CAMPINAS, BRAZIL
Campinas State University
Genetic diversity among tropical maize inbred lines as revealed by SSR markers --Laborda, PR, Oliveira, KM, Garcia, AAF, Paterniani, MEAGZ, Souza, AP Diversity of maize inbreds has a major importance to the process of maize improvement. In Brazil, little is known about the variability of this crop and an analysis of its potential is extremely necessary. The use of codominant molecular markers such as the microsatellites, or Simple Sequence Repeats - SSRs, is an appropriate and useful way of acquiring data about diversity when the genome is being surveyed. This work has the objective of (1) analyzing the diversity among 95 maize inbred lines chosen from the IAC (Instituto Agronômico de Campinas) germplasm bank and (2) dividing them into heterotic groups. This germplasm bank is composed of old lines as well as recent lines, introduced from CIMMYT.

Amplifications were carried out using 50 ng of DNA and a "touchdown" PCR with annealing temperatures varying from 65 C to 55 C. The PCR products were visualized in 4% agarose/metaphor gels stained with ethidium bromide. In order to facilitate the genotyping of each microsatellite, an "allele pattern" was established among 28 inbreds. This pattern could represent the allele diversity and was organized as a molecular weight marker, which was used later for comparison with the other 67 inbreds.

To select the best primer pairs, three sets of tests were performed: the first among three lines, the second among eight lines and the third among 28 lines. The second was the most efficient of all, being able to reveal the polymorphism between the inbreds and not wasting much effort and time. A total of 193 microsatellite primer pairs were tested, and classifications according to amplification quality and genotyping difficulty were done. Most primer pairs had a good amplification quality; nevertheless, many of them were also classified as "difficult" for genotyping, with little difference in the number of base pairs among the amplification products obtained from the 95 inbreds. From the 193 microsatellites tested, 14 were chosen for the diversity analysis.

Results in Table 1 show the number of alleles for each locus studied and its PIC (or genic diversity) values. Among the 14 SSRs, a mean of 5.7 alleles/locus was observed and the mean for the genic diversity was 0.6. These results show the great discriminatory abilities of the loci analyzed and suggest large diversity among the germplasms being studied. The dendrogram presented in Figure 1 shows the genetic distances as provided by the modified Rogers coefficient (TFPGA and NTSYS/UPGMA 2.02 used). This index was chosen due to its Euclidian behavior and due to the fact that it may be compared to other measures of this kind, since it is calculated using means among the loci studied. Further, it considers allelic frequencies, which are extremely important when working with codominant markers. The cophenetic value was 0.57, which indicates that the schematic representation of the dendrogram was not able to show with confidence the distance matrix provided by the index. Moreover, it was not possible to visualize defined groups within the dendrogram, which may be caused by the insufficient number of loci analyzed until now. In order to provide more confident schematic representation, the genetic distances in the distance matrix were grouped and represented in Figure 2. Notably, the majority of the distances are between 0.6 and 0.9, which indicates great divergence among the inbreds.

The results presented in this work suggest that the tropical germplasm under analysis is an excellent material for hybrid breeding programs. A greater characterization of this germplasm will provide a larger understanding of the potential tropical maize has and will provide quality data for the maize improvement programs in Brazil.

Table 1. Number of alleles obtained for each SSR locus and the respective PIC values.
 
Locus Number of Alleles PIC
bnlg1621b 10 0.8614
bnlg1724 6 0.4676
phi022 3 0.5199
umc1069 8 0.7076
umc1122 4 0.5058
umc1221 9 0.7981
umc1230 6 0.7898
umc1252 2 0.3006
umc1357 4 0.6409
umc1395 3 0.5203
umc1416 2 0.4943
umc1639 5 0.7521
umc1804 15 0.8754
umc1943 4 0.3166
Mean  5.7 0.6107

Figure 1. Dendrogram showing the genetic distances according to the modified Rogers coefficient and to the UPGMA clustering method. Cophenetic value = 0,5767.

Figure 2.
 
 


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