both chromosomal arms (Figures 1 A and 2 A).

All results obtained in this work allowed us to identify cytogenetically the maize races studied.           Then, the variations of the patterns for number, position and sequence composition of the heterochromatic knobs are useful markers for a proper cytogenetic characterization of maize races.

The cytogenetic characterization of different Argentinean races of maize will contribute to the knowledge of the genetic variability within native materials, useful for its integration in future breeding plans and biodiversity conservation.

 

 

 

Tupi  Amarillo

 Rosado

Karyotypic formulae

6m + 4 sm

6m + 1m-sm + 3sm

% of heterochromatin

   19,64

  11,7

Range of number of Knobs

16-22

10-11

A1

0,35

0,27

A2

0,18

0,26

 

 

 

 

 

 

 

 

Table 1: Karyotypic parameters of Tupi Amarillo and Rosado maize races. Ref.: A1: intrachromosomal asymmetry index. A2: interchromosomal asymmetry index. m: metacentric. sm: submetacentric.

 

 

Figure 1: A. Idiogram of maize Rosado. The white blocks represent the coincident DAPI-positive band and 180-bp and TR-1 FISH signals. B. DAPI-banding.  C. FISH using 180-pb as probe on mitotic metaphase chromosomes. Ref.: M: metacentric. SM: submetacentric. IC: centromeric index.  Bar 10 µm.

 

 

Figure 2: A. Idiogram of maize Tupi Amarillo. The white blocks represent the coincident DAPI-positive band and 180-bp and TR-1 FISH signals. B. DAPI-banding.  C. FISH using TR-1 as probe on mitotic metaphase chromosomes. Ref.: M: metacentric. SM: submetacentric. IC: centromeric index.  Bar 10 µm.

 

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