Maize Genetics Cooperation Newsletter 80. 2006.

VIÇOSA, BRAZIL

Universidade Federal de Viçosa

Flow cytometry analysis of DNA content in diploid and autotetraploid maize with B chromosomes

--Carvalho, CR; Saraiva, LS; Mendonça, MAC

Flow cytometry methodology was used to study the DNA content in maize diploids and tetraploids, with and without B chromosomes. The diploid with B chromosome genomes had a 22.4 % increase in DNA over the diploid without B chromosomes, and the autotetraploid with B chromosomes showed only a 3.2% increase in DNA content as compared with the autotetraploid without B chromosomes. Flow cytometry methodology resulted in differentiated histogram peaks to resolve the presence at one B chromosome level, being a useful and highly sensitive methodology for comparative analysis of DNA content differences in maize genomes, as well as for plant B chromosome evolution studies.

Diploid (2x) and diploid plus B chromosome (2x+B) seeds of Black Mexican Sweet Corn, autotetraploid (4x) of N102A, and autotetraploid plus B chromosome (4x+B) of N102E stocks, were kindly supplied from the Maize Genetics Cooperation Stock Center, University of Illinois, USA. As internal standard (5.43 pg-DNA), maize diploid seeds (2x) cv. CE–777 were kindly supplied by Dr. Jaroslav Dolezel (Lab. Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Czech Republic).

Fresh leaves (0.5 cm²) were chopped with a razor blade in a petri dish with 0.5 ml nucleus-isolation buffer (Partec ©). After 1 min, the suspension was passed through a 50 mm mesh nylon filter with 2 ml of 4’-6-diamidino–2 phenylindole (DAPI) solution (Cy-StainUV, Partec®) and stored in the dark for 3 min. Test and standard nuclear materials were processed using a PAS-Partec ® cytometer, previously calibrated and tuned to an excitation wavelength band for DAPI, and analyzed by FlowMax® software. The DNA picogram values were calculated by conversion of data from G₁ peak channels. For each sample, approximately 5000 nuclei were analyzed and those with a coefficient of variation (CV) higher than 3.0 were discarded.

The methodology resulted in a high-resolution histogram (Fig. 1) showing the 2x plant G₁ peak running at channel 100 and the 2x+B plant at channel 122.4. The 5.37 pg DNA value of the 2x plant was determined previously in this work (histogram not shown) using a maize DNA internal standard (5.43 pg). The 2x plant (5.37 pg) was then used as a parameter to calculate DNA content of 6.57 pg in the 2x+B plant. These data show the contribution of maize B chromosomes to the DNA amount to be 1.2 pg (22.4%) in this Black Mexican genome.

The 4x plant had the DNA value of 10.48 pg, determined previously in this work using maize internal standard (histogram not shown). The histogram for 4x and 4x+B plants shows G₁ peaks at channels 200 and 206.4, corresponding to 10.48 pg and 10.82 pg DNA values, respectively (Fig. 2). This genome size difference in tetraploids corresponds to 3.2% due to B chromosome presence in

Figure 1. DNA histogram of the nuclei DAPI stained from leaves of diploid (2x) and diploid plus B (2x+B) chromosomes of Black Mexican Sweet Corn. The G₁ peak of the 2x was set to channel 100. The G₁ peak of nuclei from 2x+B was at channel 122.4, corresponding to 5.37 and 6.57 pg DNA values, respectively.

Figure 2. DNA histogram of the nuclei DAPI stained from leaves of autotetraploid (4x) and autotetraploid plus B (4x+B) chromosomes of N102A and N102E maize stocks, respectively. The peak G₁ of 4x plant nuclei running at channel 200 (10.48 pg DNA) and very close to the G₁ peak of 4x+B at channel 206.4 (10.82 pg DNA).

the N102E stock.

Ayonoadu and Rees (Heredity 27:365-383,1971) studied the genome size in individuals without B and with 8B chromosomes in the diploid Black Mexican Sweet corn line, and they calculated that each additional B increases the DNA content by 5%. In the present study, the Black Mexican Sweet sample shows 4B chromosomes, as described by Carvalho et al (MNL 77:80, 2003). According to this data, the genome size difference analyzed by flow cytometry shows a 22.4% increase in DNA amount for the 2x+B in relation to the 2x, giving an average of 5.6% for each B chromosome. The 3.2% DNA content due to the B chromosome in the 4x+B N102E stock suggests the presence of only one B chromosome.

This methodology was able to detect DNA content differences in genome sizes with B chromosomes at the diploid and tetraploid levels. It was concluded that each B chromosome increased DNA amount approximately 5.6% in 2x+B Black Mexican Sweet, and only 3.2% in 4x+B N102E samples.

Flow cytometry methodology resulted in differentiated histogram peaks to resolve the presence at one B chromosome level, being a useful and highly sensitive methodology for comparative analysis of DNA content differences in maize genomes, as well as for plant B chromosome evolution studies.

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